DYNAMIC ASPECTS OF BIOCHEMISTRY DYNAMIC ASPECTS OF BIOCHEMISTRY BY ERNEST BALDWIN, B.A.,Ph.D. Professor of Biochemistry at University College fn the University of London, formerly Fellow of Si John's College, Cambridge THIRD EDITION CAMBRIDGE AT THE UNIVERSITY PRESS 1960 PUBLISHED BY tSG SYNDICS OP TUB C4MBRIDQE UNIVERSITY PRESS Bentley House. 200 Eoaton Road, London. N.Y?.l American Brunch: 32 EMt S 7th Street, New York 22. N.Y. © CAMBRIDGE UNIVERSITY PRESS 1957 firtt Edition 5017 Reprinted J011 JfaMian translation 1D1S Rtprinltd 1049 Italian translation 1051 Spanish translation 1052 •Second Edition 1052 Reprinted ) 953 Japanese translation 1053 German translation 1957 Third Edition 1057 Reprinted 1059 10CU Serbo-Croat translation 1 0CO Polish translation 1000 Portuguese translation in preparation Awarded (he. Cortina-Ulitie Prize 1052 JVifiled in Great Britain at the University Press. Cambridge (Broole Crvichley, University Printer) HOPPY (Sir Frederick Gowland Hopkins, 031., F.B.S.) WITH RESPECT, ADMIRATION AND AFFECTION 'Tho difference between a piece of stone and en atom is that an atom is highly organised, whereas the stone is not. Tho atom is a pattern, and tho molecule is a pattern, and the crystal ia a pattern; but tho stone, although it is mado up of these patterns, is just a mere confusion. It’s only when lifa appears that you begin to got organisation on a larger scale, life takes tbo atoms and molecules and crystals; but, instead of making a mess of them liko tho etone, it combines them into new and more elaborate patterns of its own.’ Atnocs Hcxlkv, Tima must have a Stop CONTENTS Tablet Figures and Diagrams Preface, to the Third Edition Preface to the First Edition A [knowledge-men Is page xii xiii XV xvii PART I. ENZYMES CHAPTER I. THE GENERAL BEHAVIOUR AND PROPERTIES OF ENZyjiES Introduction I Nomenclature and classification of enzymes 7 Specificity 8 The chemical nature of enzymee 14 Summary 22 CHAPTER II. THE NATURE OF THE CATALYTIC PROCESS The union of the enzyme with its substrate 24 Influence of concentrations of the enzyme and its substrate 20 Competitive inhibition 33 Activation of the substrate 35 Activators and coenzymes 33 Prosthetic groups 43 Quantitative characterization of enzymes 45 Summary 43 CHAPTER III. BIOLOGICAL ENERGETICS The concept of free energy Breakdown and synthesis in biological systems Reversibility of biological reactions Energetics of synthetic reactions CONTESTS Properties and functions of adenosine triphosphate page 62 The biological energy cycle 66 The etorago of high-energy phosphate 68 Summary 70 CHAPTER IV. HYDROLASES AND ADDING ENZYMES General introduction 71 Peptidases 72 Carbohydrasee 85 Lipasea and esterases 05 Other hydrolytic enzymes 100 Adding enzymes 102 CHAPTER V. TRANSFERRING AND I80MER1ZING ENZYMES General introduction 108 Transfer reactions catalysed by hydrolytic enzymes 109’ Transphosphorylation 114 Transglycosylntion 1 ] 8 Transpeptidation 124 Transamination 127 Transamidination 129 Transcaibaroylation 129 Transmethylation 130 Trnnsthiolation 131 Transacotylation 131 Transketolation 132 Tramaldolation 134 Isomer izing enzymes 135 CHAPTER VI. OXIDIZING ENZYMES Tlie oxidation of organic compounds 140 Oxidases 146 Warburg'a respiratory enzyme and the cytochrome system 160 Accessory carrier systems 172 tiii CONTENTS CHAPTER VII. DEHYDROGENASE SYSTEMS Dehydrogenases and co-dehydrogenases page 175 Flavoproteins and the reduction of cytochrome 182 Reversibility and coupling of dehydrogenase systems 197 Intracellular organization of respiratory enzymes 201 Tissue respiration and the capture of free energy 205 PART n. METABOLISM CHAPTER VIII. METHODS EMPLOYED IN THE INVESTIGATION OF INTERMEDIARY METABOLISM General principles 211 Studies on normal organisms 213 Studies on abnormal organisms 210 Studies on perfused organs 219 Use of physiological salines 221 Use of tissue Blices 222 Us© of breis, homogenates, extracts, etc. 223 Uso of isotopes 227 CHAPTER IX. FOOD, DIGESTION AND ABSORPTION Food 230 Digestion and absorption of proteins 239 Digestion and absorption of carbohydrates 241 Digestion and absorption of fats 245 CHAPTER X. GENERAL METABOLISM OF PROTEINS AND AMINO-ACIDS Functions and fate of proteins and amino-acids 25 1 Fate of a -nmino-ni tr ogen 256 Deamination 257 Transdeamination 261 Storage of amino-groups 264 CONTENTS CHAPTER XI. SPECIAL METAHOLISM OF THE AMINO- ACIDS General: glycogenosis and ketogenesis page 268 General: bacterial attack and detoxication 270 Optical isomerism and nomenclature 272 Specific metabolism 276 CHAPTER XII. EXCRETORY METABOLISM OF PROTEINS AND AMINO -ACIDS Nature of the nitrogenous end-products 208 Synthesis of the end-products: urea 303 Synthesis of the end-products: urio acid 316 Synthesis of other end-produets 322 CHAPTER XIII- SOME SPECIAL ASPECT8 OF "NITROGEN METABOLISM Distribution of choline, tetraroethyl ammonium hydroxide, tri- methylanuno and trimcthylamino oxide 325 Distribution of betaines 328 Distribution of bases related to guanidine 330 Distribution of iminnzole bases 338 Distribution of othor nitrogenous compounds 339 CHAPTER XIV. METABOLISM OF PURINE DERIVATIVES Nucleaproteins 341 Nucleic acids 343 Digestion of nucleoproteina 346 Formation of tho nitrogenous bases 346 Breakdown and formation of nucleosides and nucleotides 348 Functions of nucleosides and nucleotides 352 Metabolism of purino bases 357 Metabolism of uric acid , 359 CHAPTER XV. CARBOHYDRATE PRODUCTION IN THE GREEN PLANT Introduction 362 Photolysis of water 303 X CONTENTS Fixation of carbon dioxide page 364 Production of starch 369 Production of sucrose 370 Production of other carbohydrates 370 Formation of amino -acids 371 CHAPTER XVI. ANAEROBIC METABOLISM OF CARBOHYDRATES: ALCOHOLIC FERMENTATION Introduction 372 Alcoholio fermentation of glucose 37 C Alcoholic fermentation of other sugars 368 Energetics of fermentation 389 Fermentation by living yeast 391 Fermentative manufacture of glycerol 393 Production of fusel oil 395 CHAPTER XVII. ANAEROBIC METABOLISM OF CARBOHYDRATES: MUSCLE AND LIVER Introduction 397 Formation of lactic acid 403 Parte played by ATP and phosphagen 405 Chemical events in normal contraction 410 Glycolysis in tissues other than muscle 412 The liver: glycogenosis, glycogenolysts and glyconeogenesis 410 CHAPTER XVIII. AEROBIC METABOLISM OF CARBOHYDRATES Introduction 424 Origin of respiratory carbon dioxide 427 Oxidative metabolism of pyruvate 420 The mechanisms of oxidative decarboxylation 430 General metabolism of pyruvic acid 439 Aerobio metabolism of carbohydrates 44 ] The citrio acid cycle 447 Energetics of carbohydrate oxidation 403 OONTENTS CHAPTER XIX. THE METABOLISM OF FATS Transport and store go of fats page 466 Functions of fati constant and variable elements 469 Metabolism of fata: general 470 Desaturation 470 /7-Oxidation 477 Mechanisms of fatty acid metabolism 479 Formation, of acetoacetio acid 485 Oxidation of acetoacetio and /J-hydroxybutyrio acids 489 Synthesis of fatty acids 491 Conversion of fat to carbohydrate 494 Bibliography 406 Index of subject* G01 TABLES 1. pH Optima of some enzymes 46 2. Miehaelia constants of some enzymes 47 3. Calculated and observed heat outputs of dogs 67 4. Approximate standard free energies of hydrolysis of some phosphorio acid derivatives 63 6. Some high-energy phosphate derivatives 64 6. Action of pepsin upon synthetic peptides 78 7. Action of pepsin and chymotrypsin upon syntboti© peptides 79 8. Linkages attacked by amylolytio enzymes 86 9. Fhosphokinnses 116 10. Linkages formed by enzymes synthesizing amylase, araylo- pectin and glycogen J21 11 . Classification of dehydrogenating enzymet 140 12. Cocnzymo requirements of dehydrogenases 180 13. Turn-over numbers of some enzyme systems 197 14. Approximate free energies of some respiratory carriers 208 1 6. Composition of mammalian blood somm and Krebs's physio- logical saline 221 16. Catalytic functions of some members of the B group of vitamins 232 17, Absorption of monosaccharides from the small intestine of rata xii 243 CONTENTS 18. Absorption of fatty acids page 248 19. Nutritional status of amino -acids 252 20. Fatos of amino-acids administered to a diabetic or phlor- rhizinized dog 255 21. Rates of deamination of amino-acids by sliced rat kidney tissue 258 22. Oxidative deamination of amino-acids by kidney extract 250 23. Deamination of amino-acids of the naturally occurring L-eeries in animal tissues 270 24. Nitrogen excretion of some cbelonion reptiles 305 25. Nitrogen excretion of vertebrates in relation to water supply 309 20. Distribution of arginase in liver and kidney of vertebrates 310 27. Arginoso contents of various tissues 313 28. Distribution of arginine and creatino in the animal kingdom 334 29. Nucleotides and derivatives of catalytic importance 35 L 30. End-products of purine metabolism 360 31. Enzymes concerned in pbotosynthetio carbon-fixation 360 32. Alcoholic fermentation: enzymes, coenzymos and inhibitors 387 33. Phosphorus partition in striated muscle 409 34. Concentration of phosphagen in various tissues 413 35. Glycolysis: enzymes, coenzymes and inhibitors 416 36. Enzymes involved in the citric acid cycle 457 37. Enzymes involved in breakdown of fats and carbohydrates 474 FIGURES AND DIAGRAMS 1. A typical ‘progress curve'; tiyptio digestion of casein 15 2. Influence of temperature on digestive proteinase of Tethyum 1 8 3. Influence of pH on the activity of some enzymes 10 4. Influence of small, increasing concentrations of silver ions on 22 activity of yeast saccharose 6. Influence of substrate concentration on activity of yeast Baccharaso 27 6. Theoretical curve for Briggs & Haldane’s equation 30 7. Influence of enzyme concentration; yeast saccharose 31 8. Action of a group-specifio enzyme upon two different sub- strates 30 0. Influence of anions upon activity of salivary amylase 40 10. The 'energy dynamo' gg xiii ■ CONTENTS 11. Specificity requirements of endopeptidasee page 80 12. Hydrolysis and synthesis of triolein by Jtieintu lipase SO 13. Influence of urea concentration on activity of ureas© 101 14. Thunberg vacuum tubes 143 15. Absorption spectrum of the carbon monoxide compound of the Almungs/erment 102 lfl. Absorption spectrum of a haemochroraogen (carbon mon- oxide compound of a chlorocruorin) _ 103 17. Absorption spectrum of reduced cytochrome in the thoracic muscles of a beo 164 18. Absorption bands of cytochromes of various tissues 165 19. Absorption bands of the components of a typical cytochrome 165 20 Absorption spectrum of tho carbon monoxide compound of cytochrome oxidase (heart) 170 21. Absorption spectra of oxidized and reduced DI*N 178 22. Oxidation and reduction of DPN in a coupled reaction 201 23 Fractionation of ‘electron-transporting particles’ 204 24. Nitrogen metabolism in seedlings of Lupinus luletta 206 26. Nitrogenous excretion of developing chick embryo 308 20. Tho ornithine cycle 312 27. Reaction network in CO,-fixation 367 28. Schcmo to summarize reactions of alcohob’c fermentation of glucose by yeast juice 336 20. Neuberg’s three ‘forma’ of fermentation 394 30. Scheme to summarize reactions of glycolysis 414 31. Glycogenosis from common mono- and disaecharides 413 32. Relationships between glycogenosis, glycogenolysis and glycolysis 421 33. Changes of blood lactic acid during and after exorcise 420 34. Formation and fate of pyruvic acid 440 35. Summary of reactions leading from citrate to pyruvate 418 30, Outline of tho citric cycle hypothesis 449 37. Tho citric acid cycle 450 38. Outlines of metabolism of fats and carbohydrates 473 W. ^-OxiiaVivoVreakdown of 5aVty acids 483 xiv PREFACE TO THE THIRD EDITION There has been no diminution in tho rate of progress of bio- chemistry during the five years since the last edition of this book appeared. In preparing this new edition it has often been difficult to decide what new material to include and what to leave out. The book was originally planned rather as an ele- mentary than as a reference work and, in the new edition, I have tried to retain its original flavour; to give broad cover rather than a welter of detail. A considerable number of new discoveries of fundamental and general importance, e.g. those of transketolation, and trans- aldolation, the new work on photosynthesis, oxidative decarb- oxylation, purine synthesis and the like, deserve a place in any work of this kind. On the other hand there are fields no less important but in which the available information is vciy complex and in which even the experts differ, c.g. in the chemistry of nucleic acids and nucleoproteins. While these must be studied by every advanced student they cannot be dealt with at more than a superficial level in such a book as this. I accept full responsibility, therefore, for what may seem to some to be errors of judgement in deciding what to include and what to leave out. To find space for new material without sacrificing some of the old is manifestly impossible unless tho size of the book is to be increased and this, I think, is undesirable. A good deal of drastic pruning lias therefore been necessary. Some passages of a mainly historical interest have been removed bodily, others have been abbreviated or condensed. The remainder of the text I have done my best to bring up to date. I owe much to many friends and colleagues who have brought to my attention a considerable number of papers that had escaped my own notice, and in this connexion I am especially grateful to Drs D. J. Bell and K. R. Rees. Tho latter also under- took what is surely the most difficult and certainly the most tedious part of the whole task; the preparation of the index. PRECAGE Dra E. M. Crook and B. Rabin read large sections of the copy and to them I am grateful for many suggestions and criticisms. If the thermodynamic ideas expressed here aro any less exas- perating to pure chemists than they were formerly, the credit is due to the influence of these last-named colleagues. To my wife, who helped and supported me in every way and at every stage of this revision, I owe more than I can say. The Cambridge University Press, which exerted every effort to produce this new edition at high speed and co-operated in every way, deserves my wannest thanks. At times their task has been a difficult one, for the proof reading and type setting operations were separated by some 6,000 miles. E.B. LA JOLLA, CALIFORNIA n July mr AVI PREFACE TO THE FIRST EDITION In spite of war-time difficulties and restrictions, Biochemistry- lias continued to expand more and more rapidly each year, in stature as well as in scope. It has been impossible for many years past for any one worker to read more than a small fraction of the new output, even when foreign journals were available. Without the invaluable aidoftheAnnttaZ -Reviews o/BtocAejntsfry, to whose authors and editors I and every other biochemist must pay high tribute, the preparation of this new book would have been impossible. Even with their help, some sections of the book will probably be out of date by the time it appears in print, and may well be out of date already in certain respects. But the last few decades have seen the establishment of a considerable body of information which, though it may change considerably in detail, will perhaps not change significantly in substance during the next few years. I venture to hope that a new edition may be called for before the present contents have become wholly archaic, so that there will be opportunity to correct the many faults which have doubtless escaped notice and to bring the whole volume up to date. The subjeot-matter of biological chemistry, like that of biology itself, can be roughly divided into two parts; the static, or morphological, and the dynamic, or physiological. Knowledge of the latter demands as an essential pre-requisite a knowledge of the former, and it is therefore a matter for rejoicing that many organio chemists of the present day are devoting their attention to the chemical constitution and configuration of the organio Bausteine that form the material basis of living cells. In some fields, notably in that of protein chemistry, the interdependence and collaboration between the organic and the biochemist are so intimate that it is impossible to say which is the organic chemist and which the biochemist. If any differentiation were necessary it could best be made, probably, in terms of their respective attitudes towards tho Bausteine. For the organic chemist the main focus of attention is the structure and xvii PREFACE configuration of these materials "while, for the biochemist, the main problems are those of the behaviour and function of these substances in organized, biological systems. The more static aspects of these Bausleine are already fairly well covered by monographs and review articles, some of which I have indicated in the bibliography; the essentially dynamic aspects, on the other hand, have hitherto been but inadequately described and, in view of their wide importance and interest, I believe that a real demand exists at the present time for a book of this kind. Elementary Biochemistry is taught in this University in two courses. The first and older of these. Chemical Physiology, forms part of the course in mammalian Physiology, and caters primarily for the needs of medical and veterinary students- For these there already exists a wealth of text-books, which the present volume neither hopes nor desires to supplant. In the second course, much more recently introduced, Biochemistry is taught os an independent scientific) discipline, and without that emphasis on clinical problems with which it has usually been associated in the past, and which properly finds a place in Chemical Physiology. For students taking this second course there exists no suitable text-book, and it is primarily with their needs in mind that the present book has been written, I hope, howevor, that it will also help to open up new horizons to those whose interest in Biochemistry is primarily that of the organic chemist or the clinician in training. Perhaps it will serve too as an introduction to others who, wishing to take an advanced degree course here or elsewhere, find it difficult at the present time to discover suitable elementary reading. With the needs of such students os these in mind I have included a short bibliography of review articles and books, mostly of recent date and written by experts in their respective fields. In Biochemistry, as in any young but rapidly expanding branch of science, there are fields in which faota are scanty, evidence contradictory and speculation rife. I have tried to avoid such topics, but where this has not been possible I have attempted to give a critical account of the facts but, at the same time, to speculate a little. I cannot wholly subscribe to the doctrine that speculation is out of place in an elementary text- xviii TBEFACE book, for there aro many gaps in the subject, and unless these can in some way bo bridged it is difficult or impossible to give a coherent account. My experience as a teacher has been that coherence is essential in an elementary exposition. Speculation plays and has always played an important part in the advance* ment of scientific knowledge, for no research worker gropes blindly after he knows not what; he invariably begins with certain reasonable possibilities in mind. In short, he speculates. To speculate unreasonably is worse than not to speculate at all, but providing certain tests of reasonableness and compatibility are applied beforehand, speculation is a valuable tool, and one which finds a place in every scientific workshop. The danger is that speculation is not always recognized as what it is, and I have, therefore, tried to distinguish clearly between fact and fantasy, hoping in this way to steer a middle course between unbridled imagination on the one hand, and an equally undesirable hyper- trophy of the critical faculty on the other. A word of explanation is perhaps necessary for the use in these pages of somewhat novel and certainly unorthodox methods of writing the equations of certain chemical reactions and groups or sequences of reactions. I have adopted them only after a long period of trial. They givo a distinctively pictorial representation of chemical events, and many students find such a picture more easily comprehended and remembered than the more formal representations usually adopted. I trust that the reader will exercise the little patience necessary to become familiar with these ‘whirligigs for they have great advantages in cases where a long chain of successive chemical events has to be described briefly and as a whole. The writing of this book has been largely a spare-time occupa- tion, and there has been little enough spare time during the war years.. 1? tog-ess has often, been slow., therefore,, and. the task baa nearly been abandoned more than once. I owe the fact of its eventual completion to the kind encouragement given to me by ray friends and colleagues. Particular thanks are due to Dr D. 3 . Bell and Dr E. Watchom, who have read the whole of the manuscript, and to Prof. A. C. Chibnall, who read the proofs. I am glad also to acknowledge the help I have had from Miss V. Moyle. These, and others who have read particular PREFACE sections and chapters, have all given precious advice and valuable criticisms. Sly task has been simplified in many ways by Prof. J. B. S. Haldane’s Enzymes and by Hr D. E. Green’s Mechanisms of Biological Oxidations, and particular thanks are due to Dr Malcolm Dixon, who has given me much from his great personal store of information. I should also like to record my thanks to Dr J. C. Boursnell, who heroically undertook the preparation of the index, to Mr H. Howl, who prepared the drawings for Fig. 14, and to members of the Cambridge Part I Biochemistry Class of 1945-6, who have allowed me to make use of some of their experimental data in the preparation of Figs. 1, 3 and 7. To my wife, who prepared the work for publication, and to all departments of the Cambridge University Press I wish to express my humble and hearty thanks for their patience, consideration and expert workmanship. E.B. OAUDKIDOE January 19{G ACKNOWLEDGEMENTS The author’s thanks are due to the following for permission to reproduce figures : the Cambridge University Press for Figs. 2, 30, and 24; Dr H. Fraenkel-Conrat and the Journal of Biological Chemistry for Fig. 3B; Drs F. Schlenk and F. Lipmann and the University of Wisconsin Press for Figs. 21, 22 and 31 ; Messrs Longmans Green &. Co., Ltd., for Figs, 4, 5, 0 and 13; and Messrs W. HcfFcr & Co. Ltd. for Fig. 12. PART I ENZYMES CHAPTER I THE GENERAL BEHAVIOUR AND PROPERTIES OF ENZYMES INTRODUCTION Wherever we turn in the world of living things we find chemical changes taking place. Green plants, together with cer- tain bacteria, are capable of fixing solar energy and synthesizing complex organio substances of high-energy content from very simple starting materials, namely, water, carbon dioxide and small amounts of inorganic substances such as nitrates and phosphates. Other living organisms possess tho ability to de- compose these complex materials and to exploit for their own purposes the energy that i3 locked up within them, and it is in this way that animals, for instance, obtain the energy thoy expend in the discharge of their bodily functions ; reproduction, growth, locomotion and so on. Now it is a significant fact that nearly all the chemical changes that go on in living tissues are changes which, in themselves, proceed too slowly to be measurable or even, in many cases, detectable. How, then, does it happen that living animals can obtain energy and expend it as fast as they do? The answer is that living organisms possess numerous catalysts which speed up chemical reactions to tho rates achieved in biological systems. Whether we consider diges- tion, metabolism, locomotion, fermentation or putrefaction, chemical changes are going on, and these chemical changes are catalysed. It is the purpose of this hook to give some account of these changes and of the various mechanisms at present known to participate in their catalysis. A catalyst, in the classical definition of Ostwald, is ‘an agent * 1 BDA CATALYSIS which, affects the velocity of a chemical reaction without appearing in the final products of the reaction’. Examples of catalysts are familiar to every student of chemistry, and perhaps the most striking is that commonest of all chemical reagents, water. As is well known, hydrogen and chlorine react together with explosive violence if exposed to sunlight, and yet, as H. B, Baker showed, perfectly dry hydrogen and perfectly dry chlorine fail to react together at all. Baker found that numerous familiar reactions do not proceed except in the presence of traces of water, and that water is, in fact, a very important catalyst. Finely-divided metals, such as platinum, nickel and palladium, are also capable of catalysing a wide range of reactions, and Wicland, for instance, found that on the addition of colloidal palladium to aqueous solutions of various simple organic com- pounds, a catalytic oxidation (dehydrogenation) of the com- pounds ensues. Many more examples could be cited. Thus the hydrolysis of esters is a process that goes on only slowly in neutral solutions but is greatly accelerated by traces of strong acids or alkalis. Again, chemical reactions as a wholo proceed more rapidly at higher than at lower temperatures. But living organisms do not have at their disposal the strong acids and alkali, the high temperatures and the other artifices which aro available to a chemist working in a laboratory, yet the synthetic ability of living cells and tissues far surpasses that of the chemist. We must know something about simple catalysts and their mode of action before turning to the more complex catalysts and catalytic systems that we find in living tissues. There aro many resemblances between catalysis as effected by more or less com- plex chemical reagents on the one hand end by biological systems on the other, but differences also exist. In tlio first place, a cata- lyst, of whatever kind, only affects the rale of the reaction which it catalyses. This fact is particularly well illustrated in the case of a reaction such as the hydrolysis of on ester, which is re- versible. If we take ethyl acetate, fdr example, and heat it with water, the ester is slowly hydrolysed, but the reaction stops before the hydrolysis is complete. On the other hand, if wo start with equivalent proportions of ethyl alcohol and acetic acid and heat these together, we find that they react to form ethyl CATALYSIS acetate, but once again the reaction stops before reaching com- pletion. Indeed, from whichever side we start, the composition of the final reaction-mixture is always the same, the system attaining a state which can be represented by the following equilibrium: CjTIjOH + CHjCOOH ?=* CH,COO.C,H, + n,0. If we employ dilute mineral acid as a catalyst we again obtain a reaction-mixture of the same composition, while if a biological catalyst such as liver esterase is used, the final composition is the same once more. These facts point to several important conclusions: first, that only the reaction velocity, and not the extent to which the reaction proceeds, is affected by the catalyst, and secondly, that in the case of a reversible reaction (and on theoretical grounds it is usual to assume that all reactions are reversible) the catalyst influences the reaction velocity equally in both directions. The direction in which such a reaction will proceed is determined, of course, by mass-law considerations and by the availability of free energy. We must therefore suppose that a catalyst which accelerates the decomposition of a given substance must also be capable of catalysing its synthesis. But it does not by any means follow that the necessary conditions can be experimentally realized at the present time. A second important feature of the phenomenon of catalysis is that the effect of the catalyst is normally out of all proportion to the amount used. A minute quantity of colloidal platinum is sufficient to catalyse the decomposition of an unlimited amount of hydrogen peroxide, provided nothing happens to interfere with its catalytic properties. In practice, however, it frequently happens that catalysts are inhibited (‘poisoned ’) by the presence of extraneous material. Thus, in the example just given, minute quantities of hydrocyanic acid, mercuric chloride or certain other substances suffice to destroy the catalytic properties of the platinum. This 1 poisoning ’ is often a serious nuisance in com- mercial processes, but in many cases the catalytio activity can be recovered relatively easily. In biological systems, too, we find that a comparatively small concentration of the catalytic material is all that is necessary, and that the catalysts are easily inhibited, sometimes reversibly and sometimes irreversibly, in a variety of ways which we shall discuss in later sections. 3 1-3 0ATALYSI8 According to Ostwald'a definition, the amount and chemical composition of a catalyst is the same at the end of its period of activity as it was at the beginning, though it is frequently found that its physical properties have been changed. Here i3 what at first sight appears to be a fundamental difference between cata- lysts such as colloidal metals and the catalytic agents we find in living tissues, but the difference is more apparent than real. Biological catalysts commonly lose much of their activity os the reactions which they catalyse proceed, but in such cases it usually appears that the catalyst has undergone inhibition by the products of its own activity, or else that its physical state has been modified in such a way that its catalytic properties have been destroyed. Another apparent difference between the two types of cata- lysts is that whereas a catalyst such as platinum black does not initiate a reaction but only accelerates one which already pro- ceeds, albeit very slowly, in its absence, biological systems do in certain cases give the appearance of initiating new processes. For example, living yeast cells catalyse an almost quantitative conversion of glucose into ethyl alcohol and carbon dioxide according to the well-known equation : C,H u O, - 20*11,011 + 2C0,. By contrast, certain bacteria, e.g. Streptococcus faecalis, catalyse the conversion of glucose into lactio acid : C,n u O, - 2CJI,CII(OH)COOH. Other organisms again yield yet other products. Now glucoee itself does not show any propensity to decompose spontaneously into either alcohol or lactic acid. Nevertheless, there is no real theoretical difficulty here. As is well known, it is the rule rather than the exception in organic chemistry that side-reactions take placo, indicating that organic substances tend to decompose or react in more ways than one. Let us suppose, therefore, that glucose can decompose into a series of different products, A, B, C, D and so on, each product arising by its own series of inter- media to reactions. Under ordinary conditions the conversion of glucose into A, B> C, otc., proceeds only at imperceptible speed, but, under the influence of the catalysts of yeast, one of the possible modes of breakdown is selectively accelerated to such an •l SPECIFICITY extent that it is followed almost quantitatively. The catalysts of <5. faecalis, by contrast, selectively accelerate another and a different mode. This last case serves to illustrate what is perhaps the most striking featnre of biological catalysis. Whereas a catalyst such as platinum black can catalyse any of a rather wide range of reactions, it is characteristic of biological catalysts that they catalyse only one kind of reaction and even, more often than not, one particular reaction and one only. But this is a difference only in their degree of specificity, or exclusiveness, and cannot be reckoned as evidence that biological catalysts differ essen- tially or fundamentally from catalysts of other kinds. Although the effects of biological catalysts have long been familiar, and although they have been deliberately used by man- kind sinco the dawn of history for the production of cheese, alcoholic beverages and the like, it is only in comparatively recent years that we have acquired any knowledge or under- standing of their mode of action. The celebrated Italian physio- logist, Spallanzani, was perhaps the first to make a deliberate study of one of these catalysts, and this he did by feeding hawks with pieces of meat enclosed in wire cages, which were later regurgitated. In this way he demonstrated that the gastrio juice of hawks contains something which brings about the liquefaction of meat. But as yet the nature of the responsible agent, which we now know under the name of pepsin, could not even be guessed. It was Louis Pasteur who laid the foundations of our present knowledge. In the course of his famous researches on fermenta- tion he demonstrated that solutions of organic materials such as glucose are perfectly stable if carefully sterilized and stored in sealed vessels. If, however, air was allowed to gain access to the solutions, fermentation set in, and this, Pasteur showed, was duo to contamination with living yeast cells which came in with tho air. So long as these micro-organisms were carefully excluded no fermentation took place . Similarly, Pasteur showed that the souring of wine, a troublesome phenomenon which he was commissioned by the then government of France to investi- gate, was attributable to the presence of certain other micro- organisms. These and other observations of a like kind led 5 ZYMASE Pasteur to concl ude that processes such as alcoholic fermentation, the souring of wine and milk arc duo to, and inseparable from, the vital activities of certain particular micro-organisms, which ho accordingly named ‘ferments'. Pasteur’s views received a severe blow when it was discovered by the brothers Buchner that if yeast is macerated with sand and submitted to high pressures, a juice can bo expressed from it wlu'ch contains no living cells whatever, but is nevertheless capablo of fermenting sugar with the production of alcohol and carbon dioxide. The Buchners, in fact, succeeded in demonstra- ting what Pasteur regarded as an impossibility, the fermentation of sugar in the complete absence of living cells. Yeast juice clearly contains the catalyst or catalysts by means of which living yeast accomplishes the alcoholic fermentation of sugar, and to describe this catalytic agent the term 'enzyme* was coined, from the Greek & Zvpfl, literally ‘in yeast’. When other similar catalysts were later discovered and studied, the term enzyme was taken over as a collective title and the yeast-juice enzyme received the distinguishing name of zymase. The discovery of zymase was a fundamental advance. It had hitherto been possiblo to study fermentation and kindred pro- cesses only in the presence of living cells, but living colls multiply, die off, use up some chemical substances and produce others eo that, superimposed on fermentation proper, there are many other chemical processes. With the newly discovered yeast juice, how- ever, the chemistry of fermentation could bo studied in isolation, quite apart from all the other chemical operations carried out by the intact organism. As wo now know, * zymase ’ is not a single enzyme or catalyst, but rather a complex system of catalysts, and similar juices can be prepared from many kinds of cells. The Buchners made their fundamental discovery as recently as 1 897, and progress thereafter was rapid. The Crst enzyme to bo ob- tained in the pure, crystalline state was isolated only some 30 years later, in 1020, and since that time about 100 others twti been, purified and isolated. Certain important discoveries were made comparatively early in the rather meteoric history of enzyme chemistry. Thus it was found that zymaso loses its activity completely if boiled, and that if it is dialysed its activity is similarly lost. After P. NOMENCLATURE AND CLASSIFICATION dialysis, though not after boiling, activity could bo restored by adding the dialysate, i.e. the small-molecular materials removed in the process of dialysis, or by the addition of a little boiled yeast juice. These observations show that, in addition to the thermolabile, non-dialysable enzymes, yeast juice also contains thermostable, dialysable factors in the absence of winch fermen- tation cannot go forward. Thus there arose the conception of enzymes as thermolabile substances of high molecular weight, and of a second group of catalytic materials, called co-enzymes, which consist of small, thermostable molecules. Both are neces- sary if fermentation is to take place. Just as wo now know that zymase is in reality a complex mixture of enzymes, so, too, the dialysable complement is known to contain more than one coenzyme, and wo shall have a great deal to say about this particular case in a later chapter. Even this brief review has revealed a number of the most important properties which characterize enzymes. They are col- loidal materials of high molecular weight, are thermolabile and highly specific, and can usually be extracted from the cells in which they are produced. NOMENCLATURE AND CLASSIFICATION OF ENZYMES Enzymes may be classified in any of several ways. All enzymes so far as we know, are produced inside living cells, and the majority of them do their work inside the cells which produce them, though they can usually be extracted and their activity studied independently. In simple animal organisms such as Amoeba the processes of digestion are preceded by the phagocytic ingeBtion of food particles, which then undergo intracellular digestion, but in more highly organized forms of animal life it is commonly found, that digestive enzymes ace secreted into the digestive cavity, so that digestion is extracellular, at any rate in part. Thus we can distinguish between intracellular and extra- cellular enzymes. This mode of classification is often useful and is likely to be considt-ably extended in the future as more information is gained about the precise localization of enzymes within the cell, e.g. in the nucleus, mitochondria, cytoplasm and so on. 7 CLASSIFICATION Mote usually, enzymes are named and classified in terms of the reaction or reactions which they catalyse, though no prac- ticable system of rigid nomenclature has yet been devised. We can distinguish, for example, a large and important group of enzymes which catalyse the hydrolysis of their substrates, i.e. the substances upon which their catalytic influence is exerted, and these enzymes are accordingly termed hydrolases. This group includes all the extracellular enzymes concerned with digestion, and many intracellular enzymes besides. Individual enzymes are usually named by adding -use to the names of their respective substrates; for example, enzymes which catalyse the hydrolysis ofstarch are collectively called amylases (Latin amylum =etarch), and different individual amylases arc distinguished by reference to tho sources from which they arc obtained. Thus we find salivary and pancreatic amylases among tho digestive enzymes of mammals. Similarly, enzymes winch cntalyso the hydrolysis of proteins are known as proteinases, and those which act upon fats as lipases. The group of hydrolases also includes many non-digestive enzymes, such, for instance, as urease, which catalyses tho hydrolytic breakdown of urea into ammonia and carbon dioxide; and arginase, which catalyses the hydrolysis of tho amino-acid arginine into ornithine and urea. A second largo and important group of enzymes comprises those which catalyse biological oxidations and reductions. Bio- logical oxidations, as we shall see, involve tho removal of hydrogen from tho substrate undergoing oxidation, and moat of the oxidizing enzymes are accordingly known os dehydro- genases. Some of these differ from the rest in certain respects and are called oxidases, though this must not bo supposed to imply that they catalyso the addition of oxygen to their substrates. In addition to these two largo groups we know of a number of other types which may bo called adding, transferring and isomerizing enzymes. * SPECIFICITY One of tho most striking properties of enzymes is their specificity. By this we mean that a given enzyme can catalyso only a com- paratively small range of reactions, and even, in many cases, one reaction and one only. It is possible to distinguish fairly sharply 8 SPECIFICITY between a number of different degrees and types of specificity, and to make this clear we may consider first of all what is known as optical, or better, as stereochemical specificity. The majority of chemical substances formed and broken down in metabolio processes are optically active and, of the two pos- sible stereo-isomeric forms in which such substances can exist, only one is usually found on any large scale in natural materials and processes. Of the hexose sugars, for example, we normally find only the D-isomera, though it is true that their enantio- morphs are occasionally found. Thus L-galactose has been isolated from various plant materials and from the molluscan poly- saccharide, galactogen. None the less, it remains a fact that there is an overwhelming preponderance of D-sugars in nature. Similarly, of the a-amino-acids only the L-members occur extensively in nature: cases of the occurrence of D-amino-acida have been reported, in certain antibiotics for example, but are relatively rare. Perhaps, therefore, it is not surprising to find, as we do, that most enzymes show a strong and usually a com- plete selectivity for one member of a pair of optical isomerides, and aro therefore said to exhibit stereochemical specificity. The phenomenon of stereochemical specificity is well illus- trated in the case of the hydrolytic enzyme arginase. This enzyme acts upon l- but not upon D-arginine, producing n-omi- thino and urea: NH, HN»sC \ra + ♦CH.NH, dooir z^arginine NH» O + NH, (On,). •CH.NII, iooH iromithi nc Similarly, the lactic dehydrogenase of muscle can catalyse the dehydrogenation of l-(+ )- but not that of d-(-) - lactic acid to yield pyruvic acid : ch, cn, •ifl(OH) - 2H«==*to iooii iooH h-lactic acid pyruvic acid 0 STEREOCHEMICAL SPECIFICITY The same enzyme can also ■work in the reverse manner, acting upon the optically inactive pyruvic acid (asymmetric carbon atoms are marked with asterisks) and catalysing its reduction to yield L-lactic acid only- D-Lactic acid is never formed by this enzyme. In many micro-organisms, however, we find a lactic dehydrogenase which is specific for tho n-form of lactic acid, and this is true, for example, of Bacillus delbrUckii, an organism that is employed for tho commercial production of lactic acid. Stereochemical specificity of another kind is also known. The enzymo succinic dehydrogenase, for example, catalyses the oxi- dation of succinate to fumarato but never yields its geometrical isomer, maleic acid: CH.COOH ClljCOOn BOOC.CS iSrn.cooH in.cooH Us. coon maUic acid (eis-J tuccinic acid fumana acid (tnnvl Again, aconitose acts upon cts-aconitic acid, converting it into either citric or tso-citrio acid, but has no action upon tho trans- form of its substrate, by which it is actually inhibited. Over and above tho stereochemical specificity winch is to be observed in tho majority of enzymes, other types of specificity can be recognized. These other types differ mainly in tho degree of oxclusiveness. If, for the sake of simplicity, wo consider only hydrolytic enzymes for the moment, the reaction catalysed by any given enzyme can bo represented thus: a—b + n t Of=»A.on + d . n. The molecule of the substrate can bo considered as consisting of three characteristic fragments, the two parts of tho molecule itself, A and B, and tho linkage which joins them. Tliree main types of specificity con bo described with reference to these constitutional fragments. In the first typo only the nature of tho linkage is important, in the second the linkago and one-half of tho molecule must be ‘right 1 , while in tho third typo all tlireo fragments mu9t bo ‘right’. In tho first typo the precise nature of A and B is relatively unimportant, except that, if they are derived from optically active compounds they must have the appropriate stereo- chemical configuration, a condition which is already imposed by tho stereochemical requirements of the enzyme. What is 10 LOW AND GROUr SPECITIOITV important, however, is that the linkage joining A to B shall be of the right kind, i.e. it must be an ester linkage in the case of a lipaso or an esterase, a peptide link in the case of a peptidase, or a glycosidic link in the case of a glycosidase. If an enzyme is specific only towards the nature of the linkage bond it is said to exhibit loro specificity. This typo of specificity is probably very rare, for a number of what appeared to be enzymes of low specificity have proved to be mixtures, each component of which is more specific than the original complex. A close approximation to low specificity is found, however, among tho lipases, which have so far defied all attempts to fractionate them. The second type of specificity is more exclusive, for hero the enzyme can only act upon substances in which the right chemical linkage is present and in which one of tho two parts, A and B, is also of the right kind. As an example we may consider the case of the digestive enzyme usually called ‘ maltose 1 , since it catalyses the hydrolysis of maltose (glucose-4-a-glucosido). Maltose ob- tained from the intestinal juices of a mammal will catalyso other reactions however; its action upon maltose is only one example of its catalytic action upon a-glucosides in general, which may be expressed in the following manner: R-a-fflucosiJe aylucost The specificity requirements of this particular enzyme are as follows. An a-glycosidio link is required in the substrate; com- pounds containing a /9-glycosidio linkage are not attacked. Furthermore, the a-glycosidio radical must be derived from D-glu- cose, and replacement of tho glucose unit by one derived from another sugaryields a product which is resistant to this particular enzyme. Thus tho nature of the linkage and that of one-half of the molecule must be ‘right 1 in every detail, though the nature of the ‘R 1 group is a matter of relative indifference. An enzyme of this kind may be said to show group specificity, to indicate 11 ABSOLUTE SPECIFICITY that it can act upon a group of closely related Babst rates, in this case a group of a-glucosides. Strictly speaking, therefore, this particular enzyme ought not to bo called 'maltase', since its action is not uniquely confined to maltose; it is, in fact, an a-glucosidase. This kind of specificity is common among carbo- hydrasea, for there also exist ^-glucoaidasea, /?-galactosidasca and so on, each demanding its own particular kind of glycosidic linkage, together with a sugar radical of the appropriate type. The third and commonest kind of specificity is the most exclusive of all. It is well illustrated by a maltase found in germinating barley (malt). Unlike the so-called 'gut maltase’, this maltase acta only upon maltose itself, and is without action where other a-glucosidea are concerned, so that in this case the title of maltase is strictly applicable. Here both parte of the substrate molecule must bo 'right’, together with the linkage bond, and the enzyme ia therefore said to show absolute specificity. To take another example we may consider arginaso again. This enzyme requires for its action that the substrate shall consist of unmodified L-arginine (I). Many substances derived from and closely related to L-arginine have been prepared and submitted to the action of this enzyme, but it fails always to act. Thus a-JV-methyl arginine (II), d-iV-methyl arginine (HI) and agraa- tine (IV) are all unaffected by arginase. Urease similarly requires that the structure of its substrate, urea, shall be intact and unsubstituted, and none of the considerable number of derived ureas that have been tested has been found to undergo hydrolysis under its influence. =0 HN =C I \u Nra tin,), (in,), in.NH, a in.NHicii,) ioOH ioott i\.cu t (in.). iu.NH, ioon \n (W. cn,!« Most of the examples so far mentioned have been chosen from the group of hydrolytic enzymes, but similar phenomena are to bo seen in other groups. Thus succinic dehydrogenase acts only' SPECIFICITY upon succinic acid (V) and is without action upon the closely related malonio acid (VI) by which, indeed, it is strongly in- hibited, Succinic dehydrogenase is therefore absolutely specific, like malt maltase and arginase. Some oxidizing enzymes, how- ever, are group-specific, and as an example we may take the case of the aldehyde oxidase of liver. Given suitable conditions, this enzyme can catalyse the dehydrogenation of many different aldehydes, but its action does not extend to other groups of compounds such, for instance, as the alcohols. COOH COOH I ' I coon cooh V VI In conclusion we may consider a very unusual case. Milk contains the so-called Schardinger enzyme, which catalyses the oxidation of a very large number of different aldehydes to yield the corresponding acids, and is therefore group-specific. But milk also contains a factor which catalyses the oxidation of the purine derivatives hypoxanthine and xanthine to uric acid (for formulae see p. 358), and this factor has received the name of xanthine oxidase. Many purines other than hypoxanthine and xanthine have been submitted to its action and found not to be attacked, so that the specificity of xanthine oxidase is very nearly absolute. The curious fact is that the Schardinger enzyme and xanthine oxidase are demonstrably identical, so that in this case we have an enzyme which possesses two widely different ranges of specificity, one with respect to aldehydes, for which it is group-specific, and another with respect to purines, for which its specificity is virtually absolute. Indeed, it has been found in recent years that a number of enzymes can catalyse two or more totally unrelated reactions ; these are accordingly described as enzy mes of multiple function. It is clear, therefore, that the specificity of any given enzyme cannot necessarily he assigned to one or other of the types we have discussed. Low, group and absolute specificities are merely convenient standards of reference; many intermediate grades 13 ENZYME KINETICS exist, just as, in the solar spectrum, wc can distinguish between red, orange, yellow, green, blue and violet, although tho colours themselves merge into one another and form a continuous whole. Finally, and most important of all, there is the fact that speci- ficity is in reality a measure of the structural specialization that an enzyme requires in its substrate, and this must probably argue a corresponding degree of structural specialization in the enzyme itself. TUB CHEMICAL NATUHE OS' ENZYMES The fact that enzymes are not dialysablo long ago suggested that they might bo related to substances of high molecular weight such as tho polysaccharides and proteins, and even before any enzyme had been obtained in tho pure state there was a con- siderable mass of evidence that they arc proteinaceous in nature. In recent years many enzymes have been concentrated, puri- fied, and finally isolated in crystalline form, and in every case the product has proved to be a protein. Among oxidizing enzymes, there is often attached to tho protein part of tho molecule a non-protein fragment, known as a prothetio group, so that the enzyme is a conjugated protein. While there is no doubt that every enzyme bo far isolated is a protein of some kind, wo cannot Btato categorically that all enzymes are proteins, because not all of the enzymes known to exist have bo far been isolated. But even in other cases there is a good deal of indirect evidence to point to the proteinaceous nature of enzymes in general, and some of this we must consider hero. Information regarding the chemical nature of enzymes has been obtained in many different ways, and most of it from considerations of the influence upon enzymic activity of environmental conditions such as temperature, pH and tho presence of foreign materials of various kinds. (i) The measurement of enzymic activity. The activity of an enzyme can be determined by measuring the amount of chemical change it catalyses under any given set of conditions. If wo incubate an enzyme together with its substrate under suitable conditions of temperature, pH and so on, we can withdraw samples of the reaction mixture from time to timo and follow 14 ENZYME KINETICS the course of the reaction by analysing the samples. Thus, if we choose yeast saccharose (sucrase, invertase) os our enzyme and sucrose as the substrate, wo can measure the amount of chemical change at any given moment in terms of the amount of reducing sugars (glucose and fructose) that has been formed from the original non-reducing sucrose. It is usually necessary at the same time to carry out a * control ’ experiment in which the enzyme is replaced by a previously boiled samplo of enzyme ; in this way wo can correct our experimental results for any changes that are duo to spontaneous transformation of the substrate or to any other process that is not catalysed by the enzyme. Similar methods can bo devised and used for the study of other enzymes, and the results of a typical experiment are shown in Fig. 1. Fig. 1. A typical ‘progress cnrre'; tiyptio digestion of casern; data from a class experiment. Ordinate: increase in formol titre (ml. N&OH). Abscissa: time (hr.) It will bo observed that the reaction velocity soon begins to decrease and eventually the process stops altogether. Now while the reaction is proceeding, changes are taking place in the re- action mixture. Substrate is disappearing, the products of the reaction are being formed, and the forward reaction may be opposed by a reverse process. In Borne cases other factors too may be at work, such, for instance, as change of pH duo to the formation or utilization of acid or alkali. All enzymes are sensi- tive to changes of pH in their immediate environment, all enzymes are influenced by the concentration of substrate avail- able to them, and many are actually inhibited by the products of their own activity. If, therefore, we wish to obtain a reliable measure of the activity of an enzyme under any given set of conditions, it will be necessary either to ovoid these changes in 16 INFLUENCE OF TEMPERATURE the reaction mixture or else to make some suitable allowance for them. Two main procedures are available. In the first place wo can measure the length of time required to produce a given amount of chemical change. In thi3 case the amount of substrate used up, the amounts of products formed, the change of pH if any, and the extent of other changes likely to interfere with the enzyme will be the same in every experiment, so that different experi- ments will be comparable one with another, ahoaye provided that the enzyme is stable under the conditions selected. In such cases we can use time as a measure of the activity of the enzyme : actually, of course, tho reciprocal of the time will be proportional to the activity of the catalyst, since an enzyme preparation that is half as active will take twice as long to produce the same amount of chemical cliange. The second method is usually preferred, and consists in measuring the amount of chemical change taking place over a very short interval of lime from the start of the reaction. Provided that the time interval can be made short enough, the changes in the composition of the reaction mixture will bo small enough to be neglected. Ideally we should measure tho instantaneous initial velocity, which is not a practicable proposition, but many excel- lent micro-methods are now available by tho use of which we can obtain very good approximations to the instantaneous initial reaction velocity, and hence to the activity of an enzyrao tinder any given set of experimental conditions. (ii) The influence of temperature. Most chemical reactions are influenced by temperature, the reaction velocity increasing with rising and decreasing with falling temperature. Enzyme cata- lysed reactions are no exception to this general rule, but, because enzymes are very susceptible to thermal inactivation, the higher tho temperature becomes, the more rapidly are the catalytic properties of the enzyme destroyed. For any given set of experimental conditions, therefore, it is possible to find what is called an optimum temperature, i.e. a temperature, at which, tho greatest amount of chemical change is catalysed under that particular get of conditions. At euboptimal temperatures tho enzyme is relatively more stable and therefore lasts longer, but tho reaction which it catalyses proceeds more slowly. At 10 INFLUENCE OF TEMPERATURE temperatures above the optimum, on the other hand, the re* action takes place more rapidly but the catalyst is more rapidly destroyed. There has been a good deal of misunderstanding on this subject in the past, for many biologists have supposed that the optimum temperature of an enzyme is a fixed and unalterable charac* teristic. A rise of temperature has a dual effect upon an enzyme- catalysed process : it increases the rate of the reaction, but it also increases the rate of thermal inactivation of the catalyst itself. Consequently, if we work over a period of a few seconds the optimum temperature may be very high indeed, because the catalytio properties of the enzyme do not need to be Jong lived. If, on the other hand, we choose to work over a period of a few days, a much lower optimum will be found since the enzyme must now last for a much longer period. It follows, therefore, that the time factor must be taken into account when we seek to determine the optimum temperature of any given enzyme, and that time and temperature are interdependent variables. The relationship between time and the optimum temperature of the digestive proteinase of an ascidian (sea squirt), Tethyum, is shown in Fig. 2. With crude enzyme preparations such as were used in the earlier work on enzymes, the activity was usually of a rather low order, and it was therefore necessary to incubate the enzyme with its Bubstrate for an hour or more in order to get a reasonable amount of chemical change. Under conditions of this kind most enzymes show an optimum temperature of about 30-40° C. This has led to the suggestion that, when animals became homoio- theraic, they settled on a body temperature of the order of 37° C. because their enzymes would ‘work better’ in that neigh- bourhood than in any other. But consider the case of Tethyum. Over a period of 2 hr. the optimum temperature of this digestive proteinase is of the order of 50° C., which is well above the thermal death-point of this species. Tethyum normally lives at tempera- tures in the neighbourhood of 15° 0., and the digestion of its food takes about 60-60 hr. under natural conditions. If the optimum temperature is determined for a period of 66 hr. the value found is about 20° C., so that there is, after all, a nice adjustment of the enzyme to the biological requirements of the 2 17 BDA THERM tXi IKAOTrVATIOK animal. This seems fairly generally the case, for ‘there is evi- dence that the time taken for the passago of food through the gut at any normal temperature corresponds to the period which is optimal for enzymatic action at that temperature’ (Yonge). The thermal inactivation of enzymes is interesting from the physico-chemical viewpoint as well as from that of the biological behaviour of enzymes, for it yields important clues to the chemi- cal nature of enzymes themselves. For most chemical reactions Pig. 2. Influence of temj'eeeture on digwtWe proleiniuie of TilAyum. Ordinate: mg. «mino-*cid nitrogen per litre. (Snbitrate, gelatin: after BcrriU, 1S29.) we find a temperature coefficient, represented by Q Mi of approxi- mately 2; i.e. tho rate of the reaction is approximately doubled for a riso in temperature of 10° C. If wo determine the rate of thermal inactivation of enzymes in tho neighbourhood of 70- 80° 0, wo find Q n values of tho order of several hundreds. Tcm* poraturo coefficients of this order are known for reactions of only two kinds; for tho thermal inactivation of enzymes on the one hand, and for the thermal denaturation of proteins on tho other. There is here, therefore, a striking indication that enzymes may bo of protein nature, and that the process of thermal inactivation is analogous to, if it docs not actually consist of, denaturation. 18 INFLUENCE OF pH This latter point is stressed somewhat by the fact that enzymes are much less susceptible to thermal inactivation in the dry than they are in the wet state, while proteins appear to be more resistant to denaturation in the dry condition than they are in solution. (iii) The influence of pH. The catalytic powers of an enzyme are, as a rule, exercised only over a somewhat restricted range ofpH. Within this range the activity passes through a maximum Fig. 3. Influence of pH on the activity of some enzymes. A. Salivary amylut (substrate starch +NaCl). Ordinate: reciprocal of time taken to reach the acbromio point. Results of a class experiment. B. Fapain-cysttint (synthesis of benzyloxycarbonj l-glycylanilide). Ordinate : yields as % of theoretical maximum, estimated by isolation (O) and titration (A). After Bergmann & Fraenkel-Conrat (1937). C. v-Amina^icid oxidate (substrate DL-alanine). Ordinate: oxygen uptake in 10 min. ful), Results of Krebs (1935), after Green. at some particular pH, known as the optimum pH, and then falls off again. 'Fig. Z illustrates tbs activity {pH relationships of several enzymes. Generally speaking, the optimum pH is characteristic of a given enzyme, though under certain special conditions and in certain groups of enzymes the optimum pH may vary. This is true of the proteolytic enzymes, for example, and pepsin has an optimum pH that varies between 1*5 and 2-5 or thereabouts, different optima being found with different protein substrates. 16 ItfELUEHOE OE pH A given carbohydrase, on the other band, has always tho same optimum pH, even when acting upon different substrates. In its general form the pH/activity curve of ft typical enzyme closely resembles that obtained by plotting the degree of ioniza- tion of a simple ampholyte such as glycine against pH. It will be recalled that most of the properties of solutions of ampholytes such as proteins and amino-acids — such properties as solu- bility, osmotio pressure, conductivity, viscosity and so on — pass through either a maximum or a minimum at some par- ticular pH, the so-called isoelectric pH. These changes are attributable to changes in the ionic condition of tho ampholytes themselves. Being multipolar, any given protein con exist in a number of different ionic forms, and one of these, tho iso- electric form, possesses a number of special and peculiar properties. It is therefore tempting to suggest that an enzymo may bo regarded as a protein, and that of all tho ionic forms in which it can therefore exist, only one particular ionio species possesses catalytic properties, this being tho species which preponderates at tho optimum pH. A further indication of the proteinaceous naturo of enzymes is that extremes of acidity and alkalinity, which lead to the irreversible denaturation of proteins, lead also to the inactivation of the majority of enzymes. Moreover, these ore irreversible changes, unlike those which are observed in the immediate vicinity of tho optimum pH and which are for the most part reversible. Generally speaking, enzymes nro moat stable in the neighbour- hood of tho optimum pH, so that the observed optimum does not vary with time. The optimum pH of an enzyme is therefore a more characteristic feature than its optimum temperature. But if, as is sometimes tho case, the enzyme is one which is very unstable at or near its pH optimum, the value observed will, of course, vary with tho duration of the experiment. Accurate determinations of the optimum pH can only ho made in such cases by working over very short intervals of time. In tho case of arginasc, for example, the optimum pH is about 7-8 for a period of an hour or bo, but the true optimum lies at about 10, a pH at which arginaao is very unstable indeed. Tho case of arginasc, like that of pepsin, is an unusual one : most enzymes 20 INHIBITION have their pH optimum not very far from neutrality, most commonly between pH 6 and 7. (iv) The influence of protein precipitants. Enzymes are in- hibited by many different groups of chemical reagents, as well as by such physical factors as high temperatures, violent mechanical agitation, ultra-violet radiation and so on, all of which lead to the denaturation of proteins. Many protein pre- cipitants also lead to the inactivation of enz ymes. In this section special attention may be drawn to the effects of two groups of enzyme inhibitors which form insoluble salts with proteins, viz. the salts of heavy metals on the one hand and, on the other, the so-called ‘alkaloidal reagents’. The former precipitate pro- teins by virtue of the heavy, positively charged ions to which they give rise in solution, and the alkaloidal reagents, which include such substances as trichloracetic acid, tannic acid and phosphotungstic acid, act by virtue of their heavy, negatively charged ions. That all these agents are powerful inhibitors of enzymic activity strongly suggests that enzymes are protein- aceous in composition. More precise indications to the same effect are to be had by studying the effects of small concentrations of inhibitors of this kind. If it is indeed true that enzymes are proteins, we should expect to find that, in common with the proteins, they will be positively charged in acid solutions and therefore susceptible to the action of the negatively charged ions of phosphotungstic acid, for example. In alkaline solutions, on the other hand, they would be expected to be negatively charged and susceptible therefore to the action of positively charged ions, for example Ag + . This problem has been carefully investigated for a few enzymes, and the results obtained in the case of yeast sacoharase are shown in Fig. 4. It will be seen that the effects of gradually increasing concentrations of silver ions are most marked on the alkaline Bide of the optimum pH. Phosphotungstic acid produces similar effects on the acid side. These results show that the behaviour of yeast saccharose with respect to these inhibitors is consistent with the view that this enzyme is a protein. In practice, however, the concentration of silver ions required to produce complete inhibition of yeast sac- charose is much smaller than that needed actually to precipitate 21 INHIBITION: SUMMARY prof etna, and this suggests that the effect of Ag + is not a general one upon all the negatively charged centres of the protein molecule, but a localized and very specific one upon particular centres which are responsible for tho catalytic properties of the presumptive saccharose protein. There thus emerges the notion that enzymic activity is not a property of the protein molecule as a whole, but rather that it is associated with certain special ‘active * groups or centres. Fig. 1. Influence of «maJl, incroaaing concentration* of «il veriema on activity of yeut fc&ccW&se. OtJinlte: initial velocity othydrolytia of iiiirou, Abcciaw. yll. (After HsIJane, from AlyrbJck, 1020.) Curr# Cone. Ag + 1 0 2 fix KH»i 3 l(H a 4 2xl0-»x Curve Cone. Ag + 0 4xl0-*»i 6 10-*M 7 2 x 10-*M 8 10-*U SUMMARY 1. Enzymes are complex, organic catalysts of high molecular weight, produced by living cells but capable of acting indepen- dently of tho cells that produco them. They aro characteristically thermolabilo and highly specific. 2. Several kinds and degrees of catalytic specificity can bo recognized. The majority of enzymes show stereochemical epoch 22 SUMMARY fidty, but, in addition, their specificity may be low or very high with reference to the chemical constitution of their substrates. 3. Enzymes are profoundly affected by many physical and chemical factors, and determinations of their activity therefore require to be made under very closely controlled conditions. 4. Enzymes are in all probability of protein nature. Every enzyme so far isolated has proved to be either a simple or a con* j ugated protein. The behaviour of enzymes towards heat, changes of pH and protein precipitanta is consistent with the supposition that they consist of protein material. 28 CHAPTER II THE NATURE OF THE CATALYTIC PROCESS HHE OS TGE TOS ITS StJBSTP.iTE I T is difficult to imagine how a catal yst of any kind can influence the rate of a chemical reaction unless it actually participates in that reaction. Most authorities agree that catalysts do in some manner combine with the substance or substances upon which their catalytic influence is exerted, but there has been much difference of opinion as to whether the union is of a ‘physical’ or adsorptive kind, or whether it is to bo regarded as ‘chemical’. But it is difficult to maintain that there is any fundamental difference between these types of unions: rather must they be regarded as two extremes of one and the same phenomenon. In so far as it is possiblo to distinguish between adsorption and chemical combination it may bo said that adsorption is, on the whole, a less specific and more freely reversible process than chemical combination. Calcium carbonate is a good example of what we Bliould call a chemical compound, formed by the chemical union of carbon dioxide and calcium oxide. Yet at high enough temperatures the product dissociates freely, as though, by raising the temperature, we have converted a chemical into an adsorptive union. While it is true that adsorption is often relatively unspecific, there is evidence in plenty that it can be very specific indeed. Thus we find that a positively charged material such as mag* nesium oxide will adsorb negatively charged dyes like eosin from aqueous solution, but fails to take up a positively charged dye such as methylene blue. Similarly, a protein will take up nega- tirely charged dyes in solutions acid to its isoelectric pH, in which it is positively charged, while on the alkaline side it takes up positively but not negatively charged dyes. At or very near the isoelectric pH it will usually take up a little of both, since, being a zwittcrion, it carries an equal number of positivo and negative charges at one and the same pH. 24 ENZYME -SUBSTRATE UNION Clearly, therefore, several factors have to he taken into account when we are considering adsorption. The nature of the surface at which the adsorption takes place is certainly of importance. Carotenoid pigments, for example, are adsorbed at a magnesium oxide/petrol ether interface but not at a magnesium oxide/alcohol interface. Charcoal can be used to adsorb coloured impurities of many kinds from aqueous solution, but is relatively useless in chloroform, and so on. The second important factor is, of course, the chemical nature of the material being adsorbed. It is not difficult to understand that a given surface may be so specialized, whether by virtue of its charge or for some other reason, as to be capable of taking up, i.e. reacting with, substances of one particular kind. Nor, if we allow for its possible topographical specialization, is it difficult to imagine that a particular surface may be capable of reacting with one particular substance and one only,> There is nothing inherently improbable in the idea that an enzyme actually unites with its substrate, and it is difficult, indeed, to imagine how the facts of enzyme specificity could otherwise be accounted for. Studies of the kinetics of enzyme- catalysed reactions have made it clear that tho assumption of a union is in fact warranted. Keilin has provided direct evidence for tho formation of an enzyme-substrate complex between peroxidase and hydrogen peroxide. If peroxidase is added to its substrate in the presence of a suitable hydrogen donator such as pyrogallol, a vigorous reaction ensues, in which the pyrogallol is oxidized and the hydrogen peroxide reduced. In the absence of any hydrogen donator, however, the hydrogen peroxide does not undergo reduction. Now peroxidase is an iron-porphyrin derivative and as such has a strong absoiption spectrum, displaying four bands at 645, 583, 548 and 498 m/t respectively. If hydrogen peroxide is added to a strong solution of the enzyme there is a sharp change in colour and tho spectrum changes completely. Only two bands at 561 and 530-5 m p respectively can now be seen. This can only mean that some kind of reaction has taken place between the enzyme and its substrate. Moreover, the amount of hydrogen peroxide required just to convert the whole of tho enzyme into the new compound is equivalent to exactly one 25 ENZYME -SUBSTRATE UNION molecule of hydrogen peroxide for each atom of peroxidase-iron. Another compound, with bands at 5S3 and 645-5 m/*, is formed when the amount of hydrogen peroxide is increased to about 100 molecules per atom of peroxidase-iron. Somewhat similar observations have been made with catalase. If hydrogen peroxide is added to catalase a violent reaction takes place, the substrate being converted into water and molecular oxygen. If, however, the enzyme is first treated with sodium azide, which inhibits its nctivity, there is only a alow reaction when hydrogen peroxide i3 added. The cata- lase-arido complex has a strong absorption spectrum with bands at 624, 644 and 606-5 m/z, changing on addition of hydrogen peroxide to a spectrum with two bands at 588 and 647 m/i. The original spectrum reappears when all the hydrogen peroxide has been decomposed, but the addition of more sub- strate then restores the two-banded spectrum. These observa- tions show that the (inhibited) enzyme reacts in some way with its substrate. INFDUENOE OF CONCENTRATIONS OF THE ENZYME AND IT8 SUBSTRATE The rate of any enzyme-catalysed process depends, other things being equal, upon the concentrations of the enzymo and of its substrate, and an examination of the effects of these and other factors is very important for any understanding of enzymic catalysis. In the vast majority of cases we find that, with a fixed quantity of enzyme, the initiat reaction velocity increases with increasing substrate concentration until a limiting value is reached. Tig. 6 shows the results of a typical experiment carried out along these lines. The magnitude of the limiting velocity finally attained depends upon the concentration of the enzyme used and is, in fact, proportional to that concentration. These observations can bo accounted for in terms of tho theory first brought forward by Michaclls andlater developed and expanded by Briggs & Haldane. For the purposes of tho argument it is assumed that the enzyme and its substrate react together in some way to form on unstable complex, which then breaks down to yield the reaction 26 BRIOOS-nALDANE-MIOHAEIiia THEORY products. If we choose a case such as the hydrolysis of sucrose by eaccharase, these assumptions can be expressed in the following equations : (i) E + S v* ES, (e~P) <*) ip) (u) ^S+H,0->^+P+g. ( P ) The enzyme is represented hero by E, tho substrate by S and the intermediate enzyme/aubstrate complex by ES, while P and Q are the products of the process. Fig. 5. Influence of *ubitrate concentration on activity of yeast saccharose. Ordinate: initial Telocity of hydrolysis. Abscissa: molar concentration of sucrose. (After Haldane, from Kuhn’s data, 1023.) If we represent the total enzyme concentration by e it follows that, since an amount p is bound up in the form of ES, tho concentration of free enzyme will be equal to (e— p). The reaction velocity, which we will call v, is the rate at which the products are formed, and this will clearly be proportional to the concentra- tion, p, of the unstable complex ES. Wo arc now in a position to apply the principles of the mass law to our equations and in this way to make predictions which, if they prove to be in accordance with experimental observations, will provide evi- dence of the soundness of tho assumptions epitomized in equations (i) and (ii). It is necessary, before going further, to realize clearly that the concentrations represented by e, p, x and (e —p) must, if we 27 BBiaOS-nALDAlTE'MlCrrAELlS THEORY are to apply the mass law, be expressed as molecular and not as percentage concentrations. This fact is doubly important hero because wo are dealing with enzymes, which are colloidal materials, having very great molecular weights. Let us for a moment consider the enzyme E as aprotein with the compara- tively modest molecular weight of about 00,000, and compare it with a substrate such as urea with a molecular weight of CO. If we were to prepare 1 % solutions of the pure enzyme and of the Bubstrate, the molar concentration of the substrate solu- tion would be no less than 1000 times that of the solution of enzyme. This point is of considerable theoretical importance, as we shall see, but it serves also to emphasize the relatively enormous activity of enzymes. They occur in living cells and tissues in amounts so small that their molecular concentrations are infinitesimal, yet it is upon their catalytio activity that the life of the cells depends. Returning now to our theory we see that tho following state- ments can bo made: For equation (i) rate of forward reaction =*x(e-p) L\, rate of reverse reaction =pk t , where k\ and k 2 arc the velocity constants of tiie forward and backward reactions respectively. For equation (ii) rate of reaction =p£j and k 2 is tho velocity constant of the breakdown of tho enzyme- substrate complex. Now as long as the rato of reaction (ii) is constant the value of p will remain constant and hence therefore — — — = - 1 - )— ? « K . (A) V Hero K „ , the ratio of two constants, b itself a constant and is called tho Micliaelis constant. Its particular significance will be considered later. "Wo are very seldom in a position to evaluate either e or p, since even if we have a perfectly pure enzymo at our disposal its molecular weight is quito likely to be unknown. These terms, c and p, must therefore bo eliminated from our equations, and this can be done through the following considerations. 28 BRIGGS-HALD AHE-MICJHAELIS THEORY The reaction velocity, v, for the decomposition of ES (equa- tion (ii)) will be proportional to p and also to the concentration of water, but since the concentration of water in the system does not change appreciably we can write v~kp, (B) where k is a constant. By combining this with equation (A) we could eliminate p, but the term e would still remain, and this, like p itself, we are usually unable to evaluate. But let us con- sider a special case in which there is a large excess of substrate. This case is not a fictional invention since, on account of the great disparity of molecular weight between E and S, x will usually be much greater than e. In the presence of a largo excess of substrate, therefore, [5] will be very much greater than [.E] so that virtually all the enzyme will be converted into ES, when p=e. Now we have just seen (equation (B)) that the reaction velocity is proportional to p, and in the presence of a large excess of substrate p attains the value of e. Consequently, in the presence of a large excess of the substrate, the reaction velocity will attain a limiting value which may be called V. Wo thus have, for this special case, a third equation : V~lce. (C) Dividing (B) by (C) we get frA P» V e It is now possible to get rid of the unwanted terms from equa- tion (A). We can rewrite (A) as follows: x{e-p)^pK m , therefore ex—px=pK m , cx=*px+pK m =p{x-\-K m ), and dividing by e, x^(x + K m ). Substituting for pje from equation (D) we get: * = fa+Km'l, and hence the Michaelis or Briggs & Haldane equation: Vx V z+K m ' 29 (E) BRIGOS-HARBARE-MICKAELIS THEORY This equation allows ns to predict the .maimer in which the reaction velocity should be influenced by substrate concentra- tion. It is, in fact, the equation of a rectangular hyperbola with the following properties {see Fig. 6): (a) the limiting velocity V, is the asymptotic valuo to which the reaction velocity tends as the concentration of the substrate is increased, and Fig. C. Theoretical carve for Briggs 4 Ha Had s’* equation, Vx V “'z+K m ’ where r- initial reaction velocity, ar— concentration of substrate. Ordinate! reaction velocity. Abscissa: concentration of substrate. (6) tho Michaclis constant (A' m ) corresponds to that substrate concentration at which half tho limiting velocity is dovetoped. Tliis fact is readily understood if we substitute T'/2 for e in eqtiation (E) itself: V Vx - *+Km' therefore and 1 x 2~x+K m ' 2 x~x+K„, when tho reaction velocity is one-half the limiting velocity. 30 BRIG GS -HALDANE -HIGH AEliIS THEORY If the rectangular hyperbola of Fig. 0 is now compared with the curve of Fig. 6, which portrays the results of experimental observations, there can be no doubt that the theory is in excellent agreement with the experimental results. Moreover, we have seen (equation (C)) that, according to this theoiy, the limiting velocity att ained in the presence of an excess of substrate should be proportional to the concentration of enzyme, and this also is in agreement with the results of experimental enquiry (see Fig. 7). Atypical results are not infrequently obtained in experiments designed to test tho validity of predictions based on this theory, but these are usually due to interference by some factor or other, e.g. impurity of the enzyme or inhibition of the enzyme by the products of its own activity. When due allowance is made for this interference the corrected results are found to agree well with theoretical prediction. The agreement between theoretical requirements and experi- mental observation goes far towards justifying the assumption upon which the theory was originally based, namely, that the enzyme actually combines with its substrate to form an un- stable and correspondingly reactive complex. It may, of course, be argued that the same theoretical equation might be derived equally on the basis of different assumptions, but there is other 31 brigos-iialpake-miohaelis theory and more direct evidence for the formation of enzyme-substrate compounds to which we have already referred (p. 25). The hlichaelia constant deserves a little further consideration. Let us suppose that we havo an enzyme of low or of group speci- ficity, and let us consider its activity towards two different sub- strates, a and b. If the relationships between reaction velocity and substrate concentration are experimentally determined for both substrates wo get a pair of hyperbolic curves like those of Fig. 8. For each of the two substrates there is a K m value, and in Fig. 8. Action of a group specific enryme upon two different »a titrate*; for expUn*- tion text. Ordinate: reaction velocity. Abscissa: molarity of *nb»tr»te in arbitrary unit*. the figure it wifi bo observed that for b the value (A'^,) is greater than for a (A’ m ). This means that in order to get the same velocity out of a given concentration of enzyme, b must be taken at a higher concentration than must a. This must mean that the enzyme has a smaller affinity for b than for a : in other words a high K m is indicative of a low enzyme-substrate affinity, and vico versa. Thus, not onty does our theory furnish us with ovidcnce that the formation of an enzyme-substrate compound is an essential part of the catalytic process ; it provides at the same time a means whereby the affinity of nn enzyme for its substrate can bo numerically evaluated. As wo go on we shall seo that the behaviour of enzymes is best explained on the supposition that they react with their respective COMPETITIVE INHIBITION substrates to form reactive complexes. In addition to the indirect evidence afforded by the applicability of Briggs & Haldane’s theory there is a considerable mass of indirect evidence from other sources, as veil as the more recent direct evidence to which we have already referred. COMPETITIVE INHIBITION A great deal of information regarding the nature of enzymes and their mode of action has been gained by considering their in- hibition, and studies of tliis kind have, as we have seen, done much to confirm the view that enzymes are made up essentially of protein material. Many enzymes are inhibited by the products of their own activity, and many more by substances which are structurally related to their substrates. In many such cases the inhibition is of what is known as the competitive type. A well- known and veiy important case is found in the competitive inhibition of succinic dehydrogenase by malonate. If we take succinate together with succinic dehydrogenase we have a system in which, under suitable conditions, it is easy enough to measure the reaction velocity in terms of the rate of oxidation of the succinate. If now malonate is added the rate of oxidation promptly diminishes, but increases again if more succinate is added. Malonic acid is a dicarboxylic acid, the structure of which is closely related to that of succinic acid itself: Coon Ijn, coon aurcinic acid coon in. cooh Malonate is able to combine with the enzyme, just as does the substrate, succinate. But whereas the enzyme-succinate com- plex breaks down to yield the reaction products, the enzyme- malonate complex contributes nothing to the reaction velocity. In consequence, a part of the enzyme is bound in the form of enzyme-inhibitor complex, and so is not available for the cata- lysis of succinate oxidation, and the reaction velocity accordingly diminishes when the inhibitor is added. 33 COMPETITIVE INHIBITION This system may be more precisely described in the following manner. We Have the following equilibria and reactions to consider: E+S ?±ES-*E+F+Q, E+M^EM. Tlie rate of oxidation of the succinate is determined by [£5], and this can bo increased either by increasing [5] or by decreasing [J/j, winch indicates that these two substances ‘compote' for possession of the enzyme. Now' if the two compounds reacted at different points on the enzyme molecule there is no reason why they should not both bo accommodated at the same time, each independently of the other. The fact that they do howover compete shows that both unite with the enzyme molecule at precisely tho same point. Many other cases of tho samo kind are known. That competi- tive inhibition exists at all is an indication tlrnt tho substrato does not unite at arbitrary groups on tho enzyme molecule, but only at certain particular groups, and not elsewhere (cf. p. 22). Thus these considerations not only confirm tho view that a union ia set up between an enzyme and its substrato when the two arc brought together: they go further, by Bhowing that the union is very specific not only in nature but also in locality. It is interesting to notice in passing that tho bacteriostatic activity of sulphanilamido depends upon a competition between the drug itself and p-aminobenzoic acid. Tho latter is an impor- tant growth factor for many bacteria, and tho structural resem- blances between the drug and tho growth factor arc great enough to result in competition between tho two: aad tuiyt/vuJnnudA. If p-aminobcnzoic acid wins the bacteria multiply: if sulph- anilamido wins multiplication is checked, and the rate of multiplication depends upon tho ratio between tho concentrations of the two substances. 34 ACTIVATION OF THE SUBSTRATE ACTIVATION OF THE SUBSTRATE The combination of an enzyme with its substrate seems to be the fundamental and essential step in the catalytic process, for it is as a result of this union, apparently, that tho substrate molecule becomes more chemically reactive than it was in the free, un combined state, and is more easily split, oxidized, re- duced or whatever the case may be. Wo refer to this increase in chemical reactivity by saying that the enzyme has ‘activated’ its substrate, or that the substrate has undergone ‘activation’. We do not know what precise intramolecular changes underlie activation, but wo do know of other cases in which something of the samo sort takes place. Let us consider the behaviour of haemoglobin. This compound consists of a protein, globin, to which is attached a complex, tetra-pyrroUic ring-structure containing an atom of ferrous iron. The special and peculiar property of haemoglobin is its ability to react reversibly with oxygen, taking it up when the partial pressure is high and giving it off again at low partial pressures. It is particularly noteworthy that this performance involves no change in tho valency of tho iron, a fact which is implied by speaking of the ‘oxygenation’ as opposed to the ‘oxidation’ of haemoglobin. The important point for our present argument 13 that haem, by itself, does not possess this property. It acquires this property when, and only when, it is combined with tho appropriate protein, globin. Free haem is very insoluble in water, and reacts spontaneously with oxygen to undergo oxidation to the ferric compound, hacmatin. Globin, by combining with haem, confers upon it a large measure of solubility in water, together with the new-found property of reacting reversibly with oxygen, without at the samo time undergoing any change in the valency of tho iron. In addition to haemoglobin a number of other haem and liaematin compounds with very special and peculiar properties are known, such, for example, as cytochrome, catalase and peroxidase. In each of these the haem or liaematin system is present, but in none do we find the ability to combine reversibly with oxygen. The haem, presumably, is all right, but the protein is wrong. Thus the haem of haemoglobin possesses certain latent 35 3-3 ACTIVATORS AND OOENZYSlES properties which only become apparent when the haem nucleus is combined with the right kind of protein. While few, probably, would venture to assert that globin ‘activates ’ haem in the sense that an enzyme activates its substrate, this case does show how the properties of a given substance can be profoundly and very specifically modified when the substance concerned enters into combination with the ‘right* kind of protein. We know too little about the phenomenon of activation to be able to make any clear picture of the changes which underlie it, but it Is an interesting and highly significant fact that the hydro- lytic processes catalysed by enzymes can frequently be imitated by means of dilute acids, alkalis, or both, and sometimes merely by boiling water. Whether we treat a dilate solution of sucroso, for example, with hot, dilute mineral acid or with saccharose prepared from yeast, wo get precisely the same products, viz. glucose and fructose in equimolecol&T proportions. Saccharose, therefore, does not induce any new kind of reactivity in its substrate, but only exaggerates a tendency to react that is already inherent in the sucrose molecule. ACTIVATORS AND COENZYMES Activation of the substrate is an indispensable part of the chemical process catalysed by any enzyme, but it can tako place without necessarily being followed by the hydrolysis, oxidation or other chemical modification of the substrate. Thus if wo add peroxidase to a solution of hydrogen peroxide, the two unite to form an addition compound, as is shown by the resulting change in the absorption spectrum (p. 25). But, in tho absence of other materials, there the matter ends. If some substance capable of being oxidized is also added, AH, say, there begins a rapid transference of H atoms to the activated hydrogen peroxide so that AH, is oxidized and the peroxide reduced, thus: A\ O— II AC + ) - A + 2 IT — 0—11/ MI 6— II This example suffices to Bhovr that, while activation is an essential part of tho process of enzymic catalysis, activation only makes it possible for the reaction to take place: whether or not tho 30 ACTIVATORS AND COENZYMES reaction actually ocours often depends upon the presence of other materials, over and above the substrate and its activating protein, the enzyme. It is, in fact, true that in the vast majority of enzyme-catalysed reactions, substances other than the sub- strate and its activating enzyme-protein must also be present before any chemical change can bo brought about. In processes of hydrolysis, for instance, water molecules form an indispensable part of the reaction system. The enzyme must therefore be con- sidered as only a part, albeit the most important part from the biological point of view, of the whole reacting system. Similarly, in oxidation and reduction reactions, the majority of which are accomplished by the transference of pairs of hydrogen atoms from the substance being oxidized (the ‘hydrogen donator’) to the substance being reduced (the ‘hydrogen acceptor'), we find that both substances must be present, together with the appro- priate enzyme. It has been known for many years that a considerable number of enzymes are unable to exert their cataly tio influence except in the presence of certain appropriate materials which have become known as ‘ coenzymes ’ or ‘activators’. It will be remembered that zymase, for instance, loses its activity if dialysed, and that this loss of activity 13 attributable to the removal from the juice of certain small, thermostable ions or molecules in the absence of which fermentation cannot proceed. Recent work has shown that even such seemingly innocent substances as the ions of potassium, calcium, magnesium, chloride, phosphate and the like play indispensable parts in many enzyme-catalysed pro- cesses. In such cases it is clear that the inability of the enzyme to act in the usual way might be due to one or other of two causes. Either (a) the enzyme cannot activate its substrate because some accessory part of the enzyme itself has been removed, or (b) by contrast, the enzyme is capable of activating its substrate but no reaction takes place because some sub- stance with which the substrate ordinarily reacts has been removed. It is possible to distinguish more or less sharply therefore between two groups of accessory substances, those which are parts of the activating system on the one hand, and on the other those which are a part of the reaction system but play no part in activation. Althoughit is difficult to j ustifyany distinction 37 ACTIVATORS in many cases, tho tendency at the present time is to refer to accessory substances which are in effect apart of tho activating system and are required before the enzyme can activate its substrate, as ‘activators’. The term ‘coenzymo’, on tho other hand, tends to be reserved for substances which play some part- in the reaction catalysed by the enzyme, hut not in the activa- tion of the substrate . We shall follow this practice here . It must, however, be remembered that the activation of an enzyme by tho appropriate ‘activator’ is quite distinct from the activation of the substrate that takes place as a result of its union with the specific activating protein or enzyme. It is sometimes found that enzymes are secreted in a form in which they have no catalytic activity whatever, i.c. in the form of enzyme-precursors, or ‘pro-enzymes'. Tho classical case to consider hero is that of trypsinogen. The juice secreted by the pancreas of vertebrates contains a pro -enzyme, trypsinogen, which is devoid of action upon proteins, but when the pancreatic juice enters tho small intestine, trypsinogen is converted into tho active proteolytic enzyme, trypsin. The change is attributed to tho presence of an enzyme or enzyme-hko factor present in the intestinal juice to which the name of cntcro kinase has been given. The chango from trypsinogen to trypsin is duo to the removal from the pro-enzyme of a portion of tho molecolo which acts so to speak, ns a ‘mask’ covering the reactive centres ol trypsin itself. This ‘mask’ is a polypeptide, and its removal is due to proteolytic action on tho part of entcrokinase. This kind of activation, which for want of a better term wo maj’ refer to as ' unmasking \ is now known to occur in sereral proteolytic enzymes, and will bo dealt with at greater length when wo come to consider them in more detail. A second kind of activation, and one which is commonly encountered, might similarly bo referred to as ‘ de-inhibition'. Many enzymes are readily inhibited by mild oxidizing agents, and it frequently happens in the course of attempts to isolate a given enzyme that much of Its activity is lost in the process as the result of oxidation by atmospheric oxygen, catalysed a A a rule by traces of heavy metals present in the material, or derived from mincing machines or other metallic devices used in the preparation. In such cases the activity can very often be 38 ACTIVATORS recovered by adding reducing agents such as cysteine or reduced glutathione. It was first shown by Hopkins that in the case of succinic dehydrogenase, though the same is not true of the lactio enzyme, any treatment tending to oxidize the — SH groups of the enzyme-protein so that — S — S — cross-linkages are formed between adjacent molecules of the enzyme, results in the loss of dehydrogenase activity. These — S — S — linkages can be reduced again by means of — SH compounds, e.g. reduced glutathiono, and the dehydrogenase activity of the enzyme returns therewith. Activation by de-inhibition is a common process and may often be accomplished merely by removing inhibitory material. Cytochrome oxidase, an enzyme of central importance in respira- tory metabolism, is powerfully inliibited by carbon monoxide in the dark, and a very striking case of activation by de-inhibition can be demonstrated in this case by exposing the preparation to strong light, which causes the carbon monoxido-oxidaso com- pound to dissociate. Many other examples might be quoted. Activation by unmasking or de-inhibition is due, apparently, to the removal of material that inhibits by blocking the active parts of the enzyme. In other cases, however, it is less clear how the activator functions. Many enzymes concerned with phos- phorylation, for example, require the presence of magnesium * ions, but we do not know for certain what part these play. It is widely believed that the magnesium furnishes a means whereby the enzymo and its substrate can combine, i.e. that the enzyme- protein and the substrate react together through the magnesium ion. If this is the case, it fallows that the enzyme-protein alone is unable to activate its substrate and that the magnesium must bo regarded os a part of the activating machino. That this is so seems very probable, since one such enzyme, enolase, has been crystallized in the form of a magnesium-containing protein. Many different matallio ions are now known to function as activators of one enzyme or another and, as a general rule, the relationship between the enzyme and the metallic ion is very specific. As is well known, minute quantities of numerous ‘trace elements’, including Mn, Mo, Zn and Co for example, are indis- pensable nutritional factors for living organisms, ranging from bacteria to mammals. It is becoming increasingly clear that they play the part of specific activators for particular enzymes. In 39 ACTIVATORS general they appear to be parts of the activating system, i.e. the enzyme -protein fails to activate the substrata unless the appro- priate metallic ion is also present. Another much-quoted case is that of salivary and similar amylases. If a preparation of salivary amylase is dialysed it loses its power to digest starch at pH 6-8, -which is the optimal value under normal conditions. Activity is regained by the 4 5 0 7 8 Fig. 0. Influence of anioiw upon activity of aalivary amyla*?. Ordinalei initial velocity of hydrolyiii. AbacisM* pli. (Substrate, aoluble • larch: after Myrblek, 1028.) Curve Salt I II IH IV V VI Trace* of NaQ Nad NaBr KI Xa.NO, KOO, addition of chloride ions, but tho effect is not specific, and chloride may bo replaced by other univalent anions, though these arc less effective (Fig. 0). Here, again, there is reason to suspect that tho ionic activator is a part of the activating system, and that in its absence tho enzyme-protein cannot activate its sub- strate in the normal manner. 40 COENZYMES We have so far considered three main types of activators, those which act by unmasking tho aotive groups of the enzyme, those which remove extraneous inhibitory material, and those which perhaps act because they are, in effect, a part of the enzyme. Other cases will be dealt with later when we consider individual enzymes in greater detail. We must turn now to consider accessory substances which confer activity upon inert systems because they play a part in the chemical reaction which follows upon the successful activation of the substrate. Substances of this kind are usually spoken of as coenzymes. Of these the longest known, perhaps, is the co-carboxylase of yeast. Yeast, and the juice expressed from it by the Buchner technique, contain an enzyme known as carboxylase which, in the presence of co-carboxylase though not in its absence, cata- lyses tho decomposition of pyruvic acid into acetaldehyde and carbon dioxide: ~l — Cho COO,H Co-carboxylase enters in some way into this reaction, which is called decarboxylation, though how it does so is still uncertain. It is essential also for a more complex process known as oxidative decarboxylation, a reaction that takes place on a large scale in animal tissues and in which decarboxylation is attended by a simultaneous oxidative change: + 10 , R.CO. !coo:h ► R.COOH + CO,. This reaction, which is a good deal more complex than the 'straight' decarboxylation observed in yeast juice and involves the participation of several additional cofactors (p. 437), is responsible for the production of a very large part of the carbon dioxide formed in respiration, just as the straight decarboxyla- tion catalysed by yeast juice is the source of the carbon dioxide produced in alcoholic fermentation. In both cases we find the same Bubstance, co-carboxylase, as an essential part of the reacting system, and in both cases decarboxylation takes place. The coenzyme must therefore play some specific part in the decarboxylation reaction, though we do not at present know exactly what its role may ho. CH, io 41 C0EXZYME8 Wc are on surer ground when we consider the coenzymes involved in many oxidative processes. The majority of biological oxidations arc carried out by the transference of hydrogen atoms from the substance undergoing oxidation, the ‘hydrogen donator’, to another substance, the ‘hydrogen acceptor’. The dehydrogenases which catalyse reactions of this kind aro not only specific for the hydrogen donator but for the hydrogen acceptor as well. It follows, therefore, that in the absence of the appropriate hydrogen acceptor any given dehydrogenase, capable though it probably is of activating the hydrogen donator, cannot lead to any chemical change. This i3 true, for example, of the lactic dehydrogenase of muscle and of the alcohol dehydrogenase of yeast. In neither case does the Bubstrate undergo oxidation unless the proper, i.e. specific, hydrogen acceptor is present. In these two systems the hydrogen acceptor is a substance formerly known as coenzymo I or cozymase and to winch the somewhat misleading title of diphosphopyridino nucleotido (DPN + ) has more recently been given. (The positive sign is often omitted in common usage but is important in equations such as that which follows below.) This compound is able to take up a pair of hydrogen atoms from ft suitably activated molcculo of lactic acid or of alcohol, and the resulting reaction may bo pictured as follows, e.g. CU,CII(On)COOU + DPN* cn.cocoou + dp.yh + n\ Of the dehydrogenases at present known, the majority require DPN as hydrogen acceptor and cannot use any other known, naturally-occurring substance in its place. Even the closely related triphosphopyridino nucleotido (TPN + ) which differs from DPN only in that it contains three instead of two phos- phate radicals per molecule, cannot replaco DPN. Nor can DPN replace the TPN which is required as hydrogen, acceptor by a emallcr group of dehydrogenases, of which glucose-G-phos- phate dehydrogenase may be cited as an example. DPN and TPN arc not by any means the only compounds which can net specifically ns donators and acceptors of particular radicals or groupings. Indeed, during recent years, numerous reactions have been discovered in which particular radicals or groups are transferred from one molecule to another, and in 42 PROSTHETIC GROUPS every caso it appears that a donator or acceptor substance is involved, over and above the enzyme which catalyses the transfer. These we need not discuss in any detail here sinco we shall refer to them frequently when we deal with intermediary metabolism. DPN, TPN and a considerable number of other compounds discharging comparable ‘carrier* functions play a vital part in metabolism. Normally they occur only in very small concentra- tions in living tissues — Warburg & Christian, for example, could isolato only about 20 mg. of TPN from the red blood corpuscles of some 250 1. of horse blood — but the reactions in which they participate are very rapid indeed. Since these co-substances are essential for the occurrence of these reactions, and since they are present in such small amounts, it is clear that wo must regard them as true catalysts. Their catalytic influence is, in fact, no whit less important than that of the enzymes with which they collaborate. PROSTHETIC GROUPS In recent years a considerable number of enzymes have been greatly concentrated and finally obtained in pure crystalline form, and in many cases, notably among enzymes concerned with processes of oxidation and reduction, the enzyme molecule has been found to contain a non-protein moiety in addition to its protein component. Enzymes of this land, therefore, are con- jugated proteins, and the non-protein fragment is called the prosthetio group in each case. The question arises whether or not there is any essential resemblance between the functional behaviour of a substrate, a coenzyme and a prosthetic group. In substances such as haemoglobin, haemocyanin and the like it has long been known that the non-protein part of the moleculo is firmly attached to the protein component, and the special name of prosthetic group was coined to describe it. In more recent times we have made the acquaintance of conjugated proteins of which the prosthetic groups are relatively much more loosely attached. Thus there exists in the eggs of the lobster a green chromoprotein, ovoverdin, the prosthetic group of which can be removed by heating to 43 • PROSTHETIC GROUPS about 60° C., but reunites with the protein on cooling. Still more striking, perhaps, are the visual chromoprotems, rhod opsin and porphyrop3in, which dissociate on exposure to light, but reunite in the dark. Many such cases are now known, and the notion that a prosthetic group is necessarily firmly attached or screwed down to its protein partner has been abandoned. If we import the same notions into the field of biological catalysis we find that, in the main, it is possible to distinguish befctveen substrates and coenzymes, which are only loosely and temporarily attached to the catalytic proteins with which they co-operate, and prosthetic groups, which are relatively firmly fixed to their protein partners. The part played by the prosthetic group is precisely known in some cases. Certain oxidizing enzymes have a prosthetic group which functions as a ‘built-in ’ hydrogen acceptor, taking over a pair of hydrogen atoms from the activated substrate and subsequently passing them on to another acceptor. In such cases tho enzyme behaves as an acti- vating protein and hydrogen acceptor rolled into one, and tho functional behaviour of tho prosthetic group in such a ease is therefore analogous to that of the coenzymc of a typical dehydro- genase. The essential difference is that, whereas the partnership set up between the activating protein and tho prosthetic group of an enzyme such as catalaso is a relatively permanent affair the partnership between, say, lactic dehydrogenase and lactic acid, or between tho dehydrogenase and DPN, is only a loose and a temporary one on account of the relatively slight affinity between tho partners. Tho difference between coenzymes, substrates and prosthetic groups is therefore one of degree rather than of kind. Whether wo consider DPN os a temporary prosthetic group of lactic dehydrogenase, or haematin ns a permanent or built-in coenzyme of peroxidase, matters little so long ns the functional significance of the various parts of the system is clear. What does matter is that we shall realize that the old, sharp distinction that seemed to exist between enzymes and carriers, and between substrates, coenzymca and prosthetic groups cannot now be justified, a fact which brings a new unifying influence to bear on our knowledge of biological catalysis. 44 QUANTITATIVE CHARACTERIZATION QUANTITATIVE CHARACTERIZATION OF ENZYMES Certain features of enzymic catalysis already alluded to in the first chapter of this book may now bo considered in more detail. Enzymes in general may be considered under two headings, according as their substrates do or do not ionize. As examples of the former type we may consider the proteinoses, and of tho latter typo the carbohydrases. In all these cases tho activity of tho enzyme is profoundly affected by pH, and since the sub- strates of the carbohydrases, for example, do not ionize, tho influence of pH upon these enzymes must bo entirely due to its influence upon the catalytic proteins. Knowing that enzymes are proteins, we may infer that they carry numerous ionizable groups, tho ionic state of which depends upon the pH of the surrounding medium. Since there is some particular pH at which the enzyme is more active than at any other, we may suppose that, of all the possible ionic forms in which the enzyme-protein can exist, only one possesses catalytio properties, and that it is this form that predominates at the optimum pH. Michaelia & Davidson suggested that, since a change of pH in either direction away from the optimum leads to a diminution of catalytio activity, two kinds of ionizable groups must be involved in determining activity, the one land being acidic and the other basic in nature. The enzyme, like any other protein, must be considered as an ampholyte, and in view of tho close resemblance that exists between the dissociation curve of a simple ampholyto such as alanine on tho one hand, and the pH/activity curve of a typical enzyme on the other, Michaelis & Davidson went on to suggest that the two halves of the pH/activity ourve must correspond to tho dissociation curves of tho two particular groups or sets of groups, upon the ionic condition of which the catalytic activity of the protein de- pends. For any given enzyme, therefore, the form and position of the pH/activity curve should be constant, even if the enzyme acts upon several different substrates, always provided that the sub- strates themselves do not ionize. This seems generally to be true. If, therefore, wo determine the pK values for the dissociation of the two sots of ionizable groups, which we can do by carefully plotting the pH/activity curvo, we shall have determined in 45 QUANTITATIVE CHARACTERIZATION quantitative terms two constants that arc characteristic of the enzyme. This baa been done in only a few cases; precise deter- minations are difficult, and the results depend to some extent upon the substrate, temperature, nature and concentration of buffer employed and on the ionic strength of the medium. It is considerably easier, though less precise, to determine the maxi- mum of the resultant of the two dissociation curves, i.e. the TaBJ,E I. pll OPTIMA OP SOME ENZYMES (Frxm Haldane’s Tables) Enzyme R-ptiii Trypsin Amy Use Source Stomach Pancreas Satin Pan cr p.i3 Malt Got Yeast yS-GIacoaidaao Saccharose Lipase Succinic dehydrogenase Xanthine otldaso Argmaao Car boxy Use p-Amlnu-acid oxidase Almond Malt Out Ycaat Liver Pan crew /.VlBVJ Muscle Etth. toll Milk User Yeast User; kidney Substrate Various proteins Starch ( + chloride) Stsrcb Maltose Metbyl-a-D glitcoside Various /f-gloeoslde* Otlobioso Sucroso Ethyl Luiyrote Tnbutyrin Succinate Xanthine L-Argiuino Pyruvate Pt-Ahuuno Optimum pH 1-5-25 8-11 < 17-0 8 C-7-0-8 62 Gt optimum pH; a number of pH optima for various enzymes arc listed in Table J. In tlio case of enzymes that act upon ionizable substrates, the position is complicated by the fact that changes of pH will influence tho ionic conditions both of the enzyme and of its substrate. Further, if wo chnngc the substrate, tho shape and position of tho pH/activity curvo will ho expected to change too, and wc do in fact find that enzymes such as pepsin and trypsin show different pH optima when acting upon different proteins. Nevertheless, if wo stipulato some particular substrate in any particular case we can determine the optimum pH or the two pK values for that particular enzyme-substrate pair. An interesting example of tho usefulness of these ideas is found in connexion with the effect of chlorido ions upon the 40 QUANTITATIVE CHARACTERIZATION activity of salivary and pancreatic amylases. If chlorides are removed, the more alkaline of tho two pK values shifts from about 8 to 6-7, so that the pH/activity curve, which is tho resultant of the two dissociation curves, becomes shifted over towards the left and loses height at the same time (see Tig. 9, p. 40). Presumably, therefore, ono of the active groups of tho enzyme ionizes differently according as chloride is or is no t present. Table 2. Michaelis constants of some enzymes {From Haldane’s Tables) Enxyme Source Substrate K„ Pepsin Stomach Egg albumin 4 5% Trypsin Pancreas Casoin 2% Amylase Saliva Starch ( + chloride) 0-4% „ Pancreas „ „ 0-25% a Glucoaidase Yeast Methyl-a-D-glucoaide 0 03 7-0-075 M „ „ Phenylsx-n-giucosido 0 021-0 050m /J-Glucosklaae Almond Methyl-^-n-glneoaide 0 Q6Q-l*12u „ Phenyb/J-D-glucoaide 0 01 (M3 005 m Saccharose Yeast Sucrose 0 010-0 OIm ,, „ Raflinose 0-24-0 60m » Gut Sucroao 0 02 m lipase Pancreas Etbyl butyrate >0 03m » Liver Ethyl-( + ).mandelate 0-0007M ,» „ Ethyl-( - j-mandelata 0 0017 m Succinic dehydrogenase Musclo Succinate 0 001m Xanthine oxidase Jlilk Xanthine; hyposanthine >3 x 10"*M *, „ Acetaldehyde >lM n-Amino-acld oxidase Liver; kidney dl- A lanine 6 x 10“*M Carboxylase Yeast Pyruvate 0 01m Catalase Liver Hydrogen peroxide 0 025m Another characteristic property of enzymes that can be measured and expressed in quantitative terms is the Michaelis constant K m . This, it will be remembered, is that concentration of substrate at which, in tho presence of a given amount of enzyme, the reaction velocity attains half its limiting value. A list of some values is given in Table 2. Tho Michaelis constant differs from enzyme to enzyme, and varies also from substrate to substrate when the enzyme’s specificity is not abso- lute, If we have an enzyme preparation that acts upon several glycosides, for example, we commonly find a different for each substrate, but this does not tell us whether two or more different enzymes are concerned, or whether a single enzyme of group-specificity is at work. Preparations of yeast saccharose, for example, aot upon both sucrose and raffinose, and the question arises whether yeast contains a raffinase as well as a saccharose. 47 QUANTITATIVE OH AUACTERIZATION The K„ value for sucrose is about one-sixteenth as great as that for raffwose, but the ratio is always the same, no matter how the enzyme preparation may be prepared or purified. If two enzymes were concerned we should expect them to be present in different proportions in different preparations mado by different procedures, and it therefore follows that probably only one enzyme is concerned, a conclusion which is strengthened by the fact that tho pH/activity curve has the same pK values and the same optimum pH whether sucrose or raffinosc is the substrate. A fourth characteristic constant can be determined in certain cases. It will be remembered that in tho presence of a large excess of substrate, the reaction velocity of an enzyme-catalysed reaction reaches a limiting value V. If the (molar) concentration of the enzyme is known and is equal to e, then V=k.e, where & is tho velocity constant of the reaction. We can evajuato e in cases where the molecular weight of tho enzyme is known and the enzyme is available in a chemically pure form, and the value of k is characteristic for a given enzyme acting upon a given substrate. The best we can do in other cases is to deter- mine V in the presence of an arbitrarily defined concentration of the enzyme. If the enzyme is ono that acta upon more than one substrate, say on two compounds a and 6, the TatioT^/IJ will be constant if the enzyme concentration is the same in both cases. Even this second-best determination has proved itself valuablo in deciding the identity of pairs of enzymes. Consider once more tho case of yeast saccharaso. This enzyme attacks sucrose twico as fast os raffinosc, and tho ratio remains tho same from one preparation to another. If two enzymes were concerned wo should expect the ratio to vary from caso to case, but this does not happen, thus adding still more evidence of tho identity of tho presumptive raffinaso with saccharaso. To establish tho identity of ono enzyme with another, even in relatively crude extracts, a number of ways arc thus open. We can see whether the two behave in tho same manner with respect to inbibitora and activators, and we can find out whether their specificities are similar or different. Tlieso, however, are pro- perties that cannot be exactly defined or numerically expressed. 48 SUMMARY but further evidence of a perfectly quantitative nature can be obtained by measuring (a) the two pK values, given by the positions of the points of inflexion of the pH/activity curve, or in default of these, the optimum pH. In addition (6) the Michaelis constant may be determined for one or more different substrates, and (c) we may determine the limi ting velocities corresponding to known or arbitrarily defined concentrations of the two enzymes. SUMMARY Summarizing our conclusions as to the nature of the enzyme- substrate union it may be said first of all that there is every reason to believe that such a union does in fact take place. The union is a very specific one ; a given enzyme can combine with and activate only a limited number of substrates and, often enough, only one substrate. There is reason to think that the reaction takes place at certain definite points on the surface of the enzyme, rather than at any arbitrary point or points, and it seems that the specificity of an enzyme is really a measure of the extent to which the enzyme and the substrate ‘fit’ at the points through which they unite. Even when the activating protein fits the substrate well enough to allow union to take place between them, the ‘ fit ’ may be thought to be still Blightly imperfect, so that the substrate molecule is subjected to some kind of internal strain which results in the increase of chemical reactivity to which we refer as activation. The essential function of an enzyme is that of activating its substrate. It may lose the power to do this for any of a large number of reasons, but in many cases the lost activity can be recovered. But even when activation has been accomplished, the substrate does not necessarily undergo any chemical change, since substances other than the substrate and its activating protein may also be required. We have considered ways in which the power of activation may be restored to an enzyme that has lost it, and wo have considered some cases in which accessory substances or coenzymes enter into the reactions taking place after the substrate has been successfully activated. Finally, we have seen that it is possible to obtain quantitative data which are characteristic of individual enzymes. 49 >DA CHAPTER III BIOLOGICAL ENERGETICS THE CONCEPT OT FREE ENERGY Before going further it is necessary to know something about th© conditions which determine whether or not any given chemical reaction can take place, whether it bo catalysed or un catalysed. To analyse these conditions completely would require somewhat lengthy thermodynamic arguments, bat for present purposes use may be made of a simple mechanical analogy. Let us consider a perfectly smooth body standing on a per* fectly smooth piano. Thi3 body has a certain amount of gravi- tational potential energy, but this energy is not available for the performance of work of any kind unless tho plane is tilted. Suppose now that the plane is slightly inclined. The body begins to slide downwards because some of its potential energy has become available to push it down tho piano. When the body Blips, work can be dono (e.g. if tho body is attached by means of a string to some suitable motor), and tho amount of work dono will be thermodynamically equivalent to the amount of gravi- tational potential energy lost by tho body. How much work can ' be done by this system depends upon tho system itself, for while it is theoretically possible for the body to go on sliding down an inclined plane of indefinite length until the wholo of its potential energy has been converted into work, this is not a case of much practical interest. Ccnerally speaking the properties of natural systems are such that only a jxtrl of their total potential energy is available for the performance of work. A larger or smaller part of the total energy b always unavailable except in theoretical cases. Hen co wo must distinguish between tho 1 free ’ or available energy and tho total energy of the system. Now we know a a a matter of practical experience that a body will never move up the piano so long as tho system is left to itself; on upward movement can only be accomplished by 50 THEE ENERGY supplying energy to the system from an external source. The body will always slido down an inclined plane if left to its own devices, provided that the frictional forces opposing the tendenoy to slip are not too great. If these forces are reduced to zero the body will certainly slip, and work can be done by the system. It is in fact true that, in any self-operating system, the free energy always tends to decrease and, provided that frictional forces do not oppose it, the whole of the free energy lost to the system can be converted into an equivalent amount of work. We can express these ideas more precisely with reference to heat engines. Let us consider a heat engine supplied with an amount of energy equal to Q. This energy is supplied at a high temperature T x (measured on the absolute scale), and conducted to a lower temperature T 2 . It can be shown on theoretical grounds that the amount of work done by such an engine cannot exceed an amount W, where W=Q T,-T„ This equation can be transformed to give Thus the maximum available work will always be less than the total possible work by an amount given by Q{T 2 jT x ). It follows that no work can bo done by an engine of this kind unless the heat can be conducted from a higher to a lower temperature since, if T x ~T 2 , the second term becomes arithmetically equal to Q but opposite in sign. This point is of great importance for biochemical systems, which usually operate at virtually constant tempera- ture. Furthermore, an engine of this kind can only convert its total energy into useful work if QiTJTJ^O, i.e. when T s = 0, and the ‘exhaust’ or condenser of the engine is maintained at the absolute zero of temperature. At all other temperatures a part of the total energy will be unavailable, and the magnitude of the unavailable energy at any temperature T is determined by the product of that temperature and the factor (Q/2J). The latter is known as the entropy of the system and is usually repre- sented by S. Entropy measures the extent to which the total 51 4-2 FREE ENERGY energy of a system is unavailable for the performance of useful work. Similar considerations apply to systems other than heat engines and, in fact, for any self-operating system working at a temperature T we can write the following general equation: F=H-T.S, where F represents the free energy of the Bystem (represented by G in the British usage), i.o. the amount of energy available for the performance of useful work, II is tho total heat energy of the system and S is its entropy. It is not possiblo to determine tho absolute values of these variables apart, of course, from the temperature. We can, however, observo the changes that they undergo when tho system passes from its original state into a new condition represented by F'~H’-T.S\ Subtracting from tho former equation we got (F - F‘) ~(U- IF) - T (S - S'), or, in the usual terminology, AF~AH-T.AS. In a chemical os in any other kind of system there is always a tendency for free energy to decrease when any change takes place in tho system. A chemical change that is accompanied by a fall of free energy can therefore proceed without external ossis- tanco. On tho other hand, a reaction involving an increase of free energy can only proceed if it is in some way coupled to a suitable source of free energy, which may itself be another chemical reaction. Now chemical changes as a whole arc accompanied by thermal changes, and are most commonly exothermic. But the heal evolved during an exothermic process docs not correspond to the change of free energy, for tho change of entropy has also to bo reckoned with. In an ordinary chemical reaction, therefore, all we can measure direct I3' is All , the change in total heat energy ; AF can only be arrived at indirectly. This is not tho place to deal with the procedures whereby values for AF can bo determined or calculated, but one method of 52 FREE ENERGY particular import ance may be mentioned in passing. This depends upon accurate measurements of the equilibrium constant, K, of a reversible reaction, say A+B^G+D. K is defined by the equation K _ [A] [3 ] 10] W In such a case, AF ~ — RT log e K, where R is the gas constant and T the absolute temperature. Sometimes the change of entropy is small, so that A F is approximately equal to A/7, but it is equally possible to have a reaction in which the change of entropy is very large indeed, so that the reaction is actually endothermic but results never- theless in a fall of free energy. We see then, that the total heat change of an exothermic or endothermic reaction gives no indica- tion as to whether or not that reaction can take place under its own power or must be assisted by other processes going on in the environment; the only reliable guide is a knowledge of AF. If A F is zero the system is in chemical equilibrium: if it is positive (i.e. free energy goes into the system) the reaction cannot take place except with external aid: but if it is negative (i.e. free energy comes out of the system) the reaction can proceed of its own accord. Processes which are attended by a decrease of free energy (i.e. AF < 0) are said to bo exergonic, while those which involve an increase of free energy (i.e. AF > 0) are known as endergonic changes. A chemical reaction is thermodynamically possible, then, if it is exergonic, i.e. is attended by a decrease of free energy. Whether or not it actually takes place, however, depends upon other factors. A body will not elide down a rough plane if the frictional forces are greater than the forces exerted by its free gravitational potential energy. Similarly, while a chemical re- action can take place if it entails a fall of free energy, it will not actually do so if the ‘frictional forces’ tending to oppose it are too large. In other words, a chemical reaction requires for its accomplishment that the molecules shall be in a reactive state. This requirement is taken care of in biological systems by the enzymes 53 KATABOLISM AND ANABOLISM they contain. For example, we may keep a neutral, aqueous solution of sucrose almost indefinitely without appreciable hydrolysis, for although hydrolysis is thermodynamically pos- sible, it does not actually take place because the molecules are not sufficiently reactive. If we add a small amount of saccharose or, alternatively, a little dilute mineral add, the Bugar is hydro- lysed, By activating the molecules these catalysts overcome the 'frictional forces’ opposing hydrolysis. It follows from all this that no catalyst can initiate or accelerate a reaction that is not already possible on energetic grounds: all that it can do is to influence the velocity at which a thermodynamically possiblo reaction actually takes place. BREAKDOWN AND SYNTHESIS IN BIOLOGICAL SY8TEMS The numerous and diverse chemical processes which underlie tho activities of a living organism collectively constitute its melabotism. For purposes of discussion the overall metabolism of a cell or tissue can be considered as consisting of two parts, kntabolism, which involves the chemical degradation of complex materials into simpler products, and anabolism, which involves the elaboration of complex products from simpler starting materials. Katabolism and anabolism are usually defined in terms of chemical complexity, but this is a somewhat unsatis- factory variable because wo have no quantitative means of measuring or expressing it. But, in a general kind of way, increases in chemical complexity are associated with increases of free energy, and for our present purposes we may con- veniently define as katabolio those processes which involve a fall of free energy and are therefore exergonio, and as anabolic those in which the free energy increases and which are therefore endergonic. Now, animal and plant tissues &3 a whole are known to contain intracellular enzymes which can be extracted and shown to catalyse tho katabolio breakdown of proteins and other high- molecular materials into simpler units. It seems improbable that this can bo their solo function in the cell. After death these enzymes do in fact lead to the digestion of much of the tissue 54 DYNAMIC EQUILIBRIUM substance, a process known as autolysis, and this is why game and certain kinds of meat aro allowed to ‘hang’ before being cooked. This same process of autolysis is the first stage in the decomposition of dead organisms; bacteria and the biblical worms come into the picture considerably later. During life, however, anabolic {i.e. synthetic) processes are numerous and of very great importance, especially during growth and in tissue repair after injury. They include the syn- thesis of catalytic and tissue proteins from amino-acids; poly- saccharides from simpler sugars ; oils, fats and waxes from their constituent alcohols and fatty acids, to make no mention of the elaboration of other more or less highly specialized products such os hormones, pigments and so forth. But, in addition, we have good reason to believe that the tissue constituents them- selves are not permanent, statio structures, but rather that they are constantly in a state of breakdown balanced by synthesis.* This is true even of hard structures such as the bones and the teeth. It seems, therefore, that wo must envisage the possibility that intracellular enzymes aro concerned both with the break- down and with the synthesis of proteins, fats, carbohydrates and other cell constituents. On theoretical grounds we must believe that an enzyme which can catalyse the hydrolysis of its substrate, for example, must also be capable of catalysing the condensation of its products, and in a number of instances such a reversibility of action can readily be demonstrated, e.g. among lipases and esterases (6ee p. 06 ; Fig. 12). In many other cases, however, attempts to demonstrate synthesis by an enzyme known to catalyso the corresponding degradation have yielded only negative results. It must bo remembered, however, that in order that a given chemical reaction shall take place it is not enough for the mole- cules concerned to l>e in a reactive state : the energy conditions also fitxisi be favourable. It is possible, therefore, that the failure of man y experiments has been duo only to inability to reproduce under experimental conditions the proper energetic circum- stances, and does not necessarily mean that the enzymes con- cerned are inherently incapable of working in both directions. ?*** COC!iei ‘ on Be * Schoenheimer'a Th* Dynamic RtaU of Hedy Conrtituent* ' London, which j„ &Wly 56 DYNAMIC EQUILIBRIUM For these reasons we might assume that in the cell, if not in the laboratory, the right energetic conditions are attainable, and that, in the cell, the earn© enzymes can catalyse synthesis and breakdown alike. But their synthetic action would be expected to require the maintenance of normal physiological conditions inside the cell because, if these break down, the necessary provisions of energy might not be forthcoming so that only the phase of degradation would be observed. The function of the intracellular ‘autolytic* enzymes might bo regarded, then, as that of maintaining equilibrium between the complex materials of the cell substance and their simpler constituents, so that a balance is struck between synthesis and degradation. And wo must not think of the equilibrium thus set up and maintained as in any sense a static affair but as an essentially dynamic system in which breakdown and rcayn- the&is are proceeding simultaneously at high hut equal velocity. We should do well to recall Hopkins’s celebrated aphorism, that life ia a dynamic equilibrium in a polyphasic system. In living cells, then, katabolism and anabolism proceed together. This raises several problems of the greatest energetio importance. First, what is the biological source of the free energy required for the essentially endergonic processes of anabolism; secondly, how is that free energy harnessed or ‘captured* by the cell and, thirdly, bow is it brought to bear upon the processes wherein it is consumed! And it should be borne in mind that free energy is expended not only in anabolic reactions, but in the performance of muscular and other kinds of biological work. For many years these problems seemed 60 complex and so wholly baffling that many biologists were content to believe them unanswerable ; that living organisms must in some way contrive to operate in defiance of the laws of thermodynamics. Although the most spectacular progress in the field of biological energetics has been made in comparatively recent years, the most funda- mental experiments were undoubtedly those performed just before the end of the last century by Ilubner, in Germany, and speedily confirmed in America by Atwater, Rosa & Benedict. These workers constructed animat calorimeters capable of con* taining dogs, and later oven men, in conditions of comparative comfort for periods of days or even weeks. The apparatus, which S6 ANIMAL CALOEIMETKY will not be described here since there are admirable descriptions in many physiological and biochemical text-books, was con- structed in Buch a way that the following measurements could bo accurately and simultaneously carried out: (а) Total energy output, measured as heat. (б) Total oxygen consumption. (c) Total carbon dioxide production. (d) Total nitrogen excretion. Numerous corrections had, of course, to be applied, e.g. for heat brought into the apparatus in hot food and for heat removed in the bodily excreta. And there were many engineering difficulties. From (6), (c) and (d) it is possible to calculate the weights of carbohydrate, fat and protein oxidized in the course of an experi- ment and hence to arrive at the total yield of energy attributable to their metabolism, the calorific values of these metabolic sub- strates having previously been determined with a high degree of accuracy by combustion in the bomb calorimeter. Table 3. Calculated and observed heat outputs or doos in Rubner’s experiments. (After Lusk ) (Kilo-col. per sq.m, of body surface per 24 hr.) Duration of exp. Diet (days) None 5 Fat 5 Meat and fat 8 12 Meat 6 7 Heat output Calculated Determined 1296-3 1305-21 1091-2 1056-0 f 1510-1 149S 3 2492-4 2483 01 3985-4 3958-4 J 2249-8 2270 91 4780 8 4769 3 1 Difference <%> -1-42 -0 97 -0-42 +0-43 Of such fundamental importance is the outcome of these ex- periments that the reader is asked to examine with more than usual care the data of Table 3, which is compiled from the results of Rubner’s experiments. These were followed up by experiments of human subjects in the Atwater-Rosa-Benedict calorimeter. Whether the subjects were resting, active or even taking fairly strenuous exercise, e.g. by riding stationary bicycles, the observed and calculated heat outputs agreed within limits even 57 BEVEBSIBILITY narrower than in Rubner’s experiments. These results provided irrefutable proof that a living organism as exalted even as man himself cannot create energy out of nothingness, and no con- vincing evidence to the contrary has at any time been forth- coming. Living organisms, like machines, conform io the taw of the conservation of energy, and must pay for all their activities in the currency of metabolism. With these results before us it is clear that there can bo but one answer to the most fundamental of our problems. Energy used in anabolism must be drawn from katabolic processes: no other source is available ,* This means that there must exist some kind of coupling between katabolism and anabolism; Bomo kind of mechanism that allows the transference of free energy from one to the other. There then remains the problem of the manner in winch the free energy to which the cell gains access in the courso of its katabolic operations can bo captured and utilized for anabolic and other purposes. In order to deal with these problems we must necessarily enquire into tho mechanisms which lmderlio the synthetic reactions that make up anabolis ra and, in particular, we must consider tho possibility that anabolic processes may bo merely reversals of katabolic changes catalysed by the same enzymes. BEVEBSIBILITY OF BIOLOGICAL REACTIONS AH reactions are reversible in theory, but because they are reversible in theory it does not necessarily follow that they arc reversible in actual practice, at any rato under biological con- ditions. Tho explosion of a hydrogen bomb is reversible in theory, but reversal of the explosive process is somctliing that is not very likely to be accomplished, at any rate in tho foreseeable future. Mach tho same is true of many enzyme - ca talyscd react ions under biological conditions; and, of course, it is with biological re- actions that we are particularly concerned here. Now enzymes, in common u itk catalysts of other kinds, influence only tho velocity and not the direction of the reactions they catalyse, and there are, as hna been pointed out already, many enzyme-catalysed * TL.U tutrmral *pp!ic*, of worse, to tntmtk. Ctwn pUnl* twrtem u»e aoUr energy for »nil>oUarn. 58 REVERSIBILITY processes that go equally freely in either direction. But many others do not. No chemical reaction is totally irreversible; on the contrary, every reaction tends towards an equilibrium, e.g. A^B+C. Equilibrium is rapidly reached in the presence of a suitable enzyme or other catalyst, and the composition of the equilibrated mixture is determined by the molar concentrations of the re- actants and by the overall change of free energy. If the intrinsic free energy of A is very large compared with that of the products, decomposition of A will predominate over its synthesis and, if the degree of predominance is very large indeed, as it often is in practice, the process will bo virtually unidirectional and may, for all practical purposes, be regarded as irreversible. In con- sidering the conditions required to reverse the process and so bring about the synthesis of A, we havo in fact to consider the conditions required to alter the final equilibrium in such a way that synthesis can become the predominant process. Let us consider now a simple biological process of a virtually irreversible kind that can bo represented as follows; A B+C; AF=— coal. Since the change of free energy associated with the process is less than zero (AF< 0) the reaction is one that is thermo- dynamically possiblo and will therefore proceed spontaneously in the presence of an appropriate catalyst. When it takes place, e cal. of free energy will become available for each g.mol. of substrate transformed and, if some suitable device were present to trap it, this free energy could be converted into work of some kind. But in the absence of such a contrivance this free energy is merely dissipated and the output of heat can then he calcu- lated from the usual equation: Since e cal. of free energy are liberated when the reaction proceeds in the forward direction, it follows that it can be made to go backwards if, and only if, an amount of free energy equal to e cal. is supplied in some way. It is useless merely to supply heat ENERGETICS OF BIOSYNTHESIS equal to Af/ to the eyetem because, as we have seen, beat cannot do work unless it is conducted from a higher to a lower tem- perature, and biological processes usually take place at virtually constant temperature. Thus, although our reaction is theoretic- ally reversible and although the enzyme concerned may be able to catalyse the forward and reverse processes alike, tbo back- ward reaction can only be realized if the requisite amount of freo energy can be provided ; and provided, moreover, under biological condition s. In the laboratory the chemist, faced with tho task of per- forming an energy-consuming synthesis will as a rule make uso of high temperatures, strong acids, strong alkalis and similar ‘powerful’ reagents. But theso aro resources that are denied to biological systems. Living cells, by contrast, operate in tho neighbourhood of pH 7 and at temperatures ranging roughly from zero to 40® on the centigrade scale, and oven under theso very ‘mild’ conditions their synthetic ability is vastly superior to that of tho organic chemist. ENERGETICS OF SYNTHETIC REACTIONS Consider again tho reaction A-+B + C; AF*»— ecal. One way of achieving tho reverse process would bo in some way to incorporate into one of the reactants, B and C, an amount of freo energy equal to or greater than e cal. per g.mol. This, indeed, is tho usual method in biological systems, and processes whereby this is accomplished aro commonly known as priming reactions. Tho usual biological procedure is that some suitable additional grouping, very commonly a phosphate radical, is introduced into one of the reactants, say B, to givo a product of higher frco-cnergy content, say B — R. In the presence of a suitable catalyst a reaction leading back to tho original starting material is then possible: B-R + C-e^l+R; AF (C)Af> 0 (B)| Whether glycogen is broken down to glucose by digestive enzymes (A) or by the liver enzymes (D + B), there is a fall from a higher to a lower free-energy level, and in neither case can the pathway of breakdown be retraced. Synthesis can only proceed by a pathway involving a priming reaction (C) whereby the glucose is phosphorylated and thus raised to a higher free-energy level. Once this has been accomplished the phosphorylated Bugar can polymerize (D) to form glycogen with little further change of free energy, so that an equilibrium is set up. We shall return presently to a more detailed examination of this process. 01 nian'ESEE + C~dl, where C represents the guanidine derivative, creatine. When this reaction takes place there is only a small change of free energy. The whole 11,500 cal. associated with the terminal phosphate radical of the ATP are transferred, along with the phosphate radical itself, to the creatine, and tho free energy of hydrolysis of the product, creatine phosphate, is about —13,000 cal. per g.mol., a difference of the order of only 1500 cal., so that the transfer is readily reversible. A very convenient shorthand VA THE HEXOKINASE REACTION notation for reactions of this hind may be used and is frequently used throughout these pages: Double-ended arrows are used to indicate the free reversibility of the reaction. As a second example we may consider an irreversible transfer reaction that takes place between glucose and ATP in the presence of the very widely distributed enzyme, hexokinase. The products are glucose-6-phosphate and adenosine diphosphate (ADP). The reaction may be written in the following manner: CH.OH Overall : AJ’- -8500 cal. If we consider only the conversion of ATP to ADP we see that thi3 part of the system suffers a fall of 11,600 cal. of free energy. The increase of free energy in the conversion of glucose to its phosphate amounts to only about 3000 cal., however, so that, in the complete system, a balance of some 8500 cal. of free energy is left over and spilled out. This reaction, unlike the Lohmann reaction, is therefore unidirectional and irreversible. 65 BDA EtJNCTlONS OP ATP Although bo much free energy is wasted, this is a reaction of fundamental importance in carbohydrate metabolism because glucose can neither be stored nor metabolized unless it is first of all pbosphoiy luted, and this, the hexohinase reaction, is the principal and perhaps the only mechanism through which this preliminary phosphorylation can be achieved; simple direct introduction of a phosphate radical is energetically impossible since A-F>0 for such a reaction. The hexokinas© reaction is typical of priming reactions as a whole and is, in fact, the first step in the synthesis of glycogen from glucose (see p. 421), Group transference is a common phenomenon in biochemistry. Enzymes are known that can catalyse the transference of methyl-, amino-, amidino- and a variety of other radicals, over and abovo the phosphatic and glycosidic groups that have provided our examples here, and it should bo home in mind that whenever a group or radical is transferred from one molecule to another, there is aheays a strong probability that it icill carry xrilh it a larger or smaller quota of free energy. THE BIOLOGICAL ENERGY CYCLE In both the Lohmann and the hexokinas© reactions, ATP loses phosphate and energy in favour of its reaction partner and in both cases an endergonic reaction is achieved by coupling it to one that is exergonic. This appears to bo a fundamental opera- tion in the synthesis of complex biological compounds from simpler starting materials, and, as far as we know at present, anabolic, synthetic operations can only be accomplished at the expense of a high-energy radical of some kind, most usually the terminal of ATP. In some cases the immediate energy source is the terminal ~© of the triphosphate of some other nucleoside, e.g. gnanosine triphosphate (GTP), but tliis rapidly recoups itself by an enzyme-catalysed reaction with ATP; (i) GTP-v GDP +(0 + energy, (u) GDP+ATP^GTP+ADP. And this i3 not all. ATP can bo split in any of a considerable variety of ways. It may yield up its terminal phosphate radical and the energy with which it is associated in a reaction of the CO FUNCTIONS ON ATP kind we have just discussed, so that energy is, as it were, ‘ forced ’ into the phosphate acceptor: or, under the influence of an adenosine triphosphatase of some kind, it may undergo a cata- lysed hydrolysis to yield ADP together with free inorganic phosphate. In this case, however, the free energy of the ter- minal phosphate group is not conserved, either in whole or in part, as it is in a transfer reaction, but is liberated. If the reaction is allowed to proceed in a test-tube or flask this free energy is dissipated largely as heat, but in an intact muscle it appears largely in the form of the mechanical energy of the concomitant muscular contraction. It is probably not without significance, therefore, that the adenosine triphosphatase of muscle is identical with myosin, which is itself a constituent of the actual contractile machinery of the muscle cell and plays the part of a ‘transformer’ which converts chemical into mechanical energy. In the electric organs of certain fishes, e.g. Torpedo, Raia spp., Oymnotus, Malaplerurus, ‘transformers’ of a different kind are present, for myosin is lacking in the most highly developed of these organs, despite the fact that they arise by the morpho- logical transformation of embryonic pre-muscular cells. The electrical energy dissipated when these organs discharge arises, according to present knowledge, from ATP once again. The light energy dissipated by bioluminescent organisms such as the fire-fly [Photinus pyralis) similarly has its origin in ATP, and energy drawn from ATP can also accomplish the performance of osmotic work, in the absorption of glucose from the small gut against large osmotic gradients, for example. Now the amounts of ATP present in most tissues are not very large, and are out of all proportion to the energy turnover of these tissues. It follows, therefore, that ATP must be used and reformed over and over again if the many and diverse activities of the tissues are to be maintained. But, as we know today, new supplies 0/ arise in the course of many katabolic trans - formations and, indeed, it would appear that katabolism as a whole is directed above all to the generation of new energy-rich from energy-poor phosphate radicals. These can be transferred to ADP left after previous breakdown of ATP molecules. It is significant, however, that newly generated ~© cannot be utilized 67 5 . 3 STORAGE OP ~<$> directly for the performance of endergonio operations: they can be put to service only through the intermediation of the ADP j ATP system. No major alternative system has so far been brought to light. It would not be profitable to discuss the processes that lead to the generation of new ~ ® until we have studied katabolie pro* cesses in some detail, but one simple example may not be out of place here. Tho synthesis of ATP from ADP calls for the provision of some 11,600 cal. of free energy per g.mol. This can be provided, for instance, by new generated in the course of the metabolic degradation of glucose or glycogen. One such radical appears when one of the numerous intermediate products, glyceric acid-2-phosphate, undergoes dehydration under the influence of the enzyme enolase to yieldj enoJ-pyruvic acid phosphate: CH,01I CJI, ino© -*> &0*-® +h«q iooff ioon The removal of the elements of water from glyceric acid phos- phate results in structural alterations within tho molecule. These are attended by a redistribution of the intrinsic free energy of the system, leading as it were to a ‘ concentration ' of free energy in the neighbourhood of the phosphate radical ; in other words, to the generation of a now high-energy phosphate group. Tho free energy, together with the phosphate radical, is then transferred to ADP and a new molecule of ATP is thereby produced. THE 8TORAOE OF HIGH -ENERGY FHOSPIIATE High-energy phosphate can be transferred from ATP to creatine, arginine and certain other guanidine bases, to form ‘phos- phagens ’ and, in certain tissues, high-energy phosphate is stored in the form of these substances. This stored energy cannot be used directly, but only through the ADPjATP system. It can, however, be transferred directly to ADP through tho Lohmann reaction, and the transfer is freely reversible. When such a tissue enters into physiological activity, ATP is broken down and provides energy in the form characteristic of STORAGE OF the particular organ. Unless or until new high-energy phos- phate is generated rapidly enough to resynthesize ATP as fast as it is used up, the tissue can draw on the energy-rich phos- phate radicals of the phosphagen reserve, transferring these to ADP and thus regenerating ATP. Later, after activity has ceased, the metabolic generators continue to run for a short time, so that ATP continues to he produced and, reacting now with the free guanidine base, resynthesizes phosphagen and thus replenishes the energy etore.These notions may be diagram- matically summarized in the so-called ‘energy dynamo’ of Fig. 10. It is interesing and important to notice that the second high- energy phosphate radical of ATP is not as a rule available as a direct source of energy for chemical synthesis, nor for the production of mechanical or electrical energy. It can, however, bo rendered available through the activity of the enzyme, myoldnase, which specifically catalyses the transference of the terminal phosphate radical from one molecule of ADP to a second molecule of the same substance, so that AMP (adenosine mono- phosphate) and ATP are produced: 2A— ~® 5=»A— <® + A— The now molecule of ATP can then be utilized in the usual manner. 09 STTMMABT SffMMABY 1. The breakdown and synthesis of biological materials may, but do not necessarily, follow a common chemical pathway. Whether the pathways can ho the same or must be different depends upon the energetic conditions that prevail. 2. Free energy can be transferred from one molecule to an- other an d its transference is attendant upon that of a phosphate, a glycosidio or some other grouping or radical. 3. The free energy required for anabolic processes and other energy-consuming operations arises from katabolism. Katabolic processes are so organized that they lead to the formation of new high-energy phosphate radicals at the expense of the intrinsic free energy of their substrates. 4. The newly generated high-energy phosphate radicals ore ' captured’ \>y trnnafcTcnvo to so that raw ATP are formed, and it is only through the intermediation of the ADP/ATP system that their energy can be utilized for biological purposes. 5. High-energy phosphate can bo stored in certain tissues in the form of pbosphagens. 0. The free energy of the terminal high-energy phosphate radical of ATP can bo utilized in one or another of the many energy-expending processes in which this remarkable substance participates. Among theso are numerous 'priming’ reactions which form essential preliminary steps in the biochemical syn- thesis of complex, liigher energy products from simpler starting materials of lower free -energy content. 70 CHAPTER IV HYDROLASES AND ADDING ENZYMES GENERAL INTRODUCTION The classification of enzymes presents & good many problems and no entirely satisfactory system has yet been devised. Some kind of classification is desirable, however, for purposes of description and enzymes will here bo arbitrarily divided into the following groups : * Hydrolases. Adding enzymes. Transferring enzymes. Isomerase3. Oxidizing enzymes. Enzymes which catalyse a hydrolytic splitting of their sub- strates are known as hydrolases. All digestive enzymes, whether intracellular or extracellular, fall into this class, and digestion itself may be regarded as an organized, orderly series of hydro- lytic reactions which result in the smooth, stepwise breakdown of large, complex molecules into smaller and simpler products. Hydrolytic enzymes are capable of carrying through the processes of digestion from beginning to end without the aid or interven- tion of enzymes of other kinds. Apart from the digestive hydrolases there are numerous intracellular hydrolases concerned with processes other than digestion. A group of exceedingly important enzymes known as phos- phorylases has been discovered in recent times: these catalyse the splitting of their substrates not by means of the elements of water but by those of phosphoric acid, a process known as pbosphorolyis. This bears a close superficial resemblance to hydrolysis: Uydrolyti* R’R' + H.OH R.OH + H.R'. Phosphorohj>i» RiP.' + H.O® R.O® + H.R'. In spite of this resemblance the phosphorylases are better classi- fied as transferring enzymes and will be so considered in the next 71 REVERSIBILITY chapter. Certain hydrolases also have been found to be capable of catalysing transfer reactions as well as hydrolysis, and this aspect of their activity will also be dealt with in the next chapter. In addition to the hydrolases we shall consider in the present chapter another group of enzymes. These catalyse a Bimplo, direct splitting of their substrates without the intervention of any other reactant : R.R' p=* R + R', Enzymes of this type are best named with reference to the reverse aspect of the reactions they catalyse. Whereas hydro- lytic enzymes catalyse processes of condensation when acting in reverse, members of this other group catalyse simple addition reactions and may therefore be called adding enzymes. They include enzymes which catalyse the addition of water, carbon dioxide or some other substance to a second molecule. Although reversibility signs are used in the generalized equa- tions given here, there are many cases in which the reactions catalysed are, for energetic reasons, irreversible in practice, at any rate under biological conditions. In what follows we shall consider the properties of a number of the most widely distributed hydrolytic and adding enzymes. If the next few chapters savour somewhat of the catalogue it is because we must necessarily know a good deal about individual enzymes before we can attempt to see how, in the living cell, tissue or organism, they are organized into the orderly catalytio systems that underlie the metabolic processes inseparable from life itself. PEPTIDASES Until comparatively recently it was usual to distinguish between two main groups of enzymes concerned with the hydrolysis and presumptive synthesis of proteins and their breakdown products. On the one hand were enzymes such as pepsin and trypsin, which were believed to act upon proteins but not upon smaller molecules such as those of the peptones and peptides. On the other baud was a group of so-called peptidases, known collectively as erepsin, and these were regarded as being devoid of action upon 72 PEPTIDASES any tut the relatively small molecules of polypeptides and, perhaps, peptones. Most of these earlier views have now been seriously modified or even abandoned. As is well known, the chemical synthesis of peptides was, until about 1932, a difficult undertaking, and of the enormous variety of possible peptides a mere handful was obtainable by synthetic chemistry. Our knowledge of the specificity of protein- and peptide-splitting enzymes was fragmentary in consequence. In more recent years, thanks at first to the ingenious methods introduced by Berg- mann, peptides of many kinds hitherto unavailable have been produced and, in the meantime, a number of the enzymes themselves have been obtained in highly purified, crystalline form. The older methods for the synthesis of peptides mostly in- volved covering the — NH 2 group of ono amino-acid and con- densing the acyl chloride of the protected product with a second amino-acid or its ester. A ‘covered’ dipeptido could thus be obtained from which, by removal of the covering group, the free dipeptide could theoretically be regenerated. The reactions involved may be written as follows, if we represent the covering group by X: R X.HX.JjH R' (1) Aoci + H.N.in iooc,n, R (2) X.HN.in R'+2H»0 — io— HN.in COOC.H, Tri- and higher peptides could be prepared by further reactions based on the same lines before removing the covering group. The use of the benzoyl radical, introduced by Curtius, made possible the synthetio production of numerous benzoylated peptides, but attempts to remove the benzoyl group by hydro- lysis resulted in simultaneous hydrolysis of the peptide bonds, so that the yields of tree peptides were negligible at best. The use of X.HN.iil R' * io— HN.in + HCl; £ooc,h. *■ X.OH + C,H,OH + R' io— HN.ill toon 73 SYNTHESIS 0E PEPTIDES other substituent radicals had little better success, and numerous other methods of synthesis have been tried. In Bergmann’a method the covering group employed is ono which can be removed by reduction, a treatment which does not at the same time open peptide links. Bergmann employed ben zyloxyca rbonyl chloride. This Teagent is made by treating benzyl alcohol with phosgene in solution in toluene: C 4 H,CH,0H + COO, - C,H,CH,O.C0.a + Ha It reacts readily with the amino-group of an amino-acid to yield an N-btrizylozycarbonyl-derivative, thus: C,H,Cn,O.CO.CI + H.N.R - C«H,CH,O.CO.ItN'.R +■ Ha Its subsequent removal is accomplished by catalytic reduction with hydrogen in thepresencoof colloidal palladium, a treatment that is without action upon peptide bonds: C.H.CH, O.CO.HS.R + H, -C.tt.CH, + CO, + H.N.R. tolutnt These benzyloxy carbonyl compounds are very stable, and can readily be converted into the corresponding acyl chlorides so as to facilitate condensation with a second amino-acid. There is, more- over, noracemization of the productundcrBergmann’s conditions. As an example of a synthesis carried out by Bcrgmann’s method we may take the relatively simple case of the prepara- tion of glycylglycino. The reactions used are os follows: (I) C,TT,CH,O.CO.CI + H.N. cn.cooil - C,H,CU,0 . CO— HN . Cn.COOII + HC1; bcnzyloiycnrbonylgl'ja %t ra, (ii) C.TI ,C 51,0 . CO— IIN . CH,CO OH ► {VbCH,o.c(>--HN.CIJ,coa.• (ui) c, ii.ai, o . co — hn . cn.coci ♦ n.N.c^coo.c.n, glycine tlhyl ttlcr - C,H,CH,0 . CO — HN CH.PO— HN.CJI, COO. C,JJ, + HCJ. Inzyloxycorbonyu/lyrylglyci-ns My I islet (Iv) C,H,CH, 0 . CO-j-HN. CH.CO— HN.CH,COO.C,II, saponification c,n,cn, + co, + h,r.ct,co— hn\cu,cooh + c,n,ou. and reduction glycylglytin* By taking suitable precautions it is possible to prepare pep- tides containing the dicarboxylic nnd dibasic amino-adds as well as mo no -a mtno-mono- carbo xy lie acids by this method. With its aid peptides of many different kinds have been made 74 DIGESTIVE PEPT1DA8ES available and a number of alternative synthetic methods have now been devised. Digestive peptidases. The proteolytic enzymes of vertebrates fall into two groups, the first of which is mainly concerned with the degradation of the large molecules of the food proteins to yield smaller fragments, the second group completing the pro- cess initiated by the first and leading eventually to the liberation of free amino-acids. It is worthy of note that denatured proteins are more readily attacked than the native materials. The first group includes pepsin, which arises from the gastrio juice, and trypsin and chymotrypsin, formed from precursors present in the pancreatic j uice. In the second group we have carboxy peptidases, contributed by the pancreatic juico, together with aminopepli- dases and dipeptidases, which are present in the intestinal secretions, probably together with other carboxypeptidases. The proteolytic (‘peptolytic’) enzymes of the intestinal juice were formerly regarded as one enzyme, to which the name erepsin was given, but it is now known that erepsin is, in fact, a very complex mixture of enzymes, each individual member of which i3 very much more highly specific than the original mixture. Activation Four of these enzymes are actually secreted in the form of enzymatically inactive precursors which undergo activation by 'unmasking'. Pepsin is secreted by the gastric mucosa in the form of pepsinogen , which is activated in the first instanco by the hydrochloric acid of tho gastric juice to yield pepsin itself. Pepsin, once formed, is capable of activating more pepsinogen, so that, once begun, activation is an autocatalytio process. Both pepsinogen and pepsin itself have been obtained in pure, crystal- line form, and it has been shown that the conversion of the inert pro-enzyme into tho active form is attended by a fall in molecular weight from some 42,000 to 38,000. A polypeptide of molecular weight 5000 or thereabouts is split off, and may be regarded as the 'masking* substance. Trypsin and chymotrypsin are similarly secreted in the form of enzymatically inert precursors, trypsinogen and ckymotryp - sinogen. All four compounds have been crystallized. Trypsinogen is activated by an enzyme-like substance called enterohinase, 75 DIGESTIVE PEPTIDASES which is present in the intestinal secretions and acts upon trypsinogen to produce trypsin, which then activates more trypsinogen bo that, as in the case of pepsinogen, once begun, activation is an autocatalytic process. In this case, however, there is only a small change in molecular weight. The masking peptide in this case has been isolated and identified os consisting of valyl-{aspartyl) 4 -lysine (mol.wt. ~ca. 700). The carboxyl group of the lysine is engaged with the amino group of an iso-leucine radical of trypsin itself, and the activation process consists in tho hydrolysis of the lysyl-iso-leucyl bond. Chymofcrypsinogen differs from trypsinogen in that it is not activated by enterokinase. It is, however, activated by trypsin. Chymotrypsin does not activate chymotrypsinogen, and in this case, therefore, activation is not autocatalytic. The activation of the enzyme precursors of pancreatic juice is thus started off by enterokinase, which activates trypsinogen with production of trypsin. The trypsin then activates more trypsinogen and chymotrypsinogen as well. Tho proteolytic enzymes of pancreatic juice do not therefore become active until they reach the amall intestine and come into contact there with enterokinase, which acts as a trigger to fire off the entire activation process. Of the other pancreatic peptidases only carboxypoptidaso requires this kind of activation: the pro-enzyme is activated by trypsin, but not by enterokinase or chymotrypsin. Amino- peptidases and dipeptidases are not activated in this way. They lose their activity if dialysed, but activity is specifically restored by the addition of traces of specific metals such as Mn, Zn, Alg and occasionally even Co, which appear to bo the natural activators for theso enzymes. Some authorities consider that these metafile activators behave as loosely bound prosthetic groups for the enzyme proteins with which they collaborate, and thus provide a means of attachment for tho substrates by forming co-ordination compounds with them. Specificity Biologically speaking it is possible to draw some sort of dis- tinction between pepsin and tho trypsins on the one hand — the proteinoses of the old nomenclature — and the group of pepti- 76 DIGESTIVE PEPTIDASES dases on the other. The digestion of the food proteins is begun by the ‘proteinoses’, and the fragmented products thus formed are further degraded by the ‘peptidases * to yield in the end free amino-acids. It was formerly believed that pepsin and the trypsins are able to attack only large molecules of the same order of size as the protein molecules of the food, and that the pep- tidases are only able to deal with molecules of the order of size found among polypeptides and perhaps peptones. More recent work, which began only when Bergmann’s method had made a wide variety of synthetic peptides available, has shown that pepsin, chymotrypsin and trypsin, as well as the peptidases, are able to act upon comparatively simple peptides, always provided that peptide linkages of the right kind are present. Bergmann therefore called them all peptidases, but it is still possible to maintain a distinction between the two groups. The peptidases of the older nomenclature are able to split only those peptide links which join terminal amino-acid residues to the main chain. Pepsin and the trypsins, on the other hand, can also act upon peptide bonds remote from the terminal units and are accord- ingly called endopeptidascs, by contrast with the exopepti- dases, i.e. carboxy-, amino- and dipeptidases. Briefly, then, pepsin, trypsin and chymotrypsin, the ‘proteinases’ of the older classification, become the endopeptidases of the newer nomenclature, while carboxypeptidases, aminopeptidases and dipeptidases, the ‘peptidases ’ of earlier years, become the earo- peptidases. Our ideas about the specificity of these enzymes have under- gone a drastic change in recent years. It was formerly held that pepsin, trypsin and chymotrypsin are able to attack peptide bonds at more or less any point in the chain. But if both pepsin and trypsin are allowed to act upon the same protein we find that both enzymes together open up more peptide linkages than either alone, and it follows that both enzymes do not act upon the same, but upon different linkages. We now know a good deal about the nature of the particular bonds attacked by the various peptidases. One general fact may be emphasized at once: with certain exceptions, the peptidases as a whole act only upon normal peptide links, i.e. links formed between the ct-amino- and a-carboxyl radicals of L-amino-acids. Enzymes capable of 77 SPECIFICITY OF ENDOPEPTXDASES attacking peptides containing amino-adds of the D-6eries have been described, but will not bo considered here. Pepsin can act only on peptide bonds of certain definite types. As Bergmann showed, it can attack a peptide link lying between an L-dicarboxylio and an L-aromatio amino-acid, given certain conditions. Theso are, first, that the second carboxyl radical of the dicarboxylic acid residue must be free, and secondly, that there must not be a free amino-group in the immediate vicinity of the peptide linkage. Thus pepsin attacks benzyloxycarbonyl- n-glutamyl-i-L-tyrosine, glycyl-E-glutamyl-j-L-tyrosme, benzyl- oxycarbonyl-L-glutamyl-j-L'tyrosino and benzyloxycarbonyl-L- glutamyl-i-L-phenylalanine. The influence of the free y-carboxyl group of the glutamic acid residue is neutralized if there is a freo amino-group nearby, for E-glutamyl-L-tyrosine and benzyloxy- carbonyl-L-glutarayl-L-tyrosino amide are resistant to pepsin. The resistance of benzyloxycarbonyl-L-glutamyl-n-tyrosine amide is not due solely to the fact that the a-carboxyl group of the tyrosino is covered, for benzyloxycarbonyl-L-glutamyl-j-i** tyrosyl-glycine is attacked, though more slowly than benzyloxy- car bony 1 -n-glutamy I -i -E-tyrosine . Replacement of the E-acids by their d - isomers makes the peptides resistant to pepsin, for benzyloxycarbonyl-D-glutamyl-L-tyrosine and benzyloxycarb- ooyl-L-glutamyi-D -pheny lalanine are not attacked. These results are summarized in Table 0. Table 6. Action or vepsin cros synthetic pettices Action of Substrate pepsin Ccnr^loxycarbonyl-L-gluUmyl. -L-tjroeine + Clyryl-L-glutarnyV-L- tyrosine + EenzylaiyMrbonyl-L-glatnmyV-L.tyrosi/io + Bttuy loxy coibooy l-L-glut&my l-J -L-phcny Ulinino t L-GIuUmyl-L-tyrosine - Benry loiycarbony 1-L-glutsmj 1-L- tyrosine amido Bciuyloxy carbonyl L-gluUmyl-.-L-tyrosylglycine + Bcnzylojycarbonyl'D.glutamyl-L-tyroeine JlcMyloxycarbonyi-L-glutatnyl-D-plienylalaniiie - Chijmotrypain resembles pepsin in attacking peptido links in which aromatic amino-acida aro involved but, whereas pepsin attacks on the amino side of the aromatic acid, chymo trypsin acts on the carboxyl side. Thus both enzymes attack benzyl- 78 SPECIFICITY OF ENDOPEPTID A8ES oxycarbonyl-L-glutamyl-:-L-tyrosyl-:-glycine amide, but do so at different points, as follows : COOH OH pepsin chymolrypsin Chymotrypsin also attacks benzyloxycarbonyl-L-tyrosyl-j-gly- cine amide and benzyl oxy carbonyl -n-phenylalanyl-: -glycine amide, for example, but its action is prevented by the presence of a flee carboxyl radical in the immediate vicinity of the peptide link. Thus benzyloxycarbonyl-L-glutamyl-L-tyrosyl-glycine, un- like its amide, is resistant to chymotrypsin, though acted upon by pepsin. On the other hand, L-gl u tamy 1-E- tyrosy 1- : -glycine amide is attacked by chymotrypsin and not by pepsin, since the effect of the free y-carboxyl radical of the glutamyl unit, which is required for the activity of pepsin, is neutralized by the free a-amino-group of the same amino-acid unit. These results are summarized in Table 7. Table 7. Action of pepsin and chymotrypsin UPON SYNTHETIC PEPTIDES Action of Chymo- Subatrato Pepsin trypsin Uenzyloiycarbonyl.L-gIutamyl-;-L-tyrosyI-;.glycinB amide + + Beniyio*ycarbonyl-L-tyrosyl- : -glycine amide - + Benzyloxycarbonyl-L-phenytalanyl-I-glycine amide - + Benzyloxycarbonyl-L-glutamyl-;-L-tyroayl glycine + — L-Glutamyl-L-tyr09yl-|-glycina amide - +■ Trypsin can act at peptide linkages adjacent to either an arginine or a lysine unit and replacement of these basic amino- acid residues by others yields resistant products. The second amino-group of the dibasio amino-acid unit must be unsubsti- tuted, for trypsin acts upon a-benzoyl-E-argininefamide and a-benzoylglycyl-L-lysinejamide, for example, but not upon a-benzoylglycyl-(e-benzyloxycarbonyl-)-L-lysine amide.in which both the amino-groups of the lysine unit are covered. 79 SPECIFICITY OF ENBOPEFTIDASES To sum up, wo may say that pepsin can act at peptide links formed behcten (he a-carboxyl group of a dicarboxylic amino- acii and the a-amino radical of an aromatic amino-acid, but requires that the second acidic group of the dicarboxylic add shall be free, and is inhibited if there is an amino-group nearby, Chyinotrypsin can ad upon peplide bonds formed from the CO OH IntuliiiMjfc/ CHTWOTKYreiX — HN.CH.CO-NH— -COOH — HH.CH.CO— NN— / Ftg. 11. Specific) ty reqoiTtmcnta of endoptpliilwp*. The eagential rwpjimnent* are printed !n heavy type: the point of attack U Indicated hy an itw« Jn each eaae. a< arboxyl group of an aromatic amino-acid, but is inhibited if there is a cnrboxj’l radical in the immediate vicinity, while irypstn can act upon peptide links fanned from the carboxyl group either of arginine or of lysine, but requires that the second amino-group of the dibasic amino-acid unit shall be- free. This enzyme appears to Ire inhibited by nearby a*amino or carboxyl radicals. It must not be supposed, however, that these enzymes cannot 80 SPECIFIOITr OP EXOPEPTIDA8E8 attack peptide linkages otter than those determined by Berg- mann, for Harington has already shown that while an aromatic amino-acid is required, the free carboxyl grouping of glutamic acid can be replaced by the sulphydryl of cysteine. Both tyrosyl-oysteine and cysteinyl-tyrosine, for example, were attacked by pepsin, though more slowly than the corresponding iV-bcnzyloxycarbonyl -derivatives. Even so, the fact remains that pepsin cannot act upon any arbitrary peptide linkage, but is restricted, probably, to linkages of only a few special types. Clearly, therefore, the endopeptidases are very exacting indeed and, contrary to earlier opinion, able to act only upon bonds of certain types. The specificity requirements established by Bergmann are summarized in Fig. 11. By their concerted action the endopeptidases divide intact protein molecules into smaller fragments, and tho stage is set for tho action of members of the group of exopeptidases, viz. carboxypeptidases and aminopeptidases. These enzymes cata- lyse the splitting only of terminal peptide bonds, with consequent liberation of tho terminal amino-acid units. Carboxypeplidase, 3 remove the terminal unit of which the carboxyl radical is free, aminopeptidases acting at the other end of the chain, where tho terminal unit has a free amino-group. Thus in n-leucyl- glycyl-L-tyrosine, for example, we have the following structural arrangement: CH I CH, I H.N.CH.CO V-Uucyl 0 — HN.CHj.CO HN.I -glyryl' jA CH, l CH.COOH The appropriate carboxypeptidase acts upon this tripeptide to liberate tyrosine, and aminopeptidaso to produce free leucine. Carboxypeptidases require a free carboxyl radical for their action to take effect, aminopeptidases requiring a free amino- group; but carboxypeptidases are unable to act if there is a free 6 81 SPECIFICITY OF EXOPEPTIDASES amino-group nearby, while aminopeptidases are similarly affected by a free carboxyl radical. Neither typo of enzyme, therefore, attacks tho dipeptide left after the other has attacked the original tripeptide, nor rail either group attack peptides con- taining amino-acid residues of the D-series. - In. these peptidases, then, we can recognize specificity require- ments which include the presence of the right terminal radical, which must ba unsnbstituted, and the absenco from the im- mediate vicinity of electrically opposite radicals, together with the usual stereochemical requirements. It is now certain, how- ever, that the requirements of these peptidases are more exacting even than this, and that there exist more than one amino- and more than one carboxypeptidose, each with special end-group requirements. Whereas the endopeptidascs split large protein units into smaller fragments and produce few freo amino-acid molecules in the process, the amino- and carbosypeptidases liberate free terminal amino-acid units one by one until only dipeptidea remain. These, as wc have seen, are not further attacked by these enzymes, but are split in their turn by dipepiidaaea. Probably there aro several of these enzymes, each specific for certain individual dipeptides or groups of dipeptides. For example, glycylglycine is hydrolysed by a specific dipeptidase which, in its turn, is rather specifically activated by Co; 3In can replace Co but is very much less efficient. Again, a specific prolidase baa been described: this attacks tho bond Unking the imino-group of proline or hydeoxyproline to an adjacent carb- oxyl radical. It is activated by Mn. Like most of the other digestive exopeptidases, dipeptidases are activated by metals and require that the constituent amino-acids of their substrates shall bo members of the L-serics, so that, of the four possible alanyl -leucines, the n-L, l-d. d-d, and d-l, only one is attacked by a dipeptidaso, namely u-alanyl-u-Icucine. In passing, the reader may bo reminded that peptidases capable of attacking peptides containing amino-acids of the D-scrics am believed to exist, but the foregoing description of the digestive peptidases holds, even though it cannot be extended to cover all kinds of peptidases. . Finally, it is interesting that some peptidases have a weak IN TB A CELLULAR PEPTIDASES esterase activity; this doe3 not appear to be due to contamina- tion, for esterase activity has been observed in highly purified, crystalline trypsin, chymotrypsin and carboxypeptidase. liennin. In the gastric juice of young mammals we find another proteolytic enzyme, rennin. Like pepsin, this enzyme i3 secreted in the form of an inactive precursor, pro-rermin, which is acti- vated by hydrochloric acid. The optimum pH for activation is considerably higher than that for pepsin. The most charac- teristic featuro of rennin is its milk-clotting power, and it is, in fact, the active principle of commercial preparations of * rennet It catalyses the conversion of the milk casein (‘caseinogen’) into another product, paracasein {‘casein the calcium salt of which is insoluble so that, in the presence of the calcium of the milk, a firm clot or curd is formed. Rennin from the abomasum (fourth stomach) of the calf has been obtained in crystalline form, but little is yet known about its specificity requirements. Like pepsin, rennin has proteolytic properties, but with a more alkaline pH optimum: its optimal pH when acting upon haemo- globin, for example, is 3*7 a a against about 2-0 for pepsin. Intracellular peptidases The presence of intracellular enzymes capable of catalysing the hydrolysis of peptides of greater or less chemical complexity and molecular weight has been demonstrated in many animals and plants. In many cases these enzymes probably have diges- tive functions, especially among the lower animals, many of which produce no digestive secretions but take particulate food by phagocytosis and digest it intraccllularly. There is a con- siderable literature on this subject, and it may bo said that enzymes resembling pepsin and trypsin, at least in their general properties, are present in some cells, but not much is known about them. Other intracellular proteolytic enzymes include powerful plant enzymes such as papain, ficin and bromelin, obtained from the sap or latex of the paw-paw, fig and pineapple respectively. Probably all of these are complex mixtures. Much more is known about the intracellular, autolytic enzymes of animal tissues, known collectively as Jcathepsin. Kidney .and spleen tissues offer good sources of kathepsin, but it is present in many INTRA0ELLT7LAE PEPTIDASES other organs and also in tumours of various kinds. Kidney and spleen kathepains comprise at least four components, the specificity of each of which has now been studied. Of these, kathepsin I is homospecifio with, i.e. ha8 the same specificity requirements as, pepsin; kathepsin II J3 homospccific with trypsin, while components III and IV are homospecifio with aminopeptidosc and carboxvpeptidase respectively. The exis- tence of these liomospecificifcies suggests that extracellular digestive peptidases may have had their evolutionary origin in intracellular, kathepsin-iike enzymes. Although quantitative as well as qualitative homospecificity has been demonstrated between the digestive peptidases and the components of kathepsin, the enzymes aro not identical. The kathepains are not activated by unmasking, as in the case of the digestive endopeptidases, nor yet by heavy metals such as Co or 3In s as in the case of dipeptidases. On the contrary, many intracellular peptidases aro inactivated by heavy metals, but can bo activated by the addition of cyanide, hydrogen sulphide, cysteine, glutathione and sometimes by ascorbic acid. It is now widely believed that the active form of papain, for example, is a complex formed between the enzyme-protein and an — SH compound, probably papain-cysteine: the inhibitoiy action of heavy metals is probably duo to their tendency to react with and block the free — SH groups. It has already been suggested that these intracellular pepti- dases most probably function to maintain a dynamic equilibrium between the cell proteins and simpler products present in the cell contents. Wo have seen that the extracellular, digestive peptidases constitute a set of tools whereby the proteins of the food can bo completely dismantled, and, if it could be shown that tbo action of enzymes of tlii3 kind is reversible, we should feel more confident that the kathepains, with which they are homo- specific, constitute an outfit capable of reconstituting os well as degrading proteins. Bcrgmann succeeded in demonstrating synthetic activity on the part of several peptidases and has shown, for example, that chymotrypsin catalyses the condensa- tion of benzoyl-n-tyrosino with glycylanflide to yield benzoyl -i,- tyrosyl-glycylanilide. The product in this case vs insoluble and is precipitated, so that the hydrolytic action of the enzyme does 84 AMYLASES not seriously oppose its synthetic performance. Other syntheses have been accomplished with, for example, papain-cysteine, i.e. papain activated by the addition of cysteine, and an example can be seen in Fig. 3 (p. 19), Other peptidases include camosinase, a dipeptidase that can be obtained from pig kidney. It acts upon the thoroughly atypical dipeptide camoaino (/?-alanyl histidine) and on a few other peptides containing histidine. A similar anserinase, present in. fish muscle, similarly hydrolyses anserine, a methylated camosine. Of rather particular interest is the K-tosin of the gas gangrene organism, Clostridium wdchii. This toxin is a collagcTW.se which, by digesting connective tissue, enables the organism to spread rapidly and extensively into the adjacent tissues. Apart from collagen this toxin is only known to attack gelatin, which is itself derived from collagen. Similar enzymic toxins are produced by other Clostridia also. OABBOHYDRA9ES Enzymes capable of catalysing the breakdown of carbohydrates are very widely distributed indeed, and occur both in digestive secretions and within the cells of animals, plants, and micro- organisms of many and perhaps all kinds. They may be con- sidered under two main headings, the polysaccharases, which act upon the large molecules of polysaccharides such as starch and glycogen, and the ghjcosidases , the substrates of which are small molecules Buch as various di- and trisaccharides, in addition to glycosides of other kinds. Amyla.es PolysaccUrases 3Iost is known about the amylases, which act upon starch and glycogen but not upon cellulose. Plant amylases have been resolved into components known respectively as a- and ^-amyl- ases, and representatives of both types have been crystallized. In the ordinary course of events t hese enzymes act together upon starch and glycogen to catalyse a more or less quantitative conversion into the disaccharide, maltose. They act, however, in rather a different manner and in order to appreciate the 85 AMYLASES differences it is necessary to have in mind & fairly clear picture of the probable structure of starch and glycogen. In considering the modes of action of the a- and ^-amylases, however, it is important to realize that the use of the prefixes a- and /?- is not meant to imply that these enzymes act upon a- and /?-gIucosidie links respectively. Since some confusion is likely to arise on this paint alternative names, endo • and exo-amylases have been proposed for the a- and /?-amylases respectively, and will be adopted here. Recent work on the purification of these two amylases has shown that they are accompanied by other enzymes, which also play a part in tho total digestion of starch, and their names are given in Table 8, together with the identities of tho linkages upon which they are believed to act. Table &. Linkages attacked »y amylolytio enzymes Eniyaso Cado-imylMa Cxo-raylaw Z-enxyjne Atnylo-1.6 glacwndMcl R-etuj-me J Starch consists of a mixture of two main components, amylose, which accounts for 20-25% of most vegetable starches, and amyloptclin. Amyloso, which gives a pure blue coloration with iodine, consists mainly of 1 :4-a -linked glucose units and can bo attacked either by tho endo- or exo-amyjascs. Pure crystalline exo-amylase, which acta at the ends of tho l:4-a-linkcd glucose chains, catalyses a 70% conversion of natural amyloso into maltose, but complete conversion requires the assistance of another enzyme, the so-called Z-enzyme, winch is usually found in association with the amylases. This Z-enzyme has no action upon l:4-90%) conversion of potato amylose into maltose and maltotriose in a ratio of 2*3:1, but stops acting when this has been achieved. Pancreatic amylase, however, is said to hydrolyse maltotriose to maltose and glucose and presumably contains other enzymic components. Thus although maltose is the main product of digestion by these two enzymes, some free glucose is also produced. It has long been known that salivary and pancreatic amylases lose their activity if dialysed, and the case is often quoted in illustration of the importance of inorganic ions in the activation of certain enzymes. This, however, is not entirely a true bill, for while it is Bure enough that dialyBed salivary amylase, for ex- ample, is no longer active under conditions which were formerly optimal, it is still weakly active at more acid pH values. There is, it is also true, a considerable loss of total activity, but the removal of chloride ions does not deprive the enzyme of its power to activate the substrate, but appears to alter its physical state in some way. In all probability the enzyme dissociates in a different manner in the absence of chloride ions (see p. 47) and, if this is so, it might perhaps be expected that the replacement oellula.se of chloride by otheT ions would lead to a displacement of the optimum pH. This does in fact happen, as is shown by the curves of Fig. 9 (p. 40). Although the enzymes that catalyse the digestive hydrolysis of starch and glycogen must, on theoretical grounds, be regarded as capable of working reversibly, it has not so far proved possible to synthesize polysaccharides with their aid because the synthesis is essentially an endergonic process. Cellulose Although cellulose forms a very large part of the food of herbivores, remarkably few animals of any kind possess any enzyme or enzymes capable of catalysing its hydrolysis. Cellu- lose-splitting enzymes have been described in the digestive secro- tion3 of a number of herbivorous gastropod snails, including terrestrial forms like Helix and aquatic species such as Strotnbus, Pierocera and Aplysia ; celluloses appear to be present also in the digestive juices of silverfish and a few wood-eating insects. The shipworm, Teredo, is an interesting creaturo from this point of view, for its digestive gland contains cells which appear to be specialized for the phagocytic ingestion and intracellular digestion of the fine particles of wood which the animal scrapes off as it bores. But in the vast majority of cases it is nevertheless true that animals do not produce cellulaso, even when they depend largely upon cellulose as a primary source of food. Tills paradox is duo to the fact that most cellulose-eating animals do not digest cellulose for themselves, but maintain in their alimentary tracts large populations of symbiotic micro- organisms, including bacteria, protozoa and yeasts, which play a very important part in their nutrition. Indeed, it has been claimed that the snail’s cellulose is not a product of the snail itself but i3 formed by symbiotic inhabitants of the crop and the intestine. Many intestinal micro-organisms are capable of degrading cellulose, and recent work on the processes of digestion in ruminants has shown that cellulose is broken down by the symbionts, with production of large amounts of lower fatty acids. Acetic and propionic acids predominate, and are accompanied by formic, butyric and valeric acid3, together with largo volumes of carbon dioxido, methane and hydrogen. The ruminant thus 00 0ELLT7I.ASE obtains fatty acids rather than sugars from the cellulose it con- sumes, and has to repay the micro-organisms ‘which carry out the conversion by providing them with Lebensraum in the form of a capacious caecum, a multiple stomach, or some other com- modious dilatation of the alimentary canal. Similar processes take place in many wood-eating insects and other herbivores. Little is known about the enzymes whereby these symbiotic organisms break down tho cellulose. Presumably they must include a cellulase, and it has indeed been shown that the free- living protozoan, Vampyrella , secretes an extracellular cellulase which attacks tho cellulose of the cell walls of tho Spirogyra which furnishes its food. Cellulases have been found in culture media in which such cellulolytic bacteria as Cdlulomonas and Clostridium thermocellum have been grown, and the secretion of extracellular cellulases must probably play an important part in the early stages of microbial attack upon cellulose, which is so insoluble as to require some extracellular comminution before it can be got into the cells for further chemical manipulation. Cellulose-splitting enzymes and enzymes capable of synthesizing cellulose certainly occur in plants and fungi, and must be of very great importance in plant economy, but we have very little information about them. It is said that preparations containing cellulase will also act upon tho animal polysaccharide, chitin, in which tho /?-linked glucose radicals of cellulose are believed to be replaced by simi- larly linked units of -acetyl glucosamine. Whether or not cellulase and chitinase are identical has not been determined. Other polysacckarases include enzymes capable of splitting polyfructofuranosides, such as inulin and levan, and enzymes that act upon polysaccharides such as tho mannans, pectins and so on. Olycosidases We know of many enzymes capable of splitting simple glyco- sides, all of which show a high order of specificity towards the glycosidie part of tho substrate molecule. Of these tho most common are the glucosidases and the saccharascs. Some of these enzymes can catalyse transfer reactions as well as hydrolyses. 01 OIAJCOSIDASES a-Glucosidases a-GIucosidoses of two types are known. The most specific of these are the ‘true’ maltose*, which act only upon a single a-glucoside, viz. maltose (glucose-4-:'a-glucoside), Enzymes of this kind occur in malt and in Asperffillus. The more widely distributed digestive ‘maltoses ’ of animals, and the ‘maltose' of yeast, are able to act upon a-glucosides other than maltose and should therefore be called a-qhicosidases rather than malt- ases. These enzymes will act upon such substances as methyl-j-a- glucostde and sucrose {/S-fructo furanosido- : -a-glucoside ) , though they arc without action upon the a-glucosidic linkages of the large molecules of starch and glycogen. The order of specificity of these a-glucosidascs is very high, for a completely unmodified a-glucosido-radical ia required m their substrates: /J-glucoaides, a-galactosides, ee-xylosidcs and a-wo-rhamnosides aro not attacked, in spite of their close structural resemblances to tho a-glucosidcs: a-w>-Thamnonrlt a ryUuUt fi-Glucosidases Many cells and tissues havo been shown to contain /?-gluco- sidases. Tlie classical source of such an enzyme ia tho ‘emulsin* of bitter almonds. The latter contain amygdalin, a ^-glucosido which, when attacked by the /7-glycosidaso component of emulsin. 02 OLtTOOSlDASES yields mandelonitrile, which is then attacked by another enzyme, mandolonitrilase, to give free hydrocyanic acid. Sweet almonds also contain these enzymes, but amygdalin is absent. /?-Glucosides are as common in nature, especially in plant materials, as their a-counterparts are rare, and among the more interesting of these we may mention salicin (salicyl-:-/?-glucoside) and a /7-glucoside of indoxyl which occurs in the indigo plant and gives rise, after hydrolysis and oxidation, to natural indigo. There are many others. In addition, a number of the simpler saccharides are /?-glucosides, notably cellobiose (glucose-4-:-/?* glucoside), which stands in the same relation to cellulose a3 maltose does to starch and glycogen, and gentiobiose (glucose- G-h/l-glucoside), which occurs naturally in combination with mandelonitrile in the form of amygdalin. /?-Glucosidases obtained from different plant and animal sources have much in common. In particular, it may be pointed out that their specificity, though high, is less marked than that of the a-glucosidases. Although * cellobiases ’ have been described from time to time there is no reason to think that they are comparable with the ‘true’ maltases of malt and Aspergillus, but rather that they are all group-specifio /?-glucosidases. These enzymes are rather less specifi c than the corresponding a-glucosid- ases, which require a completely unmodified a-glucosidic radical in their substrates. The specificity requirements of the /?-g!ucosid- ascs do not extend as far as carbon 4, for some /?-galactosides are also split. Modifications can also be made at position 0, for p-iso -rhamn osi d es and /?-xylosides are also split, though less rapidly than the normal substrates. Saccharoses are very widely distributed indeed in the digestive secretions of animals, in plants and in many micro-organisms, though not, apparently, in the cell contents of animals. Two types can bo distinguished, the glucosaccharases (in digestive secretions of animals and in Aspergillus) and thefructosaccharases (in yeast). Sucrose itself is a-glucopyranosido-:-/S-fructofuranoside, and the molecule may be attacked from cither end. The gluco- saccharases appear to bo to orthophosphates, A very important example of the polyphosphatase type is the adenosine triphosphatase of muscle. This enzyme is identical with myosin, winch, in combination with actin, is the contractilo protein which makes up tho bulk of the muscle substance. Muscle and other tissues contain other adenosine triphosphatases that are separable from myosin, and similar enzymes have been found in snake venoms, potatoes and elsewhere. Bakers’ yeast contains an interesting polyphosphatase which appears to bo wholly specific for the hydrolysis of inorganic pyrophosphates, which arise biologically in a considerable number of reactions, and is devoid of action upon adenosine triphosphate and other organic pyrophosphates. A similar enzyme has been found in potatoes. Generally speaking, phosphatases of the monoesteraso typo seem not to bo specific with respect to the nature of the alcoholic radical, and act alike on many organic phosphates such, for instance, as glycerol -j -phosphate, glucosc-O-j-monophosphato and other phosphate esters. The reactions they catalyse may bo generally expressed as follows: n-o® + u.oh - it-on + uoj>. One such enzyme plays an important part in tho ossification of cartilaginous structures such as the bones and teeth. Unossi- fled cartilage, and cartilaginous structures which do not undergo ossification, contain no phosphatase, but tho enzyme makes its appearance j ust at tho time that ossification sets in. Acting upon organic phosphates present in the blood, the enzyme is thought to catalyse a localized liberation of phosphate ions, which unite with calcium ions, also provided by the blood, to form the insoluble calcium phosphate, Ca,(P0 4 ) f . This is deposited In an ordered, crystalline manner La the cartilaginous matrix. Tho 93 BIB ON U CLE ASES more rapidly ossification proceeds the higher is the phosphatase activity of the tissue. An interesting point in this connexion is that the calcified structures of the cartilaginous fishes (Elasmo- branchii) contain an active phosphatase, just as do those of the bony fishes (Teleostci). The differences between the mechanical properties of calcified cartilage and true boue appear to be due to differences in the manner and perhaps in the amounts in which the calcium phosphate is deposited in the two cases. In addition to being present in cells of nearly every kind, phos- phatase is present in milk . If milk is pasteurized in the correct manner the treatment accorded is just sufficient to inactivate the milk phosphatase, so that the absence of phosphatase activity may be taken as an indication that the process has been properly carried out. The method used for routine testing depends upon the hydrolysis of p-nitrophenyl-j-phosphato with formation of free p-nitrophenol, which gives a colour reaction with alkalis and so can bo estimated colorimetrically. Tile group of phosphatases also includes some of the enzymes concerned in the breakdown and possibly with the synthesis of the nucleic acids. Ribonuclease , which incidentally is a heat-stable enzyme, and deoxyribonuclease, are present in and can be crystallized from pancreas tissue. Fairly crude preparations will catalyse the hydrolysis of nucleic acids to yield the constituent nucleotides, but the pure enzymes carry this degradation only part of the way. Both enzymes appear to be diesterases and a similar diesterase is present in some snake venoms. How the resistant residual products — apparently di- tri- and tetranucleotides — are split is Btill uncertain, but eventually the simple nucleotides are set free. The so-called nucleotidases, some of which are highly and others less specific, appear to be phosphatases of the mono- esterase typo and these split off phosphate to liberate the corresponding nucleosides. Among the phosphatases are many enzymes that require magnesium ions as activators. Very minute concentrations of Mg ++ are all that are required in many cases, and the mode of aotion of these ions is still uncertain; probably they provide a means through which the enzymes can combine with, their substrates. aroinase: urease In conclusion it may bo mentioned that some phosphatases of plant and animal origin can catalyse transfer reactions (pp, 110-11). OTHER HYDROLYTIC ENZYMES Arginase, an enzyme of which larger or smaller concentrations are present in most animal cells, catalyses the hydrolytic deamidination of arginine to yield urea and ornithine (sec p, 9), and plays a central part in tho mechanism whereby urea is synthesized in the mammals and other urcotelic vertebrates. It is thought to bo a manganese-containing protein, and is ono of the most specific hydrolases known. Aiginaso is very powerfully inhibited by ornithine, though not by urea, a circumstance winch suggests that it must combine with the ornithine radical. It is also inliibited by the closely related amino-acid, lysine. Its optimum pH lies far in the alkaline range, probably at about 10, at which pH it is very unstable. Urease occurs in large concentrations in certain seeds, notably in jack- and soya-beans, from which it is usually prepared. It has been found in numerous other plant tissues and in tho tissues of a few invertebrates, though it appears to bo totally lacking from vertebrate organisms. Urease had tho distinction of being tho first enzyme to be obtained in tho crystalline state. It was extracted from jack-bean meal by Sumner, purified and crystal- lized in 192G, and shown to be a protein. It catalyses tho following reaction; COtSlI,), + H,0 -* CO, + 2N1I,. Urease possesses a number of very unusual properties. Unlike most enzymes, it is inliibited by high concentrations of its sub- stmto, an effect which can bo abolished by tho addition of glycine, ns is shown in Fig. 13. This phenomenon is usually explained on tho supposition that, in addition to combining with urea to form a reactive complex, ES t which is then hydrolysed, it tends to combine with, a second, molecule of urea when the concentration of the latter is high, to form a stable complex, ES t . Another, and possibly a unique feature is that tho optimum pH of ureaso is proportional to tho logarithm of tho substrate concentration. The specificity of ureaso is very high. Its action has boon 100 DEAMINASES AND DEAMIDASES tested upon a large number of substituted ureas, none of which appears to bo attacked, with possible though dubious exceptions in. the cases of the stjm. dimethyl- and diethyl-ureas. Hydrolytic deaminases and deamidases other than urease are also known {see p. 358 for formulae of substrates). Adenose, which catalyses the hydrolytic deamination of adenine to yield liypoxanthine, and guanase, which similarly converts guanine Fig. 13. Influence of urea concentration on activity of urease ( 7 ). The inhibitory in. fluenoj of high concentrations of urea is counteracted by 0-2% glycine (O)- Ordinato: initial velocity of hydrolysis. Abscissa! concentration of urea. (After Haldane, from Kato, 1923.) into xanthine, are present in the liver tissue of most mammals, and must probably occur elsewhere. Another important enzyme concerned with the metabolism of purine derivatives is the adenylic deaminase of muscle. This enzyme, which is not identical with adenase, catalyses the hydrolytic deamination of adenylic acid to yield inosinic acid, in which the adenyl radical is replaced by that of hypoxanthine. Animal tissues contain a powerful glutaminase and plants an homologous asparaginase. These enzymes catalyse the irre- versible hydrolysis of the amides of the dicarboxylic amino- acids, e.g. Coxn, cooh in, CH, I t CH, + 11,0 — ► CU, + NH, in.NH, in.NH, ioOH -l-© glorose-l-.6-dl © + gluroie. Reactions of this kind can also bo catalysed by several other (e.g. apple) but not by all fruit phosphatases, and by phos- phatases from some {e.g. prostate) but not all animal sources. Wo have so far only considered the phosphatases as purely hydrolytic enzymes capable of catalysing reactions of the general typo: rt.o© + n.on n.oH + no.©. It now appears, however, that this is only one example of ad even more general case in which H.OH con be replaced by other hydroxylio compounds, such, for instance, os sugars and alcohols: R.o© + R'.on «— R.on + R'.O©. The overall effect of such a reaction is that the phosphate radical is transferred, not to water necessarily, hut to some alternative phosphate acceptor. If the phosphate donator is labelled with radioactive phos- phorus and incubated with the enzyme and a suitable acceptor in the presence of inorganic phosphate, no radioactive phos- phorus appears in the inorganic phosphate. This shows that the phosphate radical undergoing transference docs not have even a transient free existence. It seems probable, therefore, 11U TEAJfBFEE BY HYDROLASES that the donator first hands over its phosphate radical to the enzyme E: R.O© + E 5=^. R.OH 4- E®. The product might then react in one of two ways, either (a) with water, to undergo hydrolysis, or (6) with some alternative acceptor (e.g. glycerol) to give a new ester: {a) E® + H.OH *=* E + H.O®. or (6) E® + R'.OH .=* E + U'.O©. Now it is, as we have seen in our discussion of energetics, a universal observation that free energy always tends to diminish when a chemical reaction takes place. Since the decrease in free energy is greater in hydrolysis than in transfer, it might have been anticipated that hydrolysis rather than transfer would always result. Apparently, however, the available free energy is not lost all at once and in a single reaction ; rather does it seem that free energy tends when possible to diminish in steps or stages. Other examples of this tendency are known and exam- plify the so-called ‘law of successive reactions’. How important these phosphatase-catalysed transfer reac- tions are in metabolism we do not know at the present time. It is important to realize that, biologically speaking, very high concentrations of the alternative phosphate acceptor, e.g. glycerol, are required in order to demonstrate the transfer reaction and, moreover, that transfer is succeeded by hydro- lysis in the courso of time, so that the newly formed products, e.g. glycerol phosphate, are only transient, at any rate in vitro . None the less, phosphate esters of glycerol, glucose and other alcoholic substances are of very general occurrence and great metabolic importance. In these phosphatase-catalysed transfers we have mechanisms for the phosphorylation of sugars, glycerol and so on whereby new low-energy phosphates can be formed from low-energy precursors without consuming the high-energy source used in, for example, the hexokinase reaction, viz. ATP. Finally, if, in fact, in vivo conditions are such that phosphatase- catalysed transfers can go on, it is always possible that the newly formed products might be drained off as fast as they are formed and metabolized before they can undergo hydrolysis. Ill TRANSFER BY HYDROLASES Transglycosylation by glycosidasas There exist numerous enzymes capable of transferring glyco- Bidic radicals from one molecule to another and glucosidic and fructosidic radicals arc among those that may bo thus trans- ferred. While these transfers are catalysed for the most part by specialized enzymes known as pbosphorylascs, -which will bo discussed presently (pp. 118 - 24 ), many such transfers can be catalysed by enzymes, e.g. the saccharoses, hero classified as hydrolases. A great number and variety of these processes are known, some leading to tho formation of di- and higher oligo- saccharides and some even to that of polysaccharides. An important difference between the reactions described in this section and those catalysed by the pho3pliorylases is that ■phosphates play no pari in transfer reaction* catalysed by the hydrolytic glycosidases. Tho saccharose s (invcrlases) act freely upon sucroso, tho double glycoside ofglucopyranoso and fructofuronoso, and yield these component sugars as hydrolysis products. At the same time, however, smallor or larger amounts of oligosaccharides are produced. These aro transient products which, liko tho original sucrose, undergo eventual hydrolysis under in vitro conditions. One Buch reaction catalysed by yeast saccharose may bo written as follows and involves transfructosylation : •ucnjao + mctoM ys fruclotjbucro*© + glutow. Glucose radicals are not transferred by this enzyme. Saccharoses obtained from moulds (e.g. Aspergillus) carry out similar reactions, in which sucrose itself can be replaced both as tho donator and as tho acceptor of fructosyl units; other sugars and sugar alcohols, and a variety of aliphatic and aro- matic primary alcohols can also act as fructosyl acceptors. Similar transfructosj’lationa aro catalysed by saccharoses from the higher plants, including those of sugar beet, broad bean, clover, cabbage, etc., but not by extracts of plants which contain no saccharose. Transfructosylation seems to bo tho invariable rule in all these cases, but some animal saccharoses, e g. that of tho honey bee, seem able to achieve tmnsglucosyJ* allon as well. 112 TB.ANSFEB BY HYDBOLASBS Maltoses from various sources can catalyse transglucosylalion on a large scale. Extracts from the intestines of dogs and other animals will build up a series of oligosaccharides at the expense of maltose; maltose + maltose , maltotriaoso + glucose, maltose + maltotriaoso 5=5 m&Uotetraose + glucose, and so on. Here again wo have an enzyme which, in addition to its indubitable hydrolytic powers, can also act in a transglycosyl- atrag capacity and, indeed, the same is probably true of many more members of the group of glucosidases. Phosphate plays no part in any of these processes. It seems likely that in all these cases, as among the phos* phatasea, the donating sugar first ‘parks’ a fructosyl or a glucosyl radical on the enzyme, the product then either under* going hydrolysis by reacting with water, or transferring the glycosyl radical to some alternative acceptor. Eventually, howover, the oligosaccharides, which are only transient pro- ducts, undergo hydrolysis, at any rate under in vitro condi- tions. Synthesis of polysaccharides by transglycosylation. Somowhat similar transglycosylations leading to the synthesis of poly- saccharides from sucrose have been observed in a variety of micro-organisms. In Leuconostoc dextranicum, for example, there is an enzyme known as dextran sucrose which acts upon sucrose to yield fructose, which is metabolized by the organism, together with a dextran built up from l:G-a-linked glucopyranos- ido units. Small amounts of sucrose are simultaneously hydro- lysed. In certain spore-forming aerobes sucrose is again split into glucose and fructose, but in tliis case the glucose is metabolized and the fructose units are built up into levans formed by a so-called levan sucrose from 2:G-linked fructofuranos- ide units. In both these cases the sugar unite not katabolized for energy production by the organism are transferred one after another to build up these specialized polysaccharides, and in neither case is phosphate in any way involved. Suorose is also the starting material for the synthesis of a glycogen-like polysaccharide produced by an enzyme, amylo- sucrase, of another micro-organism, Neisseria perjlava: here the 113 feOA TRANSFER BY HYDROLASES nowly formed glycosidio linkages arc mainly of tbo l:4-a-type and phosphate ngain plays no part in the transglycosylation process. Nor is sucrose the only disaccharide that can serve as starting material for polysaccharide synthesis by transglycosyl- ation, especially among micro-organisms. For example, certain strains of Escherichia coli yield cell-frco extracts containing an enzyme, amylomaltase, which converts maltose into glucose together with a starch' or ghjcogen-Mka polysaccbarido, without intervention by phosphate. In all these cases the enzymes concerned show some hydro- lytic activity as well as their transferring power; amylosucraso, for example, produces mainly fructose and its characteristic glycogen-Uko glucose polymer, but small amounts of glucoso are set free at the same time. It may bo that whereas the saccharoses Bhow a Btrong preference for water os their glycosyl acceptor, amylosucraso and similar enzymes have an equally marked preference for polysaccharides os acceptors for the glycosyl units which they transfer. Cyclical dextrans, amylose-, amylopcctin- and glycogen-Iiko polysaccharides aro produced by many micro-organisms of various kinds, sometimes with, but often without, the participa- tion of phosphorylatcd intermediates, but spaco will not allow us to discuss tbeso interesting syntheses in detail. Progress in this field is very rapid at the present time. TRAN SrilOSFHORYLAT JON A number of enzymes aro known which can catalyse tbo trans- ference of phosphate radicals from one to the other of a pair of molecules, adenosine di- and triphosphates being employed as the carrier system. Not all of theso enzymes havo eo far been named, however, and they might be collectively called 4 trans- phosphatases ’ or, following tho suggestion of Dixon, 'phos- phokinascs ', after hexokinase, tho longest-known representative of the group. Whichever name is used (and we shall use ‘phosphokinascs 1 here) it ought probably to bo restricted to enzymes catalysing reactions that involve the ATP/AD I‘ or some comparable BVBtora. It will bo convenient rvow to refer once more to tho Lohmann 114 fHOSPHOKINASES reaction, i.e. the transference of a phosphate radical from adenosine triphosphate to creatine, or to adenosine diphosphate from creatine phosphate. If we represent high-energy phosphate by the usual sjunbol — (g>, the structure of adenosine triphos- phate may be expressed as follows: 0 0 0 !i o n A — P — o~r — 0— P— OH OH i)H Further, it is known that creatine phosphate also contains high- energy phosphate, and its structure may accordingly be written thti3: 0 kb HO — I* ~ <1— N.CH.COOB (1h H CH, Accordingly, the Lohmann reaction can be conveniently repre- sented in the following terms: We do not know how this reaction takes place, whether by ‘parking’ the terminal phosphate radical of ATP on the enzyme to be ‘picked up’ later by creatine, or whether both substances react together on the surface of the enzyme to give a tautomerio complex; the most important feature of tills reaction is that it accomplishes the transference of a high-energy phosphate radical from one molecule to another. Phosphokinases occur widely and probably universally among living cells and tissues. Yeast, plants and some mammalian 116 8-3 pnoSPHOKINASES tissues contain hexoJcinasea catalysing tlie phosphorylation of glucose, fructose, mannose and glucosamine at the expense of ATP. Liver and muscle contain specific kinases for glucose, fructose and galactose, while a specific galactokinase is also found in yeast that has been ‘trained’ to ferment galactose. A kinase specifio for the phosphorylation of gluconic acid has been found in some bacteria and may bo present in mammalian tissues as well. Other phospliohcxokinas&s catalyso ft similar phosphorylation of fructofuranose-C- and fructofuranose-1 -phos- phates to tho l:0-dlphosphate and of gIucosc-6-phosphato to glucose-l:0-diphosphate. None of these reactions ia revcraiblo, however, presumably because none of the sugar phosphates contains high-cncrgy phosphate. Tho reconversion of glucose-G- phospliato to free glucose, end that of fructofuranose-I.*6- diphospbatc to the O-monophosphate, follow routes different from those of their synthesis, and are accomplished by hydrolysis catalysed by tissue phosphatases. Table 0. I’hosi’hokinases Reaction Creatine ps creatine phosphate Arginine ergimno phosphate Glucose -*■ glucoao-C-phosphato Fnictose — fructose- 0-phospbate Mannose -* rnannase-O-phosph&te Glucosamine -> glucosamine 6-pbcaphate Gluconia acid -* gluconic ncul-U-pbosphate Fructose -► fructose- 1 -phosphate lUbese rUmse-A-phosphite 2-Deooxyriboeo -» z-desoiyribosc-S-pbosphste Galactose -► galactosc-t -phosphate Fnictose-6 phosphate -* fructose- l:G-dipho*phate Fructose- 1 -phosphate -» fractoee-l'C -diphosphate GIucose-0 phosphate -► glucose- l;G-diphosphate Glyceric ACid-3-pbosphate glyccrio acid-l:3- diphosphate Pyruvic acid ft pliospho-cnoi pyruvate Adenosine monophosphate s* adenosine diphosphate Adenosine -* adenosine monophosphate Riboflavin -* riboflavin ubnimhat/s Dl’Jf -* TPX Eniymo Creatine phoiphokinaso Arginine phwpbokinMO JieioVinase Gluconakloase FructoVdnaoo P.ibokinase Ribolanase GaUctohrxoklnsae O-Phospliofnjctoldoase I •PhoaphofrurtoUnA.se C-rhosph ogl u cokin aso I’hosphoglyceno plioaphoklUM" IVruvie phospho kinase hfyokinose AdenosmeUnase IXavokiaasa (Unnamed) Zfyotinasc. a somewhat special phosphokinoso, catalyses tho transference of a phosphate radical from ono moleculo of adenosine diphosphate to a second, so that the products are adenosine monophospbnto, on the ono hand, and the corre- 110 FHOSPHOKrNASES sponding triphosphate on the other. In this case the reaction can be represented in the following manner: A-<©~<8 + A~®~® s=i A-©~©~® + A-©. In nearly all cases of phosphokinase activity known at the present time, adenosine triphosphate and the corresponding diphosphate are obligatory reactants. The second reactant may be any of a considerable number of substances ; the identities of some of them and the nature of the corresponding reactions are summarized in Table 9. It is quite certain that substances other than those listed in the table can also enter into transphos- phorylation reactions, and there is evidence that the enzymes concerned are as a rule highly specifio for their substrates as well as for the carrier, which plays the part of a coenzyme. In recent years it has been learned that triphosphates of nucleosides other than adenosine sometimes participate in phosphokmase-Iike reactions, e.g. guanosine triphosphate (GTP). Again a is transferred to some appropriate acceptor leaving GDP. This is promptly re-phosphorylated at the expense of ATP by a specific member of a special group of nucleoside, phosphoJHnases : Q-®~® + A -®~®~® «=* G-©~®~® + A-®-®. Several different nucleosides and their di- and triphosphates react similarly. To take a specific example, pkosphofructokinase can use ATP, ITP (inosine triphosphate) and UTP (uridine triphosphate), all three of which are about equally active with this enzyme. We shall have numerous occasions to refer to the other phosphokinases listed in Table 9, for all play important parts in the metabolism of cells and tissues. But many other phospho- kinases exist. We know, for instance, that ATP can act as an energy source in the biological synthesis of DPN, TPN, flavin- 'adenine dinueleotide, glutamine, hippurie acid, urea, glutathione and many more compounds of interest and importance. Each of these syntheses must probably involve a priming reaction in which ~(g) is transferred from ATP to one qf the reactants, the transfer being catalysed by an enzyme which must, by definition, be a phosphokinase of some kind. 117 TE AKSQI.VCOS YLATIOK TBAKSOLVCOSVLATIOK There exist numerous enzymes capable of catalysing the trans- ference of glycosidic radicals from one molecule to another. Some of these act also as hydrolases and havo already been considered (pp. 112-14). Often, though by no means always, tho starting material is a-glucose-l-phosphato and a number of tho enzymes concerned exclusively with transglycosylation are known as phosphorphses, They were originally so named by analogy with the hydrolases, to which they bear a close formal resemblance: iiydnUjiU n;n' + h.oii «=»» r.oh + n.ir, PhotjihorolytU R'R' + IIO.(g> R.O© + H.R'. Sucrose phosphonjlase . Sucrose cannot bo synthesized chemi- cally or biologically by simple reversal of its hydrolysis. It has, however, been shown that certain bacteria (e.g. Pseudomonas saccharopkila ) can catalyse a synthetic production of sucroso from cc-glucose-1 -phosphate and free fructose. Tho synthetio process, which is freely reversible, is one of ' dephosphorolysis i.o. elimination of phosphoric acid: CH.OH ch.oji Clearly, tho synthetic process can bo regarded as a condensa- tion -like reaction in which the elements of water are replaced 118 TBAUSGLYOOSYLATION by those of phosphoric acid. But it can also be regarded as a process of iransglucosy lation in -which a glucosyl radical is transferred from one aglycone, in the form of a phosphate radical, to another, which in this case happens to be glycosidio also. This second aglycone, a fructofuranosyl radical, is available in aqueous solutions of fructose, which contain considerable amounts of fructofuranoso in equilibrium with tho more stable pyranose form. The free energy required for the formation of the new gluco- sidio link is already available in that of the starting material, a-glucose-l-phosphate, and is transferred along with the glucosyl radical from the first aglycone to the second. Free glucose cannot serve as a starting material for this synthesis because its free-energy level is too low: it can, however, be raised to a high enough level through tho hexokinase reaction, in which ATP provides the necessary free energy for tho formation of glucose- 8-phosphate, from which a-glucose-l-phosphate can then be formed by the action of phosphoglucomutase. In tho end, therefore, the free energy for the synthesis of sucrose comes, in all probability, from ATP. While sucrose phosphorylase is absolutely specific with respect to the glucosidio portion of the substrate molecule, it is only group-specifio towards the aglycone. Thus the fructosidic agly- cone con be replaced by phosphate or by any of a number of other substances, including certain sugars, so that new disac- charides can be synthesized by the action of the enzyme. Other aglycone radicals can he furnished by L-sorboso, D-xyloketose and L-arabinose for example. Nor is this all. Any of these aglycone radicals can be replaced by any other without the inter- vention of phosphate ; for instance: a-glucoae-l-fructoaida + aarboae P + HO.® c ii .xii, iit.xn, ioou coon 124 FORMATION OF PEPTIDES been obtained from bacteria and from animal tissues which, if glutamic acid and ammonia are available, catalyse the synthetic formation of glutamine on addition of ATP (the reaction is shown on p. 124). Here again it has been suspected that a reactive y-acyl derivative must be formed as an intermediary, but attempts to isolate it have led uniformly to failure. Conceivably coenzyme a may play a part once again. Another reaction in which peptide bonds are formed is the synthesis of ornithurio acid, a compound formed in bird kidney as a means of detoxicating benzoic acid (p. 290), and here again NH— OC.C,H, i:H, Ah, u CH.NH-OC.C.1I, iooii omilhurie acid ATP is required as an energy source. But in none of these cases is a 'typical’ peptide bond produced, i.e. a bond formed by the union of the a-carboxyl group of one amino-acid and the a-aznino- group of a second. Two such bonds and one atypical bond are formed however in the synthesis of glutathione, which has been much studied recently and in which ATP must once more bo provided. The general need for ATP in peptide-bond formation makes it clear that free energy is required for the formation of bonds of this type, just as it is in the formation of glycosidic bonds. Transfer of peptide groups. The suggestion has now appeared that just as certain hydrolytic phosphatases can catalyse low- energy transfer of glycosyl radicals with formation of long- chain polysaccharides, so too bydroly tio peptidases may perhaps catalyse that oflongchains of peptide-bound amino-acid radicals. Intracellular peptidases are numerous and, moreover, very specific indeed, and might in fact be able to carry out highly specific fcransferringoperafcions leading to the formation of corre- spondingly specific peptides. If we assume that transpeptida* tion is possible — and there is evidence that this is indeed so— 125 TKANSPEFTIDATION there are two ways in which the transfer might be effected. These may be expressed in the following manner: Tmtftr •/ ■ - + \ > ICS.R'.COOfl H^f.R'.CO -"l friu/’s sf ealezjt fn»f 11, N R'.CO-"^ -JlX.R'COOH X + : ll r s P.’.COOH 1VC.R* OCKJH _ I .T^-HX.R'.COOU Hanes & Ishcrwood have used extracts of kidney and pancreas in experiments designed to test the possibility of amino-acid transfer between the tripeptido, glutathione, and its constituent amino-acids, glycino, cysteine and glutamio acid. After incuba- tion with tlio enzymo tlio digests were examined chromato- graphically, and spots were found which indicated the formation of now peptides. New peptides were also produced by interaction between glutathione and other amino-acids, including leucine, valine and phenylalanino. It i3 too early to speculate far about tho significance of this discovery, but it seems certain that on important new field has been opened up and is obviously of the most fundamental importance for every branch of biochemistry. At the present time research activity is intense but it is still too early to give any detailed account in a book of this kind. Wo may concludo with a quotation from Hanes & Isherwood’s preliminary’ publication: ‘The postulated transpeptidation reactions leading to the inter- convcraion of cr-peptidca by either carboxyl or amino transfer might prove to bo closely connected with protein synthesis, since they would provide a mechanism for tho rearrangement of amino-acid residues in a-peptide chain structures. Underlying tins suggestion, we liavo had in mind from tho beginning tho possibility that tho different proteolytic enzymes, with their sharply defined specificity characteristics, may catalyse such transpeptidation reactions, in addition to tho hydrolytio reac- tions by wluch they are normally recognized.' TRANSAMINATION TRANSAMINATION Glutamic transaminase is an enzymo that occurs widely among micro-organisms and in plant and animal tissues. The process of transamination, which is reversible, can bo expressed in the following manner: R R' R R' ill. Nil, + io io + ilt.NH, loon loon loon loon Work with purified specimens of the enzyme shows that one member of the reacting pair must be either L-glutamio acid or a-ketoglutario acid. L-Aspartic acid or oxaloacetic acid can replaco these but react much more slowly, and the enzyme, which is now called L-glutamio transaminase, is thus highly specific towards at least one of tho reactants. The a-kctoglutaric-glutamic acid system itself can act as an amino-group-carrying system. If, for example, we take alanine and add to it a purified sample of glutamic transaminase in tho presence of oxaloacetic acid, no direct transference of amino- groups from alanine to oxaloacetio acid takes place. If now a catalytic amount of glutamio or a-kctoglutario acid is also added, amino-groups are transferred from tho alanine to the oxaloacetic acid so that pyruvic and aspartic acids are formed. On account of the free reversibility of the system the reaction does not go to completion but an equilibrium condition is eventually attained; TBANSAMISATION Glutamic transaminase has a prosthetic group which consists of a phosphorylatcd derivative of pyridoxal, one of the B, group of vitamins. Pyridoxal phosphate and the corresponding amino, pyridoxaroino phosphate, have tiio following structures: UOS'CB&S cn KJ pyruScmil phmphaU CH.NH, pyridaxamine phottphuU It 6eems probable that the primary reaction in transamination consists in the transference of the amino-group from an amino- acid to the prosthetic group of the enzyme, pyridoxal phosphate, in which case the first stage of the reaction will probably bo os follows: Eska COO ll I Egg C1I, „ CH, ±H,0 ( ‘ — H!tt«^CO \ : \ coo n ; coon fcnsjinc | +If,K.Ri! [ tmyro^l Hero R represents the remainder of the moleculo of the phos- phate of pyridoxal or pyridoxamine. By further reaction with a-ketoglutaric acid, R.NH* could then revert to the aldehyde and yield glutamic acid. Other transaminases. Moro recently the tranaaminaso system has been re-investigated with freezo-dried enzyme preparations of heart, muscle, liver and kidney, dialysed and fortified with pyridoxal phosphate. In these newer experiments it has proved possiblo to demonstrate tho transamination of twenty-two amino-acids, over and above alanino, aspartic and glutamic acids. It seems that the glutamic-a-kctoglutnric system plays a central part in all of theso processes and that a different enzyme is probably involved for each amino-acid concerned, but ac- cording to some recent experiments a-kotoglutarato is not invariably tho primary amino-group acceptor. Sometimes it is replaced by pyruvate, which is converted into alanino and tho latter then reacts with a-kctoglntaratc. It would be difficult to overestimate tho importance of 128 TKANSA3IIDINATI0N: TRANSCARBAMYI.ATION transamination in protein and amino-acid metabolism (see pp. 201-267). Other experiments have shown that glutamine, as opposed to glutamio acid, can undergo a simultaneous deamidation and transdeamination, the amido-group being set free as ammonia while the a-amino-group is transferred to any of a large variety of a-keto-acids. In this way alanine, phenyl alanine, tyrosine and methionine have been formed from the corresponding a-keto-acids. The mechanisms and the enzymes involved appear to be quite different from the transaminases discussed in the last section. This new mechanism, together with transamina- tion — which is reversible — supply reactions capable of syn- thesizing most, if not all, of the amino-acids normally involved in protein formation, always provided that the corresponding a-keto-acids are available. Aaparlic transaminase. In Borne plants the place of the glutamic transaminases is taken by similar systems in which aspartic and oxaloacetic acids replace glutamic and a-keto- glutario acids respectively. TRANS AMIDINATION Enzymes exist that can catalyse the transference of the amldrne group of arginine to other substances, e.g. N*=c^ ,NH, 'NH NH, (in,), + H,N.CH,COOH - (in,), in.NH, icon arg mtne glycine in.NH, ioon ornithine /NH. IN=C< \NH.cn,c< Little is known at present about the enzymes involved or about . the mechanisms of transfer. TRANSGARB ABtYLATION The early stages of urea and uric-acid synthesis include transfer reactions involving the formation and transference of carbamyl groups. In the presence of certain derivatives of glutamic acid 9 129 ■ DA TRANSMETHYLATION and ATP, carbon dioxide and ammonia react together, to yield carbemyl phosphate. The carbamyl radical con then be trans- ferred to ornithine to yield citrullino (p. 314) or to aspartic acid, yielding carbamyl aspartate (p. 310}. TRANSMETHYLATION Biological methylation is now a well-known process. It has been known for many years that the administration of pyridine to dogs is followed by the excretion of iV-mothyl pyridine in the urine. Similarly, it bos been known for some time that glyco- oyamine undergoes biological conversion into creatine, and this change, too, involves methylation. The methyl groups are fur- nished by tho amino-acid methionine, which is thereby converted into homocysteine. Tho following examplo of transmethylation makc3 this clear. cu,s su in, in, /JH, ) xNH, l ns—cc * cii, - nh^cc + cn, Xvii.cir.cooH | xvcn.coon [ CII.. vil, | C1T.NTT, i cn, i coou coon qbjcocyarnint ntthumin* treat i in Aom/vyjf'iB* ATP is required for the methylation of glycooyamine and also foe that of nicotimo amide and bo, by presumption, for that of pyridine too. Tho part played by ATP in these processes is somewhat unusual since all threo phosphate radicals are removed and the residual adenosyl radical is transferred to tho sulphur atom, thus; CII, — S — D-riUrto— L, in, i CTt.SH, icon R-o4< notylnttfi ion i ■ r It b from this compound rather than from free methionine itself that the methyl group Is transferred. 130 TRANSTBIOLATJON: TRANSA CETYLATION Other important transmethylation reactions reconvert homo- cysteine to methionine, and these two compounds appear to act as an obligatory transport system in biological inethylation. Homocysteine can accept methyl groups from choline, glycine betaine and probably from other donators, and ATP is not required in these cases (see also p. 2S4). TKANSTHIOLATION In the foregoing transfer reactions the group being transferred is most usually exchanged for a hydrogen atom. The possibility that groups or radicals other than hydrogen may be exchanged is indicated by tho exchange of the — SH of homocysteine for the • — OH of serine, which takes place under the influence of cystathionase, an enzyme present in rat liver (see p. 280). TRANS ACETYLATION Acetylation is a not uncommon biological process. It is em- ployed in the detoxication of many foreign substances such, for example, as the sulphonamides. It also plays a part in the forma* tion of mercapturic acids (p. 283) and is important too in tho elaboration of acetyl choline from choline. Coil-free extracts of liver contain an enzyme or enzymes which will bring about acetylation in the presence of free acetic acid, coenzyme A (Co A) and ATP. ATP is required to provide free energy, since acetylation is an endergonio process. The acetylating agent is acetyl-coenzyme a, which can replace free acetate plus ATP plus coenzymo a itself, and is formed by the following pre- liminary reactions between ATP, the coenzyme and free acetate: (i) ATP + acetate s=i adenyl acetate + pyrophosphate, (ti) Adenyl acetate + Co a ?=* AlfP + acetyl Co a. The acetyl radical can now be transferred to choline, for example, yielding acetyl choline: •+ + ( CH,),N . CH,CH,OH + CH,CO~Co a — * (CH,),N.CfI,CH,O.OC.CH» + Co a, chotine acetylcholine or to sulphanilamide: ch,co~co a + — ► ch.co . ^so.yn. + &>*. 131 TRAHSKETOLATION Another interesting transacetylation reaction takes place in many bacteria; hero the acetyl radical is transferred between Co A and Inorganic phosphate: CH»CO~Coa + HO.® .=-» CH,CO~0® + Co a. In addition, some bacteria contain a special aeetokinaso which forms acetyl phosphate by direct phosphorylation at the expense of ATP: ATP + Cir.COOH «=* ADP + CH.CO-O®. Interestingly enough the ‘active’ acetyl radical of acetyl- coenzyme a can also react through its methyl group, e.g. with enol-oxaloacetato to form citrate: coon coon cn in, qOHjCOOH + 11,0 — ► i(OU)COOU + Co* ca t in, TBAN8XET0LAT10N Liver, yeast and spinach and, doubtless, many other tissues contain enzymes known as transketolasea. These enzymes catalyse reactions such as the following: CHO { CH» 0H } ilioil — » nion ^°*f nion ««>n in.o® ch,o® UCOH J neon \ CH,0 ■© V f CHO . I + neon iir.o® D-ffyerm/Mydr-if} Thiamine diphosphate (co -carboxylase) is involved in some way, presumably in tho capacity either of a prosthetic group or a coonzyme. 132 TBANSKETOLATIOX In effect these transketolases catalyse the transfer of a glycol aldehyde group from a ketose to an aldose sugar, and although the mechanism of the transference is unknown, it seems prob- able that the glycol aldehyde radical is first transferred to the enzyme and thence to the accepting aldose : in CH.OH I 0=0 — 4* f Eatrnri CH.OH Cm I CHO -f- ) Enzyme | B CH.OH | Eniyma] CHO B’ ch,oh C=0 -f- 1 Eniyme ) CHOJJ I B‘ A large number of substances can participate in transkefcolase reactions, including: Donators (letoses) Xyloloae-5® L-Erythulose p-Sedoheptulow-7® Hydroxypyru vi o acid D-Frncto»0-6ig) Evidently, therefore, the transketolases can catalyse the formation and decomposition of a very great variety of sugars and sugar phosphates. Indeed, their discovery has in effect opened a new chapter in carbohydrate biochemistry. Of par- ticular interest at the present, time are the two following reactions : ribose-G® + xylol ose-5® <= — eedobeptuloae-7® + glywr&ldebyda-S®, fructose-6© + glyceraldehyde-3® *==-- xyluloeo-5® + erythroae-4®. Sibose, xylulose and sedoheptufoso phosphates are important intermediaries in the photosynthesis of carbohydrates. Gly- ceraldehyde phosphate is immediately related to glyceric acid phosphate, the first labelled product when photosynthesis is allowed to proceed in the presence of isotopio carbon dioxide, while fruotose-6® is an intermediary in the photosynthesis as well as the breakdown of starch. Acceptor* (aldoses) D-Glyceraldebyde-3® L-Glyccraldehyde-3® B-Ribose-5® Glycolaldebyde Erythrose-4® D-L-Glyceraldehyde 133 TKANSAI/DOLATIOtf TnAKSALDOIiATIOK Transaldolases have been obtained from yeast, bacteria and plant sources. Liko the transketolases they require thiamine diphosphate as a cofactor but catalyse tho transference of dihydroxyacetone radicals instead of glycol aldehyde, for example : + tiro IlioR IICOII I > ucoit or,oj laflr, CH.01! I CO J noon /' J CW.ohJ / I li-o j / [±J Clio + Utxill nioK ircxiir neon cir.ofi frfl-n* 4 P IV Again tho transfer is from ketoses to aldoses. Present indications are that these enzymes are probably as ■versatile as tho transketolases and play as important a part in carbohydrates metabolism. Tho transfer of tho dihydroxy> acetone radical probably takes ploco by way of tho enzyme, forming an intermediary complex that can bo roughly forrau- cnoii Beforo leaving theso transaldolnses the reader may be reminded of tho fact that the aldolases proptr (p. 107) nro capable of catalysing a large variety of condensations as opposed to transfer reactions between aldebydic substances and cliliy* droxyflcctono phosphate. Most commonly the latter rcacta with gly eeral dchyd e -3 -ph osphft te to form fru ctoso- 1 : 6 -diphospha to \n the Wua\ way, but many other reactions are possible, to example is shown on p. 135. With tho discovery of transketolation and transaldolation a new interest has been awakened in the long familiar aldolases. Much new information lias been gained about a variety of sugars 134 I80MERIZING ENZYMES formerly considered aa rather exotic and, at the same time, much has been learned about their metabolism and the interplay between them and other more familiar Bugars and sugar phosphates. Nowhere, perhaps, is tills interplay more important than in photosynthesis. CH.O® CH.OH ! CEO e(!oh to hoAh muscle J aldolase HCOH CO + hAoh 1 hIod CH t O® I dihydroxyactUme CH.O® hAoh Ah.O® ttdi>htpt\iU>»c-lfl d* © *ryi&rcwe-4® ISOMERrZINO ENZYMES There remains to be considered a group of enzymes which cata- lyse isomerization in their substrates. There appear to be at least two types, the first, the members of which catalyse simple isomerization, being known as isomerates. Triosephosphate isomerate is the longest known of the group, and was formerly known simply as ‘isomerase *. It catalyses the interconversion of d -glyceraldehyde-3 -phosphate and dihydroxy- acetone phosphate: cn,o© cn,o© Ahoh «=* Ao AhO CH.OH It is usually found in association with aldolase, from which it has been separated and highly purified. Its mode of action is unknown, but conceivably the interconversion takes place by way of the hypothetical di-enol which is common to both substances: CH.Q© CH.O© CH.O® c.on CHQIT CH.OH CIIOH CEO y dihydroxynctlrmt di-enof glyetralde-hyde- phosphatt 3 -photpfinlt isovERiznro exzymss Aconilase, which we hare already mentioned (p. 102), acts by converting citric and iso-citric acids into a common inter- mediate, cw-aconitic acid, and it is possible that the isomernses in general act by converting the pairs of isomeric substances upon which they act into intermediate compounds which are common to both members of each pair. Phosphohexoiaomcrase (oxoisomerase) catalyses the intercon- version of glucose- and fructofurnnosc-G-phospbates: This reaction involves the conversion of an aldose to the corre- sponding ketoso sugar without changing tho position of the phosphate radical. A phosphomannengomerase. has also been described. It converts mannose-O-phosphato into fructoso- 0-phosphato. A phosphoriboieomerase, more recently discovered, catalyses a similar reaction, viz. tlio intcrconveraion of rihoflo-5- phosphatc and ribulose-C-phosphate: HO Oil HO O ribuio* Lon »— noirii uioti nion itLou huflt) Li, os Li.o® n7/nt>*#-5-X’ ryf»i>««-S-P !.*M ISOMERIZING ENZYMES Phosphoglucomutase, which is found in association with phos- phohexoisomerase, is a representative of another type of isomerizing enzymes, for it catalyses the interconversion of a 'glucose-1 -phosphate and glueose-6-phospbate; CH,OH CH,0® It also reacts with the 1 -phosphates of mannose and galactose, converting them into the corresponding 6-esters (see also the case of riboso below). In these cases the isomerization involves a shift in the position of the phosphate radical and the interconversion of an ester and a glycoside. It is usual to call isomerizing enzymes muiases when there is some shift, such as that indicated above, as opposed to a simple intramolecular rearrangement. It has been shown that this enzyme requires glucose-l;G-diphos- phato as a cocnzywe. This substance is produced by the action of a specific 6-pkosphohexokinase upon glucose-6-phosphato in the presence of ATP, and also by a transfer reaction between two molecules of glucose-l-phosphate, in which ATP is not involved : 2 a-glucose-1 -phosphate ► glucos©-l:6-diphosphate + gluecoe. It appears that each molecule of the monophosphorylated substrate can take over a second phosphate radical from the diphosphorylated prosthetic group, which thus becomes the product of the reaction, the former substrate molecule becoming the new prosthetic group. There is thus a continuous stream of traffic across the active groups of the enzyme. Probably an intermediate complex must he formed between the prosthetic group and the substrate, perhaps as follows : Prosthetic group: <3® glucose^ glocosa / glucose Substrate: ; 6 ® glucose glucoae glucose glucose.!-® + glacose-!:6-di ® glucose-l:Q-di + glucose-6-®. 137 ISOUEP.IZIKO ENZYMES The same enzyme can also ■work with ribose-l:5 diphosphate, which it produces from glucoso-l'.G-diphosphatc and ribose-i- phosphato: ribos®4-© 4 glneos® 1:C di © nTx»e-1.5-dJ © 4 glaccao-O©. Together with this new prosthetic group it can then catalyse the mterconversion of tho 1* and tho 5-phospbato esters of ribose: ribose-I-© 4 ribos®-l:5-di © * ribc**e.l:5-di © 4 ribo#®-5-©. The l- and tho 5 -esters of n-2-desoxyriboso are similarly interconvertible. Phosphoglyceromutase is another enzyme of tho mutase type. It catalyses tho interconversion of glyceric ncid-3-phosphato and the corresjKJnding 2-phosphato: cn,o© cn.ou iiion «— > iiio© ioon ioon Hero again a prosthetic group is present and consists of glyceric acid-2:3-(liphosphate, a substance of which tho biological existcnco but not tho function has long been known. Its mode of action probably resembles that of phosphoglucomuto.se. Other isomcrizing enzymes undoubtedly exist. One such, known aa ‘ galactowaldcnaso ’ or phosphogalaetosiomerase, is present in yeast, and in mammalian liver and is of rather special interest since it catalyses the interconversion of ga!actoso-l- phosphato and glucoso-l-phosphato, a change which corresponds to a Walden inversion at carbon 4. This enzyme requires a coen- zymo which is present in yeast, mammalian liver and clsowhere. Tho coenzyme lias been isolated and shown to bo uridino diphosphate glucose (UDPG): E»Urto«o-l-© * UDFClaeo*® r-— ■ » Ul>rC«Uclocoon + (To) - cn.cocootf + (M).en. Aldehydes also can bo oxidized in this manner, but in tliis case a molecule of water is involved: ch.ciio + h,o + (pd) - cif.coon + (rd).sir. Tho oxidation of aldehydes is a matter of great biochemical importance, as wo shall see, and it might havo been anticipated that their oxidation would proceed by the addition of oxygen directly to the aldehyde molecule as follows: 2R.CH 0 + O, - 2R.C00II. Wicland tested this possibility by treating chloral dissolved in dry benzeno with dry silver oxide, and found that no oxidation takes place. If, however, chloral hydrate was substituted for chloral itself, it was oxidized according to the following equation 0(2,011(010, + A*,0 - 08,00011 + SAf ♦ 11,0. 140 DEHYDBOOEKATION Evidently, therefore, the water molecule plays an integral part in the process, and it is now generally believed that, inaqueous solution, an aldehydic radical can become associated with a molecule of water, though it is only rarely, as in the cases of chloral and glyoxylio acid, that the aldehydic hydrate is suffi- ciently Btahle to be isolated. The following general equations can therefore be written to describe the oxidation of aldehydes: | R.CHO + H,0 - R.dff ; / (U) R.CH - 2H \h -n./ It must be remembered that whenever one substance is oxi- dized at the expense of another, the oxidation of the first is necessarily attended by the reduction of the second. Hence it is usual to Bpeak of an ‘oxidation-reduction reaction’, sometimes abbreviated ‘O/R reaction'. If the process is a biological one it can usually be represented in general terms by the equation: A. II, + B *==» A + B.H„ and we can define a number of terms which are in general use in discussions of reactions of this kind. Tho reductant, AH 2 , is known as the hydrogen donator, and the oxidant, B, as the hydrogen acceptor. A third factor is involved in biological oxida- tion-reduction reactions, viz. the catalyst . In Wieland’s experi- ments colloidal palladium played the part of a combined catalyst and hydrogen acceptor. Wieland also found that specimens of palladium that had become ‘charged’ with hydrogen in the manner just described can pass on their hydrogen to certain reducible substances such as the synthetic dye, methylene blue. In the case of methylene blue itself, reduction yields a colourless substance, leuco-methyl- ene blue, winch we may write for the sake of convenience as LIB . 2H. Thus, in the presence of an oxidizablc substance, AH S , together with methylene blue, palladium black catalyses two 141 DEHYDROGENATION processes, first tho dehydrogenation of AH* (reaction a), and secondly the reduction of methylene blue (reaction b): la) An, + (Pd) - A + (Pd). 211, (5) (Fd).2U + MB - (Pd) + MB. 2U. Hydrogen taken up from the primary hydrogen donator, AH„ is passed on to the dye, tho catalyst acting as an intermediary carrier of hydrogen. This significant fact becomes more apparent if we write tho equations in the following unorthodox but very descriptive fashion: (I'd) \-*r ^*-(1 By using this method of expression wo can emphasize the essen- tially cyclical manner in which the carrier catalyst acts, a Email amount being alternately hydrogenated and dehydrogenated over and over again, and thu3 participating in a very large amount of chemical change. Alternatively, if wo only knew that palladium acts catalytically, but did not know liow it docs so, wo might writo the overall process as follows: (I’d) Tho bracket is used hero to indicato that (Pd) acta as the cata- lyst, and tho Bcheme should bo interpreted as meaning that AH* and methylene blue react together under tho catalytic Influence of (Pd) with production of A and Icuco-mcthylene blue. Now living cells and extracts prepared from them by suitable methods are able to oxidize many organio compounds such, for example, as glucose, lactate and succinate. Thcso compounds are quite stable in aqueous solution, and it follows, therefore, that tbo cells contain enzymes which catalyse their oxidation. The nature and distribution of these enzymes, to which tho name of drhydrogenast-a has been given, were studied extensivtly 142 DEHYDROGENATION by Thunberg, who took advantage of the extreme ease with which methylene blue can be reduced. If methylene blue is added to a suspension of chopped muscle, brain, kidney, etc., or to a suspension of yeast or bacteria, the dye is rapidly reduced, thus revealing the presence in the cells of reducing systems. If such an experiment is tried in the laboratory it is usually found that the dye is decolorized in the bulk of the mixture but not at the surface. This is because leuco-methyleno blue is rapidly and Fig. 14. Thunberg vacuum tubea. (a) Original type, (li) newer type, with hollow stopper. (Drawings by H. Mowl.) spontaneously re-oxidized by atmospheric oxygen. To obviate this difficulty Thunberg introduced the use of vacuum tubes of the type illustrated in Fig. 34, in which is also shown a modem version in which the simple stopper is replaced by a curved, hollow device. Reagents can he put into tho hollow stoppers of these tubes and tipped into the rest of the reaction mixture ot any desired moment. Suitable tissue preparations are placed in these tubes, together with buffer, methylene blue and other appropriate reagents, the stoppers are greased and inserted, and tho tubes are then evacuated at tbe pump. When exhaustion has been completed tho stopper is turned and the tube is thus sealed. Alternatively, the tubes may be filled with an inert gas, 143 DEHTEROOBXASES each as hydrogen or nitrogen. In the presence of an active reducing system the methylene blue is reduced, and its reduction b, in effect, an indication of tho presence of such a system. This method has been used extensively in studies of tissue respira- tion. Itia quick, very convenient, and has tho additional advan- tage of giving quantitative results, sinco tho tirao taken to decolorizo a given quantity of methylene blue under standard conditions of temperature, pH and so on gives an inverse measure of the activity of tho system concerned. Other dyes such ns cresyl blue, pyocyanine, and simpler organic substances such as m-dinitrobenzene and o-quinone can replace methylene blue. With tho aid of this simple but ingenious technique, Thunberg carried out many important investigations on living tissues. If a samplo of minced musclo tissue is placed together with buffer and methylene blue in a vacuum tube, and tho latter evacuated, tho dyo is reduced very rapidly. If now the experiment is re- peated using boiled musclo the dyo is no longer reduced, indi- cating that the catalysts concerned arc thcrmolahilo and therefore probably enzymes. If a third experiment is performed in which unboiled and unwashed musclo tissue is replaced by tissue that has been minced and well washed, tho time taken for tho reduc- tion of tho dyo is much increased. Some part of tho complete reducing system lias therefore been removed by washing. If now substances such aa succinate, lactate and tho like are added to the mixture of washed tissuo, buffer and methylene blue, a rapid reduction of tho dye can again be demonstrated. Since these components do not reduce methylene blue spontaneously it follows that the tissuo must contain agents capable of cata- lysing their oxidation. Working in tills manner Thunberg demon- strated, iri cells and tissues of many different kinds, the presence of dehydrogenases catalysing the dehydrogenation of a very wide range of organic materials and, as was later shown by Stephenson, even molecular hydrogen can bo oxidized by a bacterial e«zyme.Succinatc,lactate,ma!ato,(X -glycerophosphate, glucose, aldehydes and alcohols are among tho many substances that can bo activated by tissuo dehydrogenases from ono or another source. Some dehydrogenases are relatively uncommon and can only l>o found In certain tissuca, and in such cases their individuality cannot bo doubted. Again, the aatue tissue, worked 144 DEHYDROGENASES up in different frays, can yield preparations which catalyse the oxidation of some compounds but not that of others, and by systematic work along these lines a great deal has been learned about the specificities of the dehydrogenases. We know now that there is not one single, master dehydro- genase but a considerable number of different individual de- hydrogenases. Some, of which the succinic enzyme is an example, act only upon one natural substrate, in this case succinic acid; others, such as the aldehyde oxidase (Schardinger enzyme) of milk, catalyse the oxidation of any of a wide range of substrates, in this case aldehydes, aliphatic or aromatic. The dehydrogenases thus show tho phenomenon of specificity. They are thermolabile, and their activity is profoundly affected by pH. They are sus- ceptible to the action of many enzyme inhibitors, and often to that of narcotic substances such as the higher alcohols and various substituted ureas. In short, they show all the properties characteristic of enzymes. It was realized fairly early in the history of the dehydrogenases that some members of the group require the co-operation of coenzymes because, after exhaustive washing of the tissues, the reduction time for some substrates is vezy much increased. It can, however, be shortened again by adding boiled extracts of muscle, in which the necessary coenzymes are present. In other cases, however, the addition of boiled muscle-juice does not restore activity, the reason being that some dehydrogenases are themselves soluble in water and are therefore removed by vigorous washing of the tissue. It must be emphasized that the use of methylene blue is not in any Bense a ‘natural’ procedure. The dye does not occur in nature, and is used simply os a convenient hydrogen acceptor for the visual demonstration of the existence of natural reducing systems. Methylene blue replaces the natural hydrogen acceptors of the tissues, and our next inquiries must be into the nature and identity of these substances. The first likely natural hydrogen acceptor that comes to mind is, of course, molecular oxygen, since it is at the expense of molecular oxygen that tissue oxidations are ultimately carried out. But if we set up experimental systems in which a given dehydrogenase and its substrate are mixed together in the pre- sence of oxygen instead of methylene blue, we find that while ail (O 14 5 UOA DEHYDBOGEJ? ASE8". OXIDASES the known dehydrogenase systems can reduce methylene blue, only a few actually take up' oxygen. Thus, contrary to all expectation, molecular oxygen is tiof fAe natural hydrogen acceptor for moil dehydrogenase systems, and wo must look further. In the meantime, however, wo can distinguish between two groups of dehydrogenases: those which can utilize molecular oxygen directly as a hydrogen acceptor and aro now called aerobic dehydrogenases, and the remainder, which operate through other hydrogen acceptors and are known as anaerobic dehydrogenases {see Table 11). Table 11. Classification or deuvdbooenatino enzyues !f.actf*ptMa it. 1 awl PtSydrogenMai anaerobic + - aerobic ♦ + OxMaaea arrobio - + Attention must also bo drawn to another important group of oxidizing enzymes. Like the dehydrogenases in general they catalyso the dehydrogenation of their substrates, hut, unlike Thunberg’s classical dehydrogenases, cannot reduce methylene blue and similar hydrogen acceptors and consequently escaped discovery for eomo time. Instead they uso molecular oxygen, and aro therefore known as aerobic oxidases. It may l>o doubted whether their inability to reduco synthetic dyes like methylene blue constitutes adequate grounds for regarding them as essen- tially different from the aerobic dehydrogenases, but there are other differences too. As a matter of convenience thoy will bo considered hero along with tho aerobic dehydrogenases under the general heading of oxidases. OXIDASES The oxidases aro distinguished from the other dehydrogenating enzymes by their ability to uso molecular oxygen directly as a hydrogen acceptor. Tho aerobic oxidases aro specifically con- fined to oxygen os a natural acceptor and cannot uso other naturally occurring substances, but in tho case of tho aerobic 146 AEROBIC OXIDASES dehydrogenases, molecular oxygen can be replaced for experi- mental purposes by methylene blue. The oxidases may be divided into two groups, the first of which catalyses the reduction of molecular oxygen to water; most aerobio oxidases behave in this way. The second group, comprising most of the aerobio dehydrogenases, leads to the formation of hydrogen peroxide. These reactions may he generally expressed as follows : aerobic oxidase aerobic dthydroqeruist It seems to be characteristic of these enzymes that they are conjugated proteins, although in a few cases no prosthetic group has so far been identified. In general, however, the presence of such a group has been demonstrated and its identity established in a number of cases. Among the known prosthetic materials are copper, iron, and perhaps zinc in aerobic oxidases, and flavin adenine dinucleotido, often together with metallic com- ponents, in aerobio dehydrogenases. Phenol oxidases Aerobic oxidases The phenol oxidases are a group of enzymes that catalyse the oxidation of phenolio substances. Several representatives of the group have been purified and shown to be copper-containing proteins, thus resembling oxygen-carrying pigments of the hacmocyanin type. Monophenol oxidase, isolated from mushrooms, catalyses the oxidation of mono-phenols to the corresponding o-quinoncs: 147 AEROBIC OjtlDASES The intimate details of the process are not known. It is perhaps unlikely that an o-diphenol is formed as an intermediary product, since tliis enzyme acts much more strongly upon mono- than upon diphenols. like other oxidases it uses molecular oxygen as hydrogen acceptor but cannot, however, reduce methylene blue. It is, therefore, an aerobic oxidase rather than an aerobic dehydrogenase. Polyphenol ozidasee have been isolated from mushrooms and potatoes and these too are copper compounds. They have no immediate action upon monophenols, but act rapidly upon o-di- phenols such as catechol to form the corresponding o-quinones in the first instance: o catechol o-quinone Triphenols such as pyrogallol are also attacked. Another member of this group of phenol oxidases, laccase, has been isolated from the latex of the lao tree, and tliis too is a copper protein but differs somewhat from the others in specificity. Ilxo primary oxidation is followed as a rule by further changes which are spontaneous, but before we consider these further reactions, attention may be drawn to one very important feature of the polyphenol oxidase system. The o-quinones formed can bo rapidly reduced again by ascorbic acid, and by certain reducing systems such, for example, as glucose-6-phosphate dehydrogenase together with its substrate and the appropriate coenzyme (TPN). If we represent the reducing substrate by AH, the reactions can be written in the following manner: Alt,--. tMJumon* X AHj dehydrogenase polyphenol + TKJ oxidase In this system a very small amount of the diphenol, e.g catechol, or the corresponding quinone can be alternately reduced and 148 AEROBIC OXIDASES oxidized many times and thus can act as an intermediate carrier of hydrogen between a theoretically unlimited amount of AH S on the one hand and molecular oxygen on the other. Such a system catalyses a continuous uptake of oxygen and a simul- taneous oxidation of AH 2 in equivalent amount. The amount of carrier required is very small indeed, and the carrier must, in fact, bo regarded as a catalyst in its own right. We have in this system a biological counterpart of the palladium systems studied by Wieland (p. 142), while Thunberg’s methylene-blue systems can act in a similar way under aerobic conditions: ddiydrogewuc rum-enzymic Systems of tliis kind are very interesting because they can be regarded as 'models’ of the respiratory systems of living cells. The latter contain reducing substances, represented by AH 2 , together with the appropriate dehydrogenases and co- enzymes. These pass on hydrogen to acceptors, the identity of which wo have to discuss here, and, at the other end of the chain, molecular oxygen acts as the ultimate hydrogen acceptor and is reduced in the process. Whether systems involving polyphenol oxidase play any important part in the respiration of plant tissues is uncertain, but the possibility has certainly not been excluded. Indeed, fcbe respiration of spinach leaves is greatly increased by dihydroxy- phenylalanine. This is a natural dipkenolie constituent of the leaves and is susceptible to the action of polyphenol oxidase, which is abundant in these leaves. Polyphenol oxidase systems are also involved in the * browning * of potatoes, apples and many other plant materials that takes place when a cut surface is exposed to the air. It is usually found that plants containing polyphenol oxidase also contain traces of o-diphenols, especially of catechol. Catechol, for example, is oxidized to the corresponding o-quinone through 149 AEEOBIC OXIDASES the agency of the oxidase {reaction (i)) and the remaining stages take place spontaneously: 0 Finally, (iv), the kydroxyquinone undergoes polymerization to yield complex, dark-coloured, melanic products, the constitution of which is still unknown. o-Quinone formed in reaction (i) is consumed in reactions (ii) and (iii), so that melanic products can continue to be formed only so long as o-quinone is supplied by the action of polyphenol oxidase in reaction (i). Tyrosinase, another phenol oxidase, occurs widely, in plants and animals alike. It is this enzyme that is responsible for the production of rao3t of the dark brown and black pigmentation of animals. Tyrosine itself is probably the starting material in many cases of melanin formation, but other phenolic compounds too may be used. From certain insects, for example, proto- catechuic (3:4-dihydroxybenzoic) acid has been isolated as a natural substrato; others contain 3:4-dihydroxyphenyIacetic acid and 3:4-dihydroxyphenyl-lactio acid. In albinism, where there is a characteristic and complete lack of mclanins, tyro- einaso is absent, while in piebald animals such as rabbits and guinea-pigs the dark portions of the skin contain tyrosinase, but the enzyme is absent from the white parts. In certain insects, on the other hand, melanic pigmentation is determined by localization of the substrate rather than by that of the enzyme. Again, the dark brown or black 'ink' of the squids and octopuses consiate of a fine suspension of melanin, which is very insoluble. 160 AEROBIC OXIDASES It is elaborated in n special gland, the ink-sac, the walls of which contain tyrosinase and are at the same time very rich, in copper. This was until recently the only indication we had that the tyrosinase of animals is a copper protein, but wo now know that mammalian tyrosinase is inhibited by reagents, e.g. cyanide, which form complexes with copper, and is released from this inhibition by Cu 4 "*" though not by other metallic ions. The first stage in the formation of melanin from tyrosine probably consists in the oxidation of tyrosine to the quinone of dihydroxyphenylalanine ('dopa quinone’). This is followed by spontaneous ring closure and by the possibly enzymic oxidation of the product to yield a red intermediate compound. Further changes, which are mainly spontaneous and include polymeriza- tion, give rise to melanin itself: J^jCn,Cn(NH,)COOH + 0< O=| / ^jCH l CH(NH,)C00H ■oJ J tyrosinase tyrosine dop (W^CH,CH(NH,)COOH + n Q III 0-U : quinone ' CH * - H,0 bn.cooH L H red intermediate Tyrosinase is involved at perhaps two stages in this reaction sequence, first in the oxidation of tyrosine to dopa quinone, a reaction that can also be catalysed by the monophenol oxidase of plants, and later in the oxidative production of the red compound from the corresponding o-diphenol, a process that can be catalysed by plant polyphenol oxidase. It may be, therefore, that tyrosinase acts both as a mono- and as apolyphenol oxidase. Ascorbic oxidase. Although ascorbic acid is one of the sub- stances known to bo oxidizablo through the polyphenol oxidase- catechol Byetem of plants, many plants, notably the Brassica 151 AEROBIC OXIDASES - family, are rich in an ascorbic oxidase. This enzyme catalyses the oxidation of ascorbic to dehyclroascorbic acid: CH,OH h-amijUbacid o, - y the uptake of an exactly equivalent amount of oxygen. With the isolation of the two eoenzymes it became possible to lassify dehydrogenases under three headings. Some can reduce icthylene blue and cytochrome without the addition of any oenzyme and, according to many workers, these red nee cyto- hromo b in the first instance and cytocliromo c only at second and. A second group can only reduco cytochrome c in the rcsence of DPN, whilo a third group requires the presence of PN: cytochrome b is not involred in either of these cases. m 00EN2YME REQUIREMENTS We have already seen that dehydrogenases are as specific towards their substrates as are other enzymes, such, for instance, as the glycosidases. We now know that they are specific also with respect to their hydrogen acceptors. Lactic dehydrogenase obtained from muscle, for instance, normally requires DPN and reacts more than 100 times as fast with this as with the closely related TPN. In many cases the specificity appears to be more complete even than this; for example, glucose-6-phosphate de- hydrogenase requires TPN and cannot collaborate with DPN. In only a few cases are the two coenzymes interchangeable. But the relationships between the dehydrogenases and their sub- strates are more specific than those between the dehydrogenases and their coenzymes, for whereas the same coenzymo can col- laborate with more than one dehydrogenase, a given substrate is activated only by an appropriate dehydrogenase and not by any other. A further point to be noticed is that the coenzyme itself is not a very reactive compound, for it can only be reduced by comparatively powerful reducing agcnt9 such as dithionite (hydroaulphite). Furthermore, lactic acid is not easily oxidized, and becomes easily oxidized only as a result of its union with the lactio enzyme and consequent activation. Similarly, DPN under- goes a great increase in chemical reactivity in the presence of lactio dehydrogenase, with which it is able to combine. The functional behaviour of the cocnzyme is therefore very similar indeed to that of the substrate, and the coenzyme might even be regarded as a second Bubstrate. Both combine with the dehydro- genase protein, and the affinity constants have been determined for both cases. There is now evidence that in the natural state the coenzymes are firmly bound to the dehydrogenase proteins, especially in tho mitochondria, and that they become freely dissociable only after the dehydrogenases have been extracted from the tissue substance. For convenience of reference a list of some of the most impor- tant dehydrogenases is given in Table 12 so as to show which coenzyme, if any, is required by which dehydrogenase.* Several of these enzymes have been found to contain a metal, e.g. Fe, Cu, Zn, and this may yet prove to be a general feature of * A lengthy cataloguo will be found in Annual Review of Biocfiemhtry (1955), 24, 24-7; this lists the more recently discovered dehydrogenase*. 179 CYTOCHROME* SPECIFIC DEHYDROGENASES dehydrogenases. In what follows wo shall refer to the three groups as cytochrome-specific, DFN -specific and TPH -specific dehydrogenases respectively. In the presence of their substrates, the appropriate coenzymes and other factors where necessary, all these dehydrogenases ore capable of catalysing the reduction of methylene blue and of cytochrome c, providing always that they have not been too rigorously purified beforehand. In what follows the coenzymes will be denoted by JDPN and TPIs r and their reduced forms as DPN.H S and TPN.H, respectively os a matter of convenience. Table 12. Coenzyme requirements or dehydrogenases None Co-dehydrOgensas required DPN TPN a-Glycerophosphate 1 a-Glycerophosphate* Glucose Snccinlc Lactic (muscle) L-Glatamio Lactic (yeast) Mabo Glucoee-6-pbospbsto Choline Trioecphoephate wo-Cltrio Fatty acyl Co a Butyiyl-Co a Alcohol Mabo decarboxylase /J-Hydroxybutyric Tnosephosphate (plants) L-Olotamio /Mtyihroxy tatty -acyl-Co a 1 Insoluble. * Soluble. Dehydrogenases requiring neither coenzyme a-Glyccrophosphate dehydrogenase (insoluble) occurs in animals, plants and micro-organisms alike. It catalyses the oxidation of n -a -glycerophosphate to D-glyceraldehyde-3-phosphate at the expense of a suitable acceptor, o.g. methylene blue: ch.o® cn,o® illOH + MB *=* Anon + MB. 211 iii.on ino This enzyme is specific for the D-( + )-isomer of a ioou Aooh succinic acid malic acid oxaloacetic acid malonic acid The activity of this enzyme depends upon the presence of its SH groups, and it is inhibited, this time non-competitively, by agents which oxidize adjacent pairs of — SH groups to form ' — S — S — linkages. It is also inhibited by fairly high concentra- tions of monoiodoacctate, which reacts with and blocks the — SH groups irreversibly: — SH + I.CH,COOH - — S.CH.COOH + HI. Several other dehydrogenases are similarly affected by iodo- acetate, but the succinic enzyme requires unusually high con- centrations for its effective inhibition. In common with many other 4 — SH enzymes’, succinic dehydrogenase is powerfully inhibited by many war gases, such as the arsenical smokes and vesicants, which act specifically upon — SH groups. Indeed, it ha3 even been suggested that poisoning by oxygen may be due to oxidation of the — SH groups of sulphydryl-dependent enzymes. 181 OYTO CHROME-SPECIFIC DEHYDROGENASES Laclic dehydrogenase of yeast (see also p. 106) is of special interest for two reasons, first because, unlike the lactic enzyme of muscle, it requires no coenzyme. Like succinic dehydrogenase it is an iron-containing flavopretein but flavin mononucleotide (FMN) appeara to be present instead of FADN. In addition, it has been highly purified and shown to involve a conjugated protein containing hacmatin, which corresponds to the cyto- chrome b 2 of yeast (cf. p. 1C8). It catalyses the oxidation of i«-( + )-Iactic acid to pyruvic acid: CH, CH, iflOH + MB lo + MB.2H iooii iooH d-(— )-Lactic acid is not attacked by this enzyme, nor are any of the ^-hydroxy-acids tested. Like most dehydrogenases, the lactic enzyme of yeast can act in reverse, producing from the optically inactive pyruvic acid the l- but never the D-forrn of lactic acid. Many micro-organisms other than yeast contain similar en- zymes, but in somo cases at least only the D-isomer of lactic acid can be formed and attacked. Cholint dehydrogenase, another insoluble enzyme, occurs in some bat not in all animal tissues. It catalyses the oxidation of choline to betaine aldehyde: (cn^N.CH.cn.OH + sru*— ^( ch.j.n.cu.cho + are.211. Fatly acyl-Co a dehydrogenases act upon acyl-Co a derivatives containing 4-16 carbon atoms and catalyse reactions of the following type : r.ch,ch,co.Coa + x«=*R.cn-cn.co.CoA + x.211. This introduction of an a:fi double bond is the flrst step in the degradation of the fatty acids after they havo been converted into their Co a derivatives. There are at least two such enzymes, differing in their specificities with respect to chain length, but both arc yellow flavoproteins with FADN os prosthetic group. Tins prosthetic group is reduced on addition of an appropriate substrate and, 182 DPN -SPECIFIC DEHYDROGENASES although in its reduced form it can reduce a number of synthetic dyea, it cannot reduce cytochrome c. Another factor is required {see p. 196). Butyryl-Go a dehydrogenase resembles the fatty acyl-Co A dehy- drogenases and catalyses a similar reaction but is active only for fatty acyl-Co a compounds noth 4-6 carbon atoms, especially C| . This enzyme too is a flavoprotein with FADN as prosthetic group, but the isolated enzyme is green in colour owing to the presence of copper, which is an integral part of the enzyme. In other respects it resembles the fatty acyl-Co a dehydrogenases. It would appear that the presence of flavin and metallic constituents in these enzymes absolves them from coenzyme requirements. We shall return to a fuller consideration of this group of enzymes (p. 190). Dehydrogenases requiring DPN U-Glycerophosphate dehydrogenase (soluble). Animal tissues contain two a-glycerophosphate dehydrogenases, one of which has already been described (p. 180). The soluble enzyme, unlike the insoluble, requires DPN and is specific for the L-( — )- instead of the d-{ + )-form of a-glycerophosphate. Methylene blue can- not be used directly as hydrogen acceptor in this case, while the reaction product is dihydroxyacetone phosphate, not glycer- aldehyde phosphate: CH,0© CH,0® ^HOH + DPN b> + DPN.H, in.on ka.on Lactic dehydrogenase of muscle has been crystallized. Like the lactio enzymo of yeast, this dehydrogenase catalyses the oxid- ation of E-lactate to pyruvate, again reversibly, but whereas the yeast enzyme can catalyse a direct transfer of 2H to methylene blue, that of muscle requires DPN as its immediate hydrogen acceptor: CH, CH, £hoh + DPX ««=* io + DPN.H, ioOH ioOH 183 DPI? -SPECIFIC DEHYDBOOENASEB TPN can replace DPN here but the latter reacts more than 100 times faster than the former. This system plays on important part in the metabolism of muscle and other tissues, and has been studied extensively. The equilibrium is very much in favour of the left-hand side, i.e. in favour of the hydroxy-acid, but the reaction can be forced over towards the right by adding on excess of hydroxy-acid or, alternatively, by adding some trapping reagent, e.g. cyanide, which reacts with pyruvate to form a eyanhydrin. Malic dehydrogenase is always found in dose association with the lactio enzyme, suggesting that there may be some functional association between the two. They are not identical however. The malic enzyme catalyses the conversion of L-malic acid into oxaloacetic acid: coon cooji ira, is* I + nra «-=* ] + dpjj.h. ciioh Co Aooh Aooh Once again the equilibrium conditions are in favour of the hydroxy-acid. Like the lactic enzyme, malic dehydrogenase can use TPN os well as DPN, but the latter is about fifteen times as aotive as the former. fi-ll ydroxyb uly ric dehydrogenase, occurs in many animal tissues, especially in heart, kidney and liver. It catalyses the inter- conversion of r,-/?-hydroxybutyric and acetoacetic acids: CH,cn(OH)cn,coos + dps «=^cn,cocn,cooH + DPN.n*. It haa no action upon a-hydroxy- or a-keto-acids. Tho enzyme does not act upon /?-hyciroxypropiomc acid, the next lower homologuo; whether higher homologues are attacked is not known. Alcohol dehydrogenase of yeast has been obtained in crystalline form and found to contain zinc. Like the succinic enzyme it requires the presence of its — SH groups for activity. This enzyme is very sensitive indeed to iodoaectate. A somewhat similar enzyme is present in mammalian liver but not, apparently, in other mammalian tissues. The yeast enzyme acts upon 184 DPN- SPECIFIC DEHYDROGENASES primary and secondary alcohols to yield the corresponding aldehydes and ketones: Primary: Secondary! R.CH.0H + DPN -=* R.CHO + DPN.U,; j^crroH + ura— * b“> + DPN.H,. The equilibrium is in favour of the alcohol so that, in order to study the forward reactions, it is necessary to work in the presence of an aldehyde- or ketone-fixative such as sodium bisulphite. Glucose dehydrogenase of liver catalyses the oxidation of D-glucose to the corresponding gluconio acid. In this case it is probable that the 5-lactone is formed first of all and then reacts, possibly spontaneously, with water: +DPN.H, Several sugars other than D-glucose are attacked by this dehydrogenase, which has the distinction of being able to use either DPiM or TPN as its hydrogen acceptor. It may be com- pared with the very specifio glucose oxidase of Penicillium (P- 155). ^-Glutamic dehydrogenase catalyses the conversion of L-glu- tamic acid to the corresponding imino-acid, a reaction that is followed by spontaneous hydrolysis of the imino-acid to yield the corresponding a-keto-acid together with ammonia: COOH COOH COOK ki f ki t cn, kf, + DPN —> dif t + DPN.U,; (W, + n,o in.NH, kmi A-.nh iioon ^ooh coon COOH n, H. 4- NB, io doon 185 UP.N -SPECIFIC DEHYDROGENASES DPN and TPN are interchangeable in this system. Both stages are reversible, and a-kctoglutaric acid can be red actively ami- nated by ammonia in the presence of the dehydrogenase to yield D-glutamic acid. Similar enzymes are present in yeast and in plants, the yeast enzyme requiring TPN whilo that of plant tissues requires DPN. The liver enzyme has been highly purified and shown to contain zinc. This dehydrogenase appears to be absolutely specific for t-glutamio acid and has no action upon the D-isomer nor upon other D-amino-acids. Triosephosphate dehydrogenase. The term 'triose phosphate’ as ordinarily applied refers to an equilibrium mixture of D-gly- ceraldehyde-3-phosphate with dihydroxyacetone phosphate: CH,0® CII,0® inoH *— » co ino iit , oh Attainment of this equilibrium is catalysed by triosephosphate isomerase (p. 135). The so-called triosephosphate dehydrogenase is concerned with only one of these components, namely, with D-glyceraldehyde- 3-phosphate, and acts upon this only under certain definite con- ditions. DPN i3 required and inorganic phosphate also must be present. The oxidation product that accumulates corresponds not to glyceraldehyde-3-phosphate but to glyceraldehyde- l:3-dipbosphate. Enzymes similar to the triosephosphate dehydrogenase of muscle are present in yeast and in plant tissues, and are probably very widely distributed indeed. A TPN-specific triosephosphate dehydrogenase occurs in the green tissues of plants (p. 189). The muscle enzyme is extremely sensitive to iodoacctatc, from which it may be deduced that its — SH groups are required for activity and in this case there is a good deal of evidence about the part played by — SH in the reaction. This interesting enzyme has been studied extensively largely, in the first place, with a view to discovering the part played by inorganic phosphate. It now appears that the dehydrogenase itself has glutathione as a prosthetic group through the — SH group of which it unites with its substrate: DPN-SPEOIFIC DEHYDBOOENASES CH,0® CH,0® j CHOH CHOH l/ H _ l/ H C. =F= 1 V + ^0 ^OH SH i j Enzyme | f Enzyme j CH,0® CH,0® CHOH CHOH |/> ±HO.® 1 C * 0=0 + N 0~® SH t S 1 | Enzyme | [ Enzyme | Tho enzyme-substrate complex hands over a pair of H atoms to DPN and the product is split by inorganic phosphate to yield glyceric acid-1 :3-diphosphate. The latter contains high-energy phosphate and accordingly can react with ADP in the presence of the appropriate phosphokinase: CH,o© I CIIOH 1 coo~® CH,0® I CHOH I COOH p-IIydroxy-falty-acyl-Co a dehydrogenase, plays an important part in the metabolism of fatty acids. It catalyses reactions of the following general type: R.CH(OH)CH»CO~Co a 4 DPN R.CO.CH,CO~Co a 4 DPN.H,. IR7 TPU-SPECIFIC DEHYDROGENASES Dehydrogenases requiring TPN Glucose and 'L-glutamic dehydrogenases havo already been described (p. 185). Glurxi3e-G-phosphale dehydrogenase occurs in red blood cor- puscles and in yeast. It requires TPN and catalyses the oxidation of glucose-6-phosphato to gluconic neid-G-phosphate, probably by way of the 4-lactone (cf. glucose dehydrogenase): CH,Oi£> CH,0® CH.O® 4-TPN.H, The process is reversible. This is a very specific dehydrogenase, for it has no action upon other sugar phosphates, e.g. upon fructofuranose-G-phosphate or upon fructofuranose-l:8-diphos- phate, nor does it act upon glucose itself (cf. glucose dehydro- genase). The oxidation of glucose-6-phosphatc to gluconic acid- 6-pbosphate is an important step in the formation of the pentose sugar, D-ribose, from bexose sources (p. 348). isQ-Cilric dehydrogenase is widely distributed in animal and plant tissues and is present also in many micro-organisms. It acts upon L-wo-citrata to produco oxalosuccinic (a-keto-/?- carboxyglutarie) acid, and the process is reversible: COOH COOJI in, in, in, coo ii + tpn in. coon + TPN. II, inoH io ioon ioon This is usually followed by the action of another enzyme which catalyses the /^-decarboxylation of the product and gives riso to a-ketoglutaric acid but this second reaction, unlike the dehydro- genation, requires the presence of manganese ions. In their absence the action of the dehydrogenase can bo studied 188 TPN -SPECIFIC DEHYDROGENASES independently but there is evidence that both reactions are catalysed by one and the same enzyme-protein. Malic, decarboxylase must be mentioned here. It differs sharply from malio dehydrogenase (p. 184) for it catalyses on oxidative decarboxylation of L-malate, using TPN a3 hydrogen acceptor and yielding pyruvate and carbon dioxide as products: COOH CO, CH, | + TPN v=i I + TPN.H. CHOU CO COOH ioou This enzyme thus combines the functions of a decarboxylase and a dehydrogenase but appears nevertheless to be a single entity, every effort to resolve it into simpler components having so far failed. Triosephosphale dehydrogenase of plants. Plants contain several triosephosphate dehydrogenases, one of which is specific with respect to TPN. This enzyme is absent from seeds, roots and dark-grown seedlings. It appears to be strictly localized in the green tissues, in the photosynthetio activities of which it plays an important part. TPN and ATP are involved in the reaction, which can be written in abbreviated form thus; cir.o© «blOH + H,0 + TPN + ADP + HO.® ino Probably tliis process is as complex as that catalysed by the DPN-specific enzyme (p. 186). CH,0© AhOH + TPN.H, + ATP ioon Work on reconstructed dehydrogenase systems Much of the work on dehydrogenases has been carried out with the aid of what are known as reconstructed systems. It is possible, for example, to build up a system containing suc- cinate, succinic dehydrogenase, cytochrome c, cytochrome oxid- ase and oxygen, and such a system can 'respire', i.e. can take up oxygen and oxidize an equivalent amount of succinate to fumarate. The lactic system described on p. 177 is another , 189 RECONSTRUCTED DEHYDROGENASE SYSTEBtS example of such a system. All the reactants employed are substances which occur in nature and all can be obtained from living materials. It seems probable, therefore, that they represent systems which actually participate in the respiration of living cells. But there are certain important criticisms that must be noted. The fact that a given series of operations can bo demonstrated in a reconstructed system is not positive proof that it takes place under biological conditions. As Green has written: ‘A sufficiently ingenious mechanic could separate the parts of a baby Austin and use them to make a perambulator or a pressure pump or a hair-dryer of sorts. If the mechanic was not particularly bright and was uninformed os to the source of these parts, he might be tempted into believing that they were in fact designed for the particular end ho happened to have in view. The biochemist is presented with a similar problem in the course ofliis reconstruc- tions. The materials of the cell offer unlimited possibilities of combinations and interaction, but only a few of these possibilities are realized in the cell under normal conditions. There is thus a grave element of risk in trying to reason too closely from reconstructed systems to the intact cell. The reconstruction can have no biological significance until some definite counterpart of these events is observed *n vivo/ It will bo remembered that the oxidation and reduction of the cytochromes can, in fact, be observed within living cells, while the presence of dehydrogenases in intact cells can bo demonstrated by the Thun berg method, U9ing a dye such as methylene blue. The discovery that the specificity of dehydro- genases is such that each can only reduco its own particular natural hydrogen acceptor shows that these substances, which do in fact occur in living cells of all kinds, must necessarily act as intermediates in processes of cellular oxidation. Thus the fact that the substances and the catalysts used in reconstructed systems do occur ia intact cells, the fact that certain components can be observed at work within the cells themselves, and the incontestable facts of enzyme specificity — all these taken to- gether make it seem improbable that wo shall be led into gross error by the study of reconstructed systems, provided that the results ftTO cautiously interpreted. But the further research has RECONSTRUCTED DEHYDROGENASE SYSTEMS gone in tins field the more complicated many apparently Bimple processes have turned out to be. Intact cells, in which substances such as the cytochromes and coenzyraes are present only in small concentrations, respire relatively much faster than reconstructed systems containing the same reactants at the same order of concentration. This seems at first to suggest that the two may be fundamentally different. But there are good reasons for believing that whereas events in a reconstructed system take placo in a more or less haphazard manner, the enzymes, coenzymes and other reactants of the intact cell are organized in such a way that each is present in the right place at the right time. Wo shall have occasion later to enlarge somewhat on this notion of intracellular organization. The outstanding properties of the anaerobio dehydrogenases are: their specificity towards their substrates, their specificity towards their hydrogen acceptors , and the fact that, so far as our information goes, they are all capable of acting reversibly. In addition, tlioy possess the usual properties of enzymes, viz. thcrmolnbility, dependence upon pH and so on. As wo have seen, three main groups can be distinguished, the DPN- and TPN-spccific types, and a third group which has no requirement for either of these coenzymcs. If now we take a dehydrogenase together with its substrate and the appropriate coenzyme, we have a system which can reduce methylene blue or cytochrome, always provided that the enzyme preparation has not been too exhaustively purified. But as purification is carried progressively further and further we find that the system eventually fails to reduce cytochrome and methyleno blue. This might bo due in part to some kind of damage done to the enzymes by the methods used in their purification, hut it might also mean that there must be one or more steps in the whole reaction sequence requiring catalysis by some enzyme or enzymes that are present in crude extracts but eliminated by purification. It is known, in fact, that the cyto- clirome-specific dehydrogenases cannot reduce cytochrome c directly and that one or more additional catalysts are required to establish communication. Again, in the case of dehydrogenases that operate through DPN and TPN, there is definite evidence that additional catalysts are required to accomplish the transfer 101 FLAVOPROTEINB of hydrogen from the reduced cocuzyme to cytochrome c. The nature of these additional catalysts we shall consider in the next section. FLAVOPROTEINS AND THE REDUCTION OF CYTOCHROME The flavoproteins are a group of conjugated proteins which arc characterized by the presence in the prosthetic group of ribo- flavin. This 13 a yellow substance which exhibits a strong green fluorescence even in very dilute solutions. It is identical with the flavin of milk (lactoflavin) and with that of egg-wliito (ooflavin), and occurs very widely indeed among cells and tissues, its importance in which may be gauged from the fact that it is a member of tho B 2 group of vitamins. It is derived from the nitrogenous base G:7-dimetliyl-tso-alloxftzino and tho pentahydrio sugar alcohol D-ribitol, linked together in tho fol- lowing manner: h n n o Certain points call for special comment. The substance can exist in the oxidized form shown above, but can be reduced by fairly powerful reducing agents such as dithionite, (Na^S 1 0 < ). Two atoms of hydrogen are taken up in the process, which may be described as follows: D-nbifol D-ribitol S / 192 FIiAV0PB0TElN8 Although reagents such as dithionite aro necessary to effect the reduction of the oxidized form, the reduced form is autoxidizable, i.e. it can be oxidized by shaking -with molecular oxygen, the oxygen being thereby reduced to hydrogen peroxide. Another point -worthy of notice is the presence in this substance not of the pentose sugar, n-ribose, but of the corresponding sugar alcohol, D-ribitol, so that the name riboflavin, suggesting as it does that the molecule contains D-ribose, is a misnomer: a better name would be ribitylflavin. Riboflavin occurs in the flavoproteins in the form of its 5'-phos- phate (see p. 355), a substance which resembles a nucleotide in its general structure. Strictly speaking, however, it is not a nucleo- tide since, while it does contain a nitrogenous base, it does not contain a pentose sugar, but in view of its importance in cellular metabolism, in which other nucleotides also are intimately con- cerned, it has become common practice to refer to riboflavin phosphate as flavin mononucleotide (FMN). Two other important nucleotides are adenine mononucleotide (adenylic acid) and nicotinic amide mononucleotide. The two latter are present in DPN and TPN, and the structures of all three mononucleotides should be compared (see pp. 352, 354). The flavoproteins fall into two classes. In the first of these the prosthetic group consists simply of flavin mononucleotide, in the second of flavin adenine dinucleotide (FAD or FADN), i.e. the dinuclcotide formed by the union of flavin and adenine mononucleotides through their respective phosphate radicals. The mononucleotide can be formed from free riboflavin at the expense of ATP by an enzyme obtained from brewers’ yeast and called flavokinase: riboflavin + ATP ► nboflavin-5'-phoaphate + ADP. Flavin adenine dinucleotido can then bo formed from the mono- nucleotide by another enzymatically catalysed reaction with ATP: flavin mononucleotide + ATP — -*■ flavin adenine dinucleotide + Inorganic pyrophosphate. Like free riboflavin, the free mono- and dinucleotides can be reduced by dithionite, the reduced forms being autoxidizable. Similarly, when combined in the form of flavoproteins, these 103 n FI*A Y0PR0TEIN3 nucleotides can still bo reduced, and -when in the reduced form are sometimes though not invariably autoxidizable. We have already encountered several flavoprotcins such, for example, os the Qavoproteln oxidases, the prosthetic group of which acta as a built-in hydrogen acceptor for pairs of hydrogen atoms which are taken over from activated molecules of the substrate and handed on to molecular oxygen. The fact that the prosthetic group is so readily reduced by activated substrate molecules when combined with the protein component of the oxidase shows that tlii3 protein activates not only its substrate but the prosthetic group as well, just as the dehydrogenases activate their coenzymes os well as their respective substrates. Nor i3 tliis all. Many fiavoproteins ere now known to be asso- ciated with heavy metals, in particular Fe, Mo and Cu, and it is probable that these also aro activated by the protein component. In the case of the metalloflavoprotcin oxidases it is probable that the metallic constituents establish the necessary contact between the reduced flavin component and molecular oxygen, though this i3 not proven. The known, natural flavoprotcins fall into a number of groups, and research on these compounds is very active indeed at the present time. Flavoprolein oxidases (i.e. oxidases with FADN as prosthetic group) ore fairly numerous. They aro commonly associated with heavy metals and, ns their name indicates, they can use molecular oxygen as hydrogen acceptor. Several have already been described (pp. 1G3-G0). Flavoprotein dehydrogenases (i.e. dehydrogenases with FADN as prosthetic group) have been known for some years. Unable though they aro to reduce molecular oxygen some of them can reduce cytochrome c. As in the case of the oxidases, the FADN of the prosthetic group acts os an intermediate carrier of hydrogen. The two longest known of these, formerly called diaphorases J and 11, act as spccilic dehydrogenases for the reduced form3 of DPN and TPN resx>ectively. They can use methylene blue as a hydrogen acceptor, but cannot reduce cytochrome c. It was accordingly believed that an additional carrier of eorao kind is required to bridgo the gap between the reduced form of the 104 FLAVOmOTElHQ . diaphorase on the one hand and oxidized cytochrome on the other. During the last few years work on xanthine oxidaso and the aldehyde oxidase of mammalian liver, for example, has shown conclusively that many flavo proteins are intimately associated with heavy metals. It now seems reasonably certain that the diaphorases, which act as DPN.H 2 - and TPN.H 2 -specific dehy- drogenases, are, in tho natural state, closely associated with iron in particular, but that this iron is removed by the methods formerly used for their isolation and purification. When pre- pared by methods calculated to preserve the metal association, highly purified products have been obtained which are speci- fically reduced by the reduced form of the appropriate coenzyme, and will actively reduce cytochrome c in their turn. They may therefore be regarded as reduced coenzyme-cytochrome redudases. One enzyme of this kind, with Fe-association, has recently been obtained from heart muscle and, in the highly purified form, it oxidizes DPN.H 2 and reduces oxidized cytochrome c. Removal of the iron yields a product with virtually the same properties as diaphorase I, also isolated from heart muscle, i.e. in the absence of iron it oxidizes DPN.H 2 and reduces methylene blue without however reducing cytochrome c at any appreciable rate. Perhaps it is significant that the heavy metals so far found in association with flavoproteins, iron, molybdenum, copper and perhaps manganese, are all metals that can exist in an oxidized and in a reduced form, e.g. ferric and ferrous. It may be, therefore, that the specific protein component of each of these particular flavoproteins accomplishes a threefold activation, (i) of the substrate, (li) of the flavinadenine dinuclcotide which forms the prosthetic group and (iii) of the metal with which it is also associated. We may thus be justified perhaps in tentatively representing the mode of action of the coenzyme-specifio dehy- drogenase systems as follows: •H,— ^ UPN -fy. _^*-FAl)N‘.2U — .. -la redoetd — _ - — l°i 1 ] Y mdal T tytoeifom c | ' ''' — FA DM ' s — '''♦-H.O j*olctn tompanent r>J JlanproU t* 105 .. » 3 *» FRAVOEROTBIHS The metals associated with fl apoproteins, formerly regarded as impurities or contaminants to be removed at all costs , now appear as indispensable parts of the complete enzymes and this realization has, as we have Been, enabled us to give a plausible account of the action of complete coenzyme-specific dehydrogenase systems. The picture ia more confused in the case of dehydrogenase systems requiring no cocnzyme, but the recent spurt of interest in metalloflavoprotcins has led to the eventual isolation of succinic dehydrogenase, as an amber-brown coloured substance containing flavinadenino dinucleotide together with iron. In this case, perhaps, the complete system may be represented: II,' -V ^ FADN '" V >-FADN.2J X -ous — oxidized^. >*-ir,0 Ft ] cytochrome t If (1 — ^ 10 , protein component oj euceinic. dehydrogenate In the case of the lactic dehydrogenase of yeast there is again evidence for the presence of iron, this time in haem combination. A flavin derivative is also present, apparently flavin mono- nucleotide, and this enzyme again reduces cytochrome c. Rather more ia known about fatty acyl'Co A dehydrogenase, which is a flavoprotein apparently devoid of heavy metals, and about the green, copper-containing flavoprotein, butyryl-Co a dehydrogenase. Both are reduced by an appropriate substrate, but the reduced forms are unablo to reduce added cytochrome c directly. An additional flavoprotein, which has been called the ‘electron -transporting flavoprotein' (ETF) but about which wo do not yet know very much, is required to set up communication between the reduced dehydrogenases and oxidized cytochrome. For these two enzymes we may perhaps write: H,— v ^ FADX X EADX.su- ETF Y eytoehromee T FADX '*^ /Xs ^redo»d-^ /N,s ~' oxidized -*^^*-11,0 protein component of dehydrogenate protein component of electron- transferring JtamproUin COUPLED DEHYDROGENASE 8YSTEM8 This scheme still leaves the copper content of the bufcyryl-Co a enzyme unexplained. like the rest this must be regarded as only a tentative scheme. Much still remains to be discovered and yet more new enzymes and hydrogen- or electron-carriers may be concerned. More- over, we still have a great deal to learn about the manner in which all the catalysts involved are arranged at their sites of action. REVERSIBILITY AND COUPLING OF DEHYDROGENASE SYSTEMS If we consider the complete system involved in the oxidation of lactio acid in animal tissues it is clear that this oxidation is accomplished through the repeated reduction and reoxidation of a chain or series of carrier substances. A single molecule of DPN, for example, might be reduced and reoxidized, say, a thou- sand times, and thus contribute to the oxidation of a thousand molecules of lactio acid. The oxidation and reduction of these carriers takes place with great rapidity under biological con- ditions, and Table 13 presents data for the ‘turn-over numbers’ of some oxidation catalysts, i.e. the number of times they can he reduced and reoxidized under biological conditions in 1 min. at the temperatures stated. It is precisely because their turn -over numbers are so great that very small quantities of DPN and TPN, for example, can catalyse very large amounts of chemical change. They are, in fact, catalysts, just as truly as are the enzymes with which they collaborate. Table 13. Turn-over numbers of some enzyme systems Temp. Enzyme “ C. Catalase 0 Cytochrome c 38 Cytochrome reductase 25 Amino-acid oxidase 33 Polyphenol oxidase 20 Alcohol dehydrogenase (DPN) 20 Triosephoephato dehydrogenase (DPN) 20 Carboxylase (thiamine diphosphate) SO 197 Mol. substrate transformed per mol. enzyme per min. (approx.) 25x10* 1-4x10* 4x10* 2x10* 7x10* 2x10* 2x10* lx]0* COTJEEEn »EnY»R00ESA3E SYSTEMS It is to bo anticipated that if the supply of oxygen to the lactic acid BJ’S tern 13 cut off, tho oxidation of lactic acid will cease almost immediately, since tho amount of DPN available to act as hydrogen acceptor is relatively very small. This is in fact true as far as the isolated or reconstructed system is concerned. Yet many cells and tissues, including even mammalian muBcle, are capable of functioning in complete absence of oxygen. It is known that metabolic oxidations can still go on in many kinds of cella under anaerobic conditions, and in this section wo shall outline tho mechanisms whereby these aro accomplished. It is clear that, even under anaerobic conditions, the reduced coenzymcs must bo reoxidized in some way since, so far as we know, they aro tho only biological substances that can act as hydrogen acceptors for the coenzyme-spccific dehydrogenase systems. As long ago as 1924 it was shown by Quastcl & Whethara that the lactic and succinic dehydrogenase systems of bacterial cells can bo coupled together in intact cells maintained under anaerobic conditions. Lactic acid is oxidized to pyruvic at the expense of the reduction of fumaric to succinic acid, thus: CH.COOH cfl.cutonjcooH-. cn.coou A. ch.cooit cn.cocoou^r CHjCOOH At least two dehydrogenases aro involved, tho lactic enzyme, which catalyses tho dehydrogenation of lactic acid, and the succinic enzyme, which catalyses the reduction of fumaric to succinic acid, acting in this case ’in reverso’. Neither of these requires any known cocnzyme. Investigation of the mechanisms of coupled oxidation-reduction processes of this kind showed that in extracts, as opposed to intact bacterial cells, the coupling only takes place in tho presence of a reversibly oxidizablc and reducible compound such as methylene blue. Other substances, including cytochrome e, were also tested, but the only naturally occurring compound found to replace methylene blue os on intermediate hydrogen-carrier was pyocyanine, itaelfa reversibly oxidizabte and reducible bacterial pigment. In the presence of 108 COUPLED DEHYDROOEXASE SYSTEMS methylene blue or pyocyanine it was possible to demonstrate separately the reduction of the dye by lactate in the presence of lac tic dehydrogenase, and its subsequent reoxidation by fumarate in the presence of succinio dehydrogenase. When the complete system of reactants and catalysts is taken together the condi- tions are such that a four-point equilibrium is finally attained, and can be modified in accordance with the usual mass-law principles. Thus we may write: lactic succinic dthydrogenas* dehydrogenase The natural intracellular carrier here is still unidentified. Later work showed that DPN and TPN can act as inter- mediary carriers between pairs of dehydrogenases, provided that both enzyme s are specific for the same coenzyme. A dehydrogenase that is specific for DPN cannot be coupled to one that normally co-operates with TPN. Many coupled pairs of coenzyme-linked dohydrogenases have now been investigated, and the trioso- phosphat© and lactic systems of muscle, the triosephosphate and alcohol systems of yeast, and the triosephosphate and a-glyccro- pho3phate systems, also of yeast, are three of the many pairs that can be linked together through DPN. The L-glutamic and glucose-6-phosphate systems of yeast can be similarly coupled together through TPN. Reactions of this kind are frequently hut inaccurately de- scribed as ‘dismutations’ and compared with the well-known Cannizzaro reaction. A truo dismutation involves two molecules of one and the same substance, one of which is oxidized at the expense of the reduction of the second. Where such a true dismutation is catalysed by a single enzyme the latter is some- times referred to as a mutase, and is a single entity rather than 199 COUPLED DEIIYDBOGENASE SYSTEMS o pair of cocnzyme-linked dehydrogenases. The term ‘mutase* is unfortunate since it can lead to confusion ; certain iso ra erases (pp. 137-0) are also known as mutasea though they catalyse very different kinds of chemical change. The part played by the coenzyme in a coupled pair can be very elegantly demonstrated by taking advantage of the absorption band at 3-100 A. which is shown by the reduced, but not by the oxidized, form of the coenzyme (Fig. 21, p. 178). Let ns con- sider the coupling between triosephosphato dehydrogenase (sco p. 187 for mechanism of action of this enzyme) and alcohol dehydrogenase: CH.O® IrtaxtphoajihaU alcoW dO\ijdroqcna*e dehydrogenase (direct) (in reverse) If triosephosphnte is token together with the oxidized form of DPN in tho presence of phosphate, no band is observable at 3400 A. When triosephosphato dehydrogenase is added tho co- enzyme becomes reduced, and the progress of the reaction can bo followed by measurements of the intensity of tho bond (Fig. 22). Tho coenzyrao is not completely reduced because tho reaction does not proceed to completion. The reaction mixture is now boiled to destroy the enzyme and then filtered. The intensity of tho band remains unchanged, and is unaffected by the addition of acetaldehyde. If alcohol dehydrogenase is now added the second phase of the process can bo observed; tho cocnzyroo is reoxidized and the band fades. It would be difficult to exaggerate the importance of reactions of this kind and, as wo shall see later, they are a very frequent feature of metabolism under anaerobic conditions. Whethcrthey 200 COUPLED DEHYDROGENASE SYSTEMS take place when conditions are aerobic is difficult to say with certainty. They are still possible under aerobic conditions, but the rate of reoxidation of a reduced coenzymo by the next member in the oxidative chain is ordinarily so great that these dismu- tation-like reactions are probably suppressed, if not abolished altogether. dehydrogenase added at first arrow, alcohol dehydrogenase at second; for rest of explanation see text. Ordinate: relative intensity of band at 3400 A. Abscissa: time. (Modified after Schlenk, 1042.) We have so far been at pains to think of these as essentially reversible Bystems which tend towards equilibrium. This they do in isolated systems, but when, as happens under biological conditions, one or other of the reactants undergoes some further change as fast as it is formed, the system as a whole goes in one direction only, and we shall see many examples of tliis kind in later chapters. INTRACELLULAR ORGANIZATION OP RESPIRATORY ENZYMES By grinding muscle, liver or other tissue sufficiently finely in the cold, one obtains a homogenous preparation which will, given suitable conditions, carry out most of the metabolic activities of the original tissue. Such a preparation is known as a 201 mitochondriai, organization homogenate. If now the homogenate is submitted to fractional centrifugation, again in the cold (see p. 224 for further details) the original whole cells can bo separated into a number of different fractions, e.g. cell nuclei, mitochondria, microsomea and ‘cytoplasm’. Much can accordingly bo learned about the intracellular localization of different enzymes by studying tho enzymic activities of the various fractions. The supernatant ‘cytoplasm’ remaining at the end of the fractionation procedure contains all the enzymes required for the (anaerobic) breakdown of glucose or glycogen to pyruvate or lactate, but is totally unable to oxidize these products. Indeed, this supernatant does not respire at all, because virtually all of the oxidative machinery of the cell is concen- trated in its mitochondria. A fairly typical mitochondrion is, cytologically speaking, a rod-like or spiral object about 3 fi long and some 0-6 ji in diameter and stains well with Janus Green. Metabolically speaking, however, mitochondrial suspensions prepared from different sources differ somewhat in their metabolic capabilities, but generally speaking they can catalyse tho complete oxidation of lactate and pyruvate, tho complete oxidation of fatty acids, and the capture of a large proportion of the freo energy to which they gain access in the coutso of these katabolio processes. Many synthetio operations too can be ncliieved. In recent years the interests of many cnzymologists have swung rather sharply away from individual enzymes and their individual properties to those of organized systems of enzymes; to intact mitochondria by the systematic degradation of which much is being learned about the internal enzymio organization of the mitochondria themselves. It appears, for instance, that what we may call tho terminal hydrogen- or electron -transporting systems, which wo have hitherto considered rather os a mixture than as an organized system of enzymes, ate present in close juxtaposition to each other and to the systems that feed in hydrogen atoms or elec- trons arising from their respective substrates. By breaking down intact mitochondria in a variety of ways ono might therefore hopo to be able to develop as it were an 'enzyme map * of tho internal structure of the mitochondrion. Green, working 202 MITOCHONDRIAL ORGANIZATION on mitochondria prepared from beef heart muscle, has been able to separate what he calls electron-transporting particles (ETP). These possess the power to oxidize reduced DPN and succinate very rapidly indeed and to use molecular oxygen as the terminal acceptor. No co-factors, no cytochrome and nothing but the appropriate substrate need be added. It may therefore be concluded that all the necessary enzymes, coenzymes, prosthetic groups and activators are present, bound together in some way in the structure of these particles and therefore, by inference, in the mitochondria themselves. ETP particles contain flavin, and for each molecule of flavin there are also present 5-C red and green haems, 30 atoms of non-haem iron and G atoms of copper. Further degradation of ETP by aqueous butanol, followed by differential centrifugation, gives rise to two further particulate fractions, one green in colour and containing all the copper, and the other red (see Fig. 23). The green particles have powerful cytochrome oxidase activity and contain, in addition to copper, a haem the spectral absorption bands of which correspond closely with those of cytochromes a and a^. The red particles have high succinic dehydrogenase activity and considerable dehydrogenase activity towards reduced DPN, but molecular oxygen can no longer be utilized : cytochromo c must now be added, and is reduced. Further fractionation of the red particles yields two further red fractions, one of wliich is rich in lipid material and has considerable activity with respect to reduced DPN. Tho other fraction is poor in lipids and oxidizes reduced DPN only slowly but has intense succinic dehydrogenase activity. The succinic dehydrogenase fraction contains flavin, a good deal of non-haem iron, and a red haem with absorption bands corresponding to those of cytochrome b. Further splitting with alkali at pH 11 dissolves out an amber-brown substance which carries all the succinic dehydrogenase activity with it, leaving behind an inactive particulate residue which contains the haem and some of the non-haem iron. The succinic dehydrogenase contains jlavin and some non-haem iron but no haem, and can catalyse the dehydrogenation of succinate if cytochrome c is added os hydrogen acceptor; the ability to use molecular 203 MtTOOnONDBI^L ORQAWIZA.TIOK oxygen was lost much further bach in the fractionation, it will be remembered. Finally, the succinic enzyme can be still further degraded and separated into a protein component end the flavin, and when 'ELECTRON-TRANSPORTING PARTICLES' (flavin; red and green tie ms ; non-haem Fe; Cu) GREEN PARTICLES RED PARTICLES (copper; haem) (flavin; red haem; non-haem Fc) Cytochrome oiutue Succinic and DPJJII, iehydrogm mcs RED PARTICLES RED PARTICLES (LIPID-POOR) (L1PID-IUCH) (flavin; red haem; non- haem Fe) DPNH,-«kAy is excreted without further change. Since the only apparent intermediary we shall detect in such a case is B 2 , we may be misled into believing that the whole of A is normally transformed into B 2 . It is necessary that these various possi- bilities should be kept in min d in the interpretation of results obtained in feeding or injection experiments : provided they are realized and that due allowance is made for them, valuable information can usually be obtained. Experiments of this kind havo done yeoman service in the past and will doubtless continue to do so in the future. A further method in which intact, normal animals aro used involves the chemical alteration of the substance A in such a manner that it and its products can more easily be detected and recognized. Thus Knoop, in his classical experiments on the metabolism of fatty acids, introduced a phenyl radical into the terminal position of the fatty chain and was able then to find aromatic derivatives in the urine of animals to which these w-phenylated fatty acids had been administered; w-phenyl valeric acid, for example, gave rise to hippuric acid when given to dogs : c,n, .CH.CH.cn.GH.coon — * c,n,.co— hn.ch.cooh. It was already known that the administration of benzoic acid to dogs gives rise to the appearance of hippuric acid in the urino, and Knoop was therefore able to conclude that phenyl valeric acid is converted into benzoic acid by the animal’s tissues. This method is open to a number of serious objections. First, we cannot assume that if we modify the starting material we shall not alter its fate in the organism, nor, secondly,' can we assume that by feeding abnormal material we shall not induce some completely new aeries of reactions which, in the ordinary way, play no important part in metabolism. Valuable information has nevertheless been gained in the past from work of this kind, and the method has its present-day counterpart in the use of isotopes, such as heavy hydrogen, heavy nitrogen, radioactive carbon, phosphorus, sulphur and so on, a3 * tracers These isotopes are not chemically distinguishable from the normal elements, and it may reasonably be supposed 216 USE OF ABNORMAL ORGANISMS therefore that their metabolism Trill follow normal lines. Tho isotope method is used extensively at the present time and is an enormous advance on substitution methods of the kind used by Knoop and others among the earlier workers. Many examples of tho use of isotopes will be found in these pages. STUDIES ON ABNORMAL OROANJ8MS Organisms that are intact but suffering from some pathological derangement of motabolism offer valuable experimental material for 6omo purposes. Certain very special metabolic abnormalities occur spontaneously, though rarely for tho most part. Albinos, for example, are devoid of tho enzyme tyrosinase, and may be used in Btudies of certain aspects of tho metabolism of tho aromatic amino-acids. The metabolism of tyrosine goes astray in a number of other curious genetic freaks, notably in alcapto- nuria, a disorder in which the urine becomes dark brown or black when allowed to stand exposed to tho air. The blackening is duo to tho presence of a diphenol, homogcntisic acid, wluch arises from tho aromatic araino-acids. When tho urine is allowed to stand, bacterial invasion takes place and ammonia is formed from urea by tho invading organisms. Like many other diphcnola, homogentisic acid undergoes spontaneous oxidation in alkaline solution to yield dark.colourcd products. Cases of alcaptonuria were studied in an early attempt to decido whether amino-acids undergo oxidative or hydrolytic deamina- tion. Homogentisic acid is no longer excreted if aromatic amino- acids are excluded from the diet. It is reasonable therefore to suppose that any substance which lies on the route between tyrosine and homogentisic acid will, if administered to an alcap- tonuric deprived of aromatic amino-acids, give rise to a renewed excretion of homogentisic acid. Now if tyrosine were hydro- lytically deaminated tho deamination product would bo p-hydroxyphenyl -lactic: if oxidatively, tho fust product would bo p-liydroxyphenylpyruvic acid. Both these substances were accordingly prepared and separately administered to human patients suffering from alcaptonuria. It was then found that whercasp-hydroxj'phenylpyruvicacid was almost quantitatively converted into homogentiaio acid, little or none was formed 216 USE OF ABNORMAL ORGANISMS from the corresponding hydroxy-acid. The relationships of these substances are as follows: It was concluded that the deamination of tyrosine, and by inference that of other amino-acids, is an oxidative rather than a hydrolytio process. The danger-points in this argument are, first, the supposition that because p-hydroxyphenylpyruvic acid yields homogentisic acid it necessarily lies on the pathway tyrosine -* homogentisic acid: it might equally well form homogentisic acid by somo independent and possibly abnormal route. Secondly, even if we discount the first objection and take it as established that tyro- sine is in fact deaminated with production of the corresponding keto-acid, it is exceedingly dangerous to assume that amino -acids other than tyrosine also undergo oxidative deamination, if only because, in alcaptonuria, the metabolism of tyrosine itself is seriously deranged. Particularly important among the pathological conditions of which advantage has been taken is the state of diabetes. Spon- taneous diabetes, diabetes induced by surgical removal of the pancreas or by injections of alloxan or of the diabetogenic hor- moun of the anterior pituitary, and the pseudo-diabetes, induced by injection of the drug phlorrhizin, have all been put to service, especially in studies of the metabolism of fats and carbohydrates. These animal preparations have two important features in common. First, carbohydrate metabolism is profoundly de- ranged and glucose, instead of being stored in the liver in the form of glycogen, is eliminated in the urine. Secondly, there is a large-scale excretion of the so-called acetone or ketone bodies. 217 UBE OV iBSOEHiL OBQAN1SM3 These compounds, acetoacetic and /?-hydroxybutyric acids, to- gether with acetone, arc formed from fatty sources. If a diabetic or phlorrhizinizcd animai is maintained on a constant diet, a steady rate of excretion of glucose and acetone bodies can bo established. If now substances such as alanine, lactic acid, glycerol and the like are administered, an increased output of glucose ensues, indicating that these substances give rise to or replace carbohydrate in the organism. Other compounds, such as butyric and acetic acids, together with the amino-acid leucine, increase the excretion of acetone bodies. The diabetic or phlor- rliizinized animal is thus useful as a device which allows us to detect the formation of carbohydrate and fatty materials from Bubstances of other kinds. Particularly important among the surgical preparations is the hepatcctomized animal. Total removal of the liver is a difficult operation, and the subjects do not survivo for more than a few days. An alternative procedure is to establish an Eck’s fistula, i.o. to by-pass the liver by leading the portal blood directly into the inferior vena cava. The liver plays a leading part in many metabofic processes, and in its absence these are thrown out of gear or even stopped altogether. Intermediary products tend to pile up and commonly appear in tho urine. Mention has already been mado of ono such case: ammonia produced by tho de- amination of amino-acids is normally converted into urea by mammalian liver, and into uric acid by tho liver of birds, but these synthetic operations cease with removal of the liver or establishment of an Eck’s fistula, amino-acids and ammonia accumulating instead. This tells us that urea and uric acid are formed from ammonia and that their synthesis takes place in tho liver, but gives no indication of tho mode of synthesis. It does, however, Bervo to show what particular organ wo must study in order to elucidate the rest of tho story. Tho hepatcctomized animal is of particular value on account of tho central metabolic role of tho liver, but pancreatcctomized, adrenalectomizcd, hypopliysectomizcd, thyroidectomized and other preparations have been much employed, esjwcially in attempts to discover the parts played by hormones in the regula- tion and control of metabolic processes. In all such cases the preparation is abnormal in certain known respects, but it is 218 USB OP PEEPUSBD OBOAK8 necessary to realize that processes other than those which we know to be deranged may also be thrown out of gear. Con- firmation of results obtained by one method should therefore always be sought, and usually is sought, with the aid of other methods and different preparations. STUDIES ON PERFUSED ORGANS It is often possible to study the metabolic activities of a par- ticular organ by providing it artificially with an independent circulation. The organ to be perfused may either be left in situ in the animal, or may bo removed and kept under conditions that approximate as closely as possible to those which it enjoys under normal physiological conditions. The circulating medium may be the animal’s own blood, or blood drawn from another individual of the same species; alternatively, it is possible to use certain physiological salines which we shall discuss presently. The necessary head of pressure may be obtained by means of mechanically operated pumps arranged to imitate the action of the heart, and many types of ‘artificial lungs ’ have been devised for oxygenation of the medium. The method of artificial perfusion is open to a number of serious objections. It takes time to establish the artificial cir- culation, and the animal must of course bo anaesthetized during the operation. This means that, for the first few minutes, the organ is liable to be influenced by temporary deprivation of oxygen and by the anaesthetic. The choice of anacsthetio has therefore to receive careful consideration. How much damage may be done by the temporary shortage of oxygen is not easy to ascertain but, given speed and skill, damage from this cause can bo minimized. If blood is used for the perfusion it is neces- sary to add an anti-coagulant such as heparin, and the possible action of this upon the organ lias to be reckoned with. Care must be taken to ensure that the perfusion medium is kept at the right pressure, temperature, pH and so on, and that it shall be kept well oxygenated, but all these are largely technical matters which can bo dealt with, given experience and skill on the part of the operator. There are, however, other objections that cannot so easily bo 219 USE Or EERI-USED OBOAUS countered. As long as the organ enjoys its normal blood supply it ia exposed to nervous and hormonal influences which cannot bo exactly reproduced outside the animal. Thus the liver, n favourite object of study by the perfusion method, plays a great part in the metabolism of carbohydrates, and this, as we know, is profoundly affected by a number of hormones, notably by insulin, adrenaline and certain pituitary and adrenocortical hormones. When the liver is removed from the body tho influence of these is with- drawn, and tliis may be expected to result in abnormal metabolio behaviour. Thus, from the moment at which the normal circula- tion is replaced by tho experimental perfusion system, tho organ is exposed to conditions which aro abnormal, and probably be- come progressively more abnormal as time goes on. It is always difficult to be certain that the whole of tho organ is actually bein g perfused, so that parts may bo moribund or dead long before tho whole. Nevertheless, it does seem that, for tho first hour or two, a skilfully manipulated preparation behaves in a manner which approximates fairly closely to normal, and results obtained with such preparations commonly find confirmation by other techniques. The general procedure, following the successful establishment of the artificial circulation, consists in adding substances of which tho metabolism is to be studied to the circulating medium, samples of which are withdrawn from time to time for analysis. Perfusion experiments may be done with liver, muscle, heart, kidney and so on. Largo animals are usually preferred since largo Biro facilitates the operative procedure, though it makes considerable demands upon laboratory accommodation at tho same time. As well-known examples of the successful employment of this method we may refer once more to tho classical observation that isolated, perfused dog liver synthesizes urea from added am- monia, gooso liver producing uric acid by contrast; and, in addition, to tho work of Embden, Friedmann and others on tho formation of ketone bodies from fatty- acids. 220 USE OF PHYSIOLOGICAL SALINES USE OF PHYSIOLOGICAL SALINES Perhaps the most Important single contribution ever made to physiology and biochemistry was the discovery in the early 1 880’s by Sidney Ringer that simple solutions of the chlorides of sodium, potassium and calcium can maintain the action of the perfused hearts of frog3 and tortoises. Subsequent work has shown that with the aid of sliglily more complex media the heart-beat of warm-blooded animals also can be maintained for many hours or oven days. There is reason to think that, of all the multifarious constituents of mammalian blood, many are specialized features of secondary importance, tho ionic constituents alone being abso- lutely fundamental and essential. Given a supply of well-oxy- genated physiological saline at the appropriate temperature, pH and osmotic pressure, it seems that tho fundamental physiological requirements even of mammalian tissues can be fulfilled. Table 1C. Composition op mammalian blood sebum AND KBEBS'S PHYSIOLOGICAL SALINE Na+ K* Ca + * cr ror sor ncor CO, (at 38’ C.) PH Mammalian Borura (average#) c. 320 22 10 370 10 11 54 vol. % 2-5 vol. % 7-4 Krebs's physiological saline 11*4 54 vol. % 2 5 voL % All concentrations are in mg. per 100 ml., excepting bicarbonate and carbon dioxide, which are expressed as vol. CO, per 100 ml. Glucose (0 2%) is also added before use. Numerous salines have been introduced for specific purposes. They can be used, for example, to replace blood in perfusion experiments, and have been used clinically on a large scale in the past to make up the blood volume after severe haemorrhage. Solutions containing the clilorides of sodium, potassium, calcium and magnesium, together with small amounts of phosphate, are suitable for many purposes, and are best buffered with bicarb- onate and carbon dioxide. Glucose is often added to provide 221 use or tissue slices ‘ food* for the tissues, Tbo ionic composition of one such saline is given, side by side with that of the mammalian blood it ia designed to imitate, in Table 15; this particular medium has been used extensively by Krebs and others in work involving the use of tissue slices. USE OF TISSUE SLICES In the last 20 or 25 years the somewhat messy method of perfusion, which makes considerable demands upon the surgical Bkill of the experimenter, has been practically abandoned in favour of the use of tissues in the form of tliin slices. Provided certain conditions ore fulfilled, these slices will survivo for some hours, apparently in a manner that approximates closely to the physiological, and are simple to prepare and manipulate. The size of the average cell is such that, although many cells are inevitably damaged when tlio tissue is sliced, the proportion of damaged to undamaged cells is very small, wliile tho debris of those that are damaged can bo removed fairly completely by washing Provided that tho organ to bo used is removed, sliced and washed rapidly, wo can obtain small fragments of virtually normal tissue. Their removal from the normal blood supply of coureo implies that they are removed also from tho infiuonco of tbo animal as a whole, just os ia tho case in perfusion ex- periments, but whereas an unknown and often a considerable proportion of the cells in a perfused organ is probably in a poor if not actually moribund condition, washed tissuo slices contain relatively few cells that are appreciably injured. Certain conditions must be fulfilled in tho preparation and uso of these tissue Bliccs. It is usually convenient to have fragments one or two centimetres square. Their fcliickneas must bo such that the cells in tho middle of the slices, winch can acquire oxygen only by inward diffusion from tho medium, do not suffer from lack of oxygen. Usually, therefore, tho medium is kept in equili- brium with an atmosphere containing 2-5-5 % of carbon dioxide for buffering purposes, and 07-5-95% oxygen. If such a gas mixture is used the slices must not bo more than 0-3 mm. thick. Satisfactory slices can fairly easily bo cut free-band with a sharp razor. 222 USE OF BBEIS: HOMOGENATES Tho usual procedure is as follows. Suitable vessels are filled with the appropriate saline, through which the gas mixture is bubbled, the whole being gently shaken in a thermostatic bath at body temperature. Othersamples of the medium are prepared for washing the slices. The animal is killed, by a blow on the neck for example, and the organ required is rapidly removed and placed on clean blotting-paper moistened with warm saline. Slices are rapidly cut and placed at once in the warm, oxygenated Baline until enough have been accumulated. They are next washed two or three times and then transferred to the main vessels. The substances to bo studied are added and tbe whole apparatus is shaken for a suitable period, during or after which samples of the medium are withdrawn, deproteinized if necessary, and analysed. Tho method is open to several of the criticisms that apply to the perfusion technique, though others are obviated. No anaes- thetic is necessary, and a small animal such as a rat or a guinea- pig will supply enough material for a number of experiments. It is possible that the cells may behave abnormally as a result of their exposure to the high partial pressures of oxygen required to ensure adequate oxygenation of the deeper layers of cells. The method has, however, found wide favour. On account of the small size of tho tissue fragments, the method is very suitable for the application of manometrio methods which, as is well known, can be used for a very wide range of measurements and estimations on the micro- or Bemimicro-scalo. USE OF BREIS, HOMOOENATES, EXTRACTS, ETC. Tho analysis of a complex series of metabolic events into its component reactions usually provides evidence for tho participa- tion of a number of enzymes and accessory catalysts, and for a complete analysis the discovery and identification of the function of each of these is required. To obtain this information it is necessary to separate tho enzymes one from another, to destroy some enzymes and preserve others, or in some other way so to disrupt the cellular organization that intermediate products can he discovered. Sometimes thi3 can bo done by tbe use of specific inhibitors known to inactivate particular enzymes ; in other cases ‘trapping’ reagents can be employed to fix homogenates: mitoghondkia particular Intermediates. More usually it 13 necessary to extract the enzymes, though it sometimes suffices to minco or grind the tissue. The resulting miners and ‘6 rets' contain all the enzymes of the original material, but the normal spatial relationships between them are destroyed by disruption of the cellular architecture. In recent years tho use of homogenates has found wide favour. These are prepared by grinding tho chopped tissue very finely indeed in a mill consisting of an outer tube and an inner, closely fitting, mechanically driven pestle. Tho grinding surfaces are roughened by previous grinding with carborundum powder. Complete disintegration of the cells can bo easily and rapidly achieved with a good homogenizer. All the operations are normally carried out at a low temperature and water, buffer, 0-25 m sucrose or isotonic saline are used as suspension media. Tho homogenates contain all tho enzymes of tho original tissue, and by dilution, which lowers tho concentrations of all tho substrates, coenzymcs and other co-factors, tho metabolism of the homogenates can bo reduced to a very low lovel. If cyto- chrome c and AMP, ADP or ATP are then added, tho homo- genate will oxidize added substrates. Intermediate products can frequently be caused to accumulate by working in the presence of specific inhibitors. As early os 1013 Warburg observed that the oxidative pro- cesses going on in cell-free extracts arc virtually confined to the larger granules liberated from tho cells. Now tho nuclei, mitochondria and other particulate ccll-constitutcnts which aro present intact in homogenates can bo separated one from another by differential centrifugation. A usual procedure is a3 follows, nil tho operations being conducted at 0-2° C. A 10% homogenate of tho tissue is prepared using 0-25 m sucrose as tho suspension medium. The product is centrifuged at about 000 g for U) min.; this removes cell debris and cell nuclei. Tho 8iij>ematant is centrifuged for a further 10 min. at 8000 g to sediment tho mitochondria, which are resuspended, usually at 20% on the basis of tho weight of the original tissue, and put through tho fractionation procedure once again. The uso of Bucroso Bolutions is designed to prevent osmotic swelling and bursting on the part of tho mitochondria, 224 MITOOnONDRIA: ACETONE POWDER8 After removal of the mitochondria the microsomes can be sedimented by centrifuging for a further 30 min. at 20,000- 50,000 g leaving the cytoplasmic enzymes in the supernatant solution. Suspensions of cell nuclei too can be obtained but their separation from general cell debris presents a number of special problems. Methods are also available for preparing suspensions of other particulate cell inclusions, e.g. melanin, nucleoli and Golgi apparatus. Washed suspensions of mitochondria have been much used in studies of oxidative metabolism since they contain most of the enzymes involved in cellular respiration and require only to be fortified with cytochrome c, ATP, ADP or AMP and the neces- sary co enzymes, usually Mg'*”*' and inorganic phosphate. DPN and TPN are firmly bound in the mitochondria and are not removed by washing. The addition of ADP has been used in studies of oxidative phosphorylation, i.e. the synthesis of ATP wlxich is associated with oxidative metabolism. The presence of ATP’ases, which split ATP as fast as it is formed, led to many failures in early experiments but these enzymes are strongly inhibited though not, unfortunately, totally inactivated, by fluoride. A great step forward was made with the introduction of a simple method of purifying yeast hexoldnaso. If the latter is added together with glucose, only traces of ATP are necessary to phosphorylate the glucose, the residual ADP being rephosphorylated by the mitochondria and so on. The total synthesis of ATP can then bo determined in terms of gIucose-6-phosphate formed. Many tissue enzymes can be extracted with water or saline, freed from the general cell debris by filtration or centrifugation, and later purified. Of the soluble enzymes some tolerate pre- cipitation with acetone at or below 0° C. and enzymes of this kind can be extracted with aqueous media, precipitated by means of acetone, and then extracted again with water or saline from the resulting 'acetone powders’. A fine example of the usefulness of whole, cell-free extracts is found in the case of yeast juice, which is prepared by macerating the cells with sand and squeezing the mass in a suitable press. Many of the enzymes extracted in this way require coenzymes, which can he removed by dialysis, and much of our present .5 225 PCIUFIED ENZYMES knowledge of fermentation has been gained by the use of dialysed yeast juice, often with supplementary tools in the form of sdttlivt inhibitors . Similarly, the enzymes involved in glyco- lysis can bo obtained in solution by aqueous extraction of minced muscle, for example. In the end it is often necessary to have recourse to purified enzymes. Finely divided tissue is allowed to stand with icc-cold water or isotonic potassium chloride, for example. After centri- fugation of the extract to remove the insoluble cell debris it is possible to purify many enzymes by fractional precipitation, fractional adsorption, and other more specialized procedures, go that highly concentrated preparations are obtained. By further rigorous purification, the details of which vary according to the nature of the enzyme, crystalline preparations are obtainable in many cases. It is well to remember, however, that proteins in general — and enzymes arc no exception to the rule — are very prone to tho formation of mixed crystals while still far from being chemically pure. Many important enzymes, e.g. cyto- chrome oxidase and succinic dehydrogenase, are insoluble how- ever. Until recently they were usually studied in thoroughly washed and suitably fortified suspensions of finely minced or homogenized tissue. In more recent times a number of insoluble, particulate enzymes have been isolated by a systematic degrada- tion of washed mitochondria, and a broad description of tho separation of cytochrome oxidase, reduced DPN-cytochroroc reductase and succinic dehydrogenase from mitochondria will be found on p. 204. Pctailcd knowledge of the processes catalysed by singlo en- zymes often helps us to analyso into several stages a process which seems at first sight to be a single metabolic operation, and information gained by the study of ‘built-up’ or reconstructed systems, comprising several enzymes and their appropriate ac- cessory catalysts, may give valuable indications of tho manner in which tho individual stages are organized in tho metabolic wholes. We shall come across numerous examples of this kind, but for the moment the reader may be reminded of the use of reconstructed systems in tho study of tho dehydrogenases, and Green’s warning apropos of these reconstructions may also be recalled (p. 100). 228 USE or ISOTOPES USE or ISOTOPES In biochemical as in many other kinds of research the now ready, availability of isotopes has been of immense value. Isotopic elements differ not at all in their chemical properties but are detectable by differences in their physical properties. For example, deuterium (heavy hydrogen, 2 H) is twice as heavy as ‘ordinary’ hydrogen, since, while the nucleus of the latter contains only 1 proton the heavy isotope contains 1 proton and I neutron and so has twice the atomic mass. Similarly, while normal carbon, lI C, has 0 nuclear protons and G neutrons, there also exist isotopic carbons U C and 14 C, the nuclei of which contain 1 and 2 additional neutrons respectively. The nuclei of these two isotopes are unstable and consequently radioactive. Heavy hydrogen can be detected by burning it, collecting the water formed and measuring its physical characteristics, since ‘ordinary* water and heavy water differ in a number of ways; e.g.: ‘Ordinary’ ’Heavy* water water Freezing point (“C.J 0 3 82 Doilmg point ( # C.) 100 10142 Specific gravity 1-000 1-1074 By accurate measurements of one or other of these properties it is possible to determine the amount of heavy water present in a mixture with ordinary water. The radioactive carbons and many other radioactive isotopes can readily be detected by the mere fact of their radioactivity, and quantitative determinations can be made by counting the particles they emit. But 12 C, “C and U C all have the same number (G) of orbital electrons, and it is the number of these orbital electrons that determines the chemical properties and therefore the metabolic behaviour of the element and its compounds. Of the stable isotopes perhaps 2 H, 18 0 and 1S N have been most widely used, while among those that are radioactive, l4 C, 15 C, *S and 3J P have perhaps found most employment. Many examples of the use of isotopes will be found in this book, but for sake of example we may take the synthesis of creatine, which can be formulated as shown on p. 22S. 227 15 -a USB OB ISOTOPES Reaction (1) takes place in the kidney and react ion (2) in the liver. If kidney slices are incubated with arginine together with glycine labelled with 15 N, the glycocyamino formed carries heavy nitrogen in the corresponding position. Similarly, argi- nine with 1S N in one of its own amidino nitrogen atoms yields a glycocyamino with heavy nitrogen in tho corresponding posi- tion in its amidino radical. If now glycocyamine is incubated with liver slices and trideuteromethionino, i.e. methionine, the methyl group of which contains 3 deuterium atoms, tho creatine formed has a trideutcro-methyl group. This is but one example. Isatnpia substances have been used in feeding experiments, in experiments involving tissue slices, homogenates, mitochondrial suspensions, purified enzymes and so on. (For page references to other examples seo tho Index.) In addition to their use in metaboho studies isotopes have been extensively used in analytical procedures. Suppose, for 228 USE OB' ISOTOPES example, that wo have a protein hydrolysate and wish to know how much of each of several amino-acids it contains. The quantitative separation and purification of these amino-acids by normal chemical methods is usually tedious and exceedingly difficult, and losses are apt to be high. We can, however, use the method of isotope dilution. An isotopically labelled sample of one of the amino-acids in which wo are interested is prepared and its specifio activity is measured. A known amount is then added to a known amount of the hydrolysate. A specimen of the amino-acid in question is now isolated ; there is no need to isolate the whole. The isolated sample will contain the isotope, and the ratio of its specific activity to that of the sample originally added will be equal to the ratio between the weight of the sample isolated and that of the total amount of the amino-acid in the sample of hydrolysate. Several ingenious variants on this method of isotope dilution have been devised for use in metabolic as well as in analytical procedures. 220 CHAPTER IX FOOD, DIGESTION AND ABSORPTION FOOD Living organisms can bo broadly divided into two groups. Some, lilce the green plants, only require to be provided with simple inorganic materials from which, with the aid of energy drawn from the external world, they can accomplish tho synthesis of everything required for their life, growth and reproduction. Others, like the animals, can only live and reproduce if provided with complex, energy-rich, organic materials, collectively desig- nated as food. These two groups of living organisms are known, as aulotrophea and heterolrophe a respectively. Predominant among autotrophic organisms are the green plants, winch are able to fixate and utilize tho energy of solar radiation. This is brought to bear, in a manner which is only now beginning to be understood, upon the synthesis of complex energy-rich materials ; and, os raw materials for the synthesis, carbon dioxide, water, salts, and some Bimple source of nitrogen such.for instance, as ammonia or nitrate are all that is necessary. The key substance in photosynthesis, chlorophyll, finds counter- parts in the specialized bacterial pigments upon which pholo- synthetic bacteria rely for a comparable fixation of solar energy. In tho remaining group of autotrophic organisms, the chemo- synlhetic bacteria, energy is not obtained from the sun but by harnessing the chemical energy of some inorganic process such as the oxidation of ammonia to nitrite or nitrate, the oxidation of hydrogen sulphide to elemental sulphur, or that of ferrous compounds to the ferric state. The autotrophea are in every case competent to synthesize all the structural, catalytic and storage materials they need for growth, maintenance and reproduction: everything their life requires can be produced from thesimplest of starting materials, the necessary energy’ being collected from the external world. Heterotrophic organisms stand in sharp contrast to the auto- . 230 AOTOTROPJIES AND HETEROTROPHES trophes, for not even the most versatile of heterotrophio forms can live except by exploiting the industry and synthetic in- genuity of other organisms. Only by fermenting, oxidizing, or in some other way degrading complex organic materia! can the heterotrophes obtain the energy required to maintain them- selves. It may thereforo be said that all heterotrophes require ‘food 1 , that is to say, oxidizable or fermentable material by the breakdown of which free energy can be released and harnessed for locomotion, chemical synthesis, and other energy-consuming processes. Many heterotrophio forms of life such, for example, as the free-living bacteria and yeaels , can live and reproduce in very Bimple media. Apart from water, salts and some simple source of nitrogen, they need only to be provided with some fairly simple organic compound such, say, as lactate. Given these substances the free-living bacteria can accomplish de novo the synthesis of everything their life requires. But many micro- organisms are more exacting. The presence of certain particular compounds in the habitual environment of a given species can lead in the end to the loss within that species of the ability to synthesize the substances in question. Thus many milk-souring bacteria, such as are cultivated for the manufacture of cheese, cannot live or multiply except in media containing riboflavin. Tree-living organisms are able to synthesize tins important sub- stance for themselves, but these cultivated milk-sourers have lived for bo many generations in milk, which is a fairly rich Bource of riboflavin, that their ability to synthesize it has been lost for lack of employment. For these organisms, riboflavin has become an indispensable accessory food factor; in other words, a vitamin. The nutritional requirements of many micro-organisms have been carefully investigated in recent years, and there is now abundant evidence that some degree of synthetic disability is a common feature among them. Yeasts which have been carefully nursed and pampered in vineyards and breweries require the provision of a number of tho factors that free-living forms can make for themselves, while among bacteria and protozoa many forms, includingnumerous highly pathogenicstrains and species, have been found to have nutritional requirements that are very 231 NUTRITIONAL REQUIREMENTS exacting indeed. Loss of synthetic ability seems to be a step- wise process, for certain bacteria, given /^-alanine or nicotinic amide, can synthesize pantothenic acid or DPN respectively, but in other cases it is necessary that these more complicated substances should be given intact; even the ability to join together the constitutional fragments has been lost. The work of Beadle, Tatum and their collaborators and followers on mutant strains of the bread-mould, Ncurospora crassa, has revealed many examples of step-by-step loss of synthetic ability. This loss of Synthetic ability has gone even further in certain micro-organisms than it has in animals, for some of them are unable even to synthesize haematic. By contrast with green plants, or even with free-living micro- organisms, animals are very exacting creatures indeed. In addi- tion to water, salts, and ‘food ' in the sense of energy-yielding organic substances, animals of every kind need to be provided with certain amino-acids, and with a number of the other indis- pensable accessory food factors collectively known as vitamins. These include thiamine, riboflavin and nicotinic amide, all three of which are constituents of essential coenzymes. This implies the inability of animals to synthesize many of the ti33ue con- stituents and catalysts which they require. More recently evidence has come forward to point to catalytic roles for most of the B group of vitamins including, in addition to those already mentioned, pyridoxal, biotin and pantothenic acid (see Table 10). Table 16. Catalytic vunctionb op boise members or the B oboup or vitamins Formula Present in See p. Co-car boiylsse; coeniyme of oxidaliva do- 345 earboxylation, transLstoUUon and trsns- aldolation DPN and TPN 351 Prosthetic groups of all flavoproteuw 355 Prosthetic groups of transaminases, decar b- J23 oxyLees, serin o and threonine deaminases Geeeayc ao A 3S& Co-factor involved in fixation of CO, — (bacterial These general notions help greatly in the interpretation of the food relationships that exist between living organisms of dif- ferent kinds. In the words of Charles Elton, ‘Animals are not Compound Thiamine Nicotinamide Riboflavin Pyridoxal NUTRITIONAL REQUIREMENTS always straggling for existence. They spend most of their time doing nothing in particular. But when they do begin, they spend the greater part of their lives eating. The primary driving force of all animals is the necessity of finding the right kind of food and enough of it.’ The ‘right kind of food’ is largely determined, of course, by the animal’s ability to capture and kill other organ- isms, but on the chemical side we can say that the ‘right kind of food ’ is that which provides the eater with the energy require- ments of its kind and, at the same time, with whatever Bpecial materials are essential to the species in consequence of its synthetic disabilities. In any natural animal and plant community we can trace out what are known as food chains. A food chain typically begins with green plants, which are exploited by herbivorous animals and these, in their turn, by carnivores. These become the prey of larger and more powerful carnivores and so on until, in the end, wo arrive at an animal so large and powerful that it has virtually no enemies except, perhaps, that ubiquitous animal, man. Always in these food chains the starting-point is with autotrophic organ- isms. Herbivorous animals rely at first hand, and carnivores at second or third hand, upon the autotrophes for supplies of the numerous essential substances which they require, as well as for a sufficiency of complex, energy-yielding organic foodstuffs. Gathered together in the first instance by herbivorous beasts, these essential materials are passed stage by Btage along the food chains. The same general ideas are also valuable in the interpretation of food relationships of other kinds. Parasitism , for example, presents many problems which a thorough knowledge of the nutritional requirements of parasitic organisms may go far to- wards solving. We have already seen that the requirements of a given organism are liable to be influenced by the availability of particular substances in the environment to which the organism is accustomed, quite apart from the general physico- chemical properties of the habitat. Many micro-organisms are now confined to particular habitats and have, in fact, become absolutely dependent, i.e. parasitic, upon those habitats because they hare lost the ability to synthesize certain substances and can therefore only survive in environments in which those 233 PARASITISM AND SYMBIOSIS substances are to be found. Possibly the same trill prove to bo true of other parasites, such, for instance, os the tape -worms and round-worms that are such a common feature of the intestinal fauna of animals of every kind. Another important type of nutritional association is symbiosis. A cow may harbour largo numbers of parasitio worms in addition to the multitude of symbiotic micro-organisms that inhabit its rumen, b^t tho relationships between the cow and the worms on tho one hand, and between the cow and its symbionts on the other, are very different. The cow acts virtually as a food- collecting machine for both groups of organisms, but gets nothing in return from its parasitic inhabitants. The symbionts, however, repay their host by breaking down cellulose and other cow- indigestible materials, from which they produce short-chain fatty acids which the cow can utilize. Similar arrangements are found in herbivores of many kinds, from cows to cockroaches. But the host member of the pair stands to gain yet further rewards for hospitality rendered. Some at least of tho symbionts can synthesize from very simple materials all the amino-acids and vitamins that they themselves require. These compounds become incorporated in the first instance into the substance of the symbionts, but these organisms are cot immortal. When they die and undergo eventual autolysis or digestion by the host’s enzymes, their essential amino-acids and vitamins become avail- able to the host, at any rate in part. There can be little doubt that some herbivores depend largely upon their intestinal flora and fauna for supplies of essential accessory food materials, though it may be doubted whether supplies from these sources are over sufficient by themselves. Provided that their somewhat exacting nutritional require- ments are fulfilled and that sufficient energy-yielding substances are available, heterotrophic organisms such as the mammals are capable of dismantling their food materials and rearranging the component parts in a very versatile manner. From its food pro- teins, for instance, an animal can build up the species-specific proteins that are characteristic of its own tissues and secretions; carbohydrates and even fate can be produced from the deami- nated residues of superfluous amino-acids; carbohydrates can be converted into fate and so on. Herbivorous animals can lay 234 DIGESTION down both carbohydrate and fat at the expense of the short- chain fatty acids produced by the exertions of their intestinal symbionts. The first steps in this direction consist in the digestion and absorption of the food, and these are followed by storage or metabolism of the ingested materials. In human feeding cookery plays an important part. Cooking denatures the food proteins and bursts the granules of natural starch, thus facilitating their attack by the digestive enzymes. By digestion we mean the hydrolytic breakdown of food materials, which consist pre- eminently of relatively large molecules, into simpler compounds from which a given organism can build up its own tissues and food reserves. This definition is one that can be widely inter- preted, for the food may be the food eaten by an animal, on the one hand, or, on the other,, it may comprise the materials provided in a seed or an egg for the embryonic development of a plant or an animal. A seedling plant is as heterotrophio as any animal until it reaches the daylight and can begin photo- synthetic activities on its own account. The seeds of plants contain considerable reserves of organic foodstuffs from which new plants can develop. At or before the time of germination, enzymes are present which may be said to have digestive functions, since they Berve to dismantle the food materials into simpler components wliich the young plant then oxidizes or rearranges in its own characteristic manner. The seeds of the castor-oil plant, Bicinus, are rich in oils and contain a powerful lipase. Barley, which is rich in starch, contains power- ful exo- and endo-amylases and a maltase at germination. More is known, perhaps, about the enzymes of the jack-bean than of any other 6eed, if only because it has been so much exploited as a BOiirce of urease. A veritable army of enzymes has been described here, for apart from urease itself, this bean contains an amylase, a lipase and a pectinaso, together with peroxidase, catalase and several others. Among animals, digestion may be accomplished in either of two main ways. The food may be phagocytically ingested and then intracellularly digested, or it may undergo extracellular digestion beforo being absorbed. Often both mechanisms are used side by side in one and the same animal. Intracellular 235 DIGESTION digestion is probably more primitive than its extracellular counterpart, for phagocytosis is only possible for particles up to a certain order of size. That extracellular digestion arose as an adaptation to the necessity of breaking up relatively large food masses prior to absorption seems very likely, and it is probably significant in thi3 respect that the peptidases involved in the extracellular digestion of proteins in the mammals are known to be qualitatively and quantitatively homospecific with the intracellular peptidases or kathopsins. In some animals it is possible to observe what seems to be a transitional process that is neither entirely intracellular nor wholly extracellular. In certain platyhelminth worms, for instance, the gut is lined with ceils endowed with considerable amoeboid activity. When food i3 taken, these cells absorb water from it and swell up, sending out processes which form a syncytial network that fill3 the gut cavity and enmeshes the food mass. Digestion takes place within the syncytium, which is later withdrawn. In some cases, how- ever, a syncytium is formed but withdrawn before digestion has proceeded very far, and digestion continues even in its absence. Here it would appear that the function of the syncytium has been discharged once the enzymes it can provide have been liberated. In many organisms, notably among the protozoa and sponges, phagocytosis followed by intracellular digestion is the only mechanism available for assimilation, but in other phyla it is not uncommon to find both the intracellular and extracellular modes of digestion used together. It usually appears in cases such as these that if an animal is carnivorous its extracellular enzymes are those which act upon proteins ; if it is herbivorous the extracellular enzymes are those that act upon carbohydrates. Thus among the coelenterates, which are mainly carnivorous, protcinases are secreted by the walls of the coelenteron while non-protein materials are digested intracellularly. Similarly, the only extracellular digestive enzymes found among lamellibranch molluscs, which are almost exclusively herbivorous, are amylases . The general disintegration of the food mass that results from the action of extracellular enzymes is usually facilitated by mechanical movements of the walls of the digestive cavity, so that the mass ia eventually reduced to a particulate dispersion fine enough to allow of phagocytosis, and digestion is subse- DIGESTION quently completed within the cells. But when we come to animals as complex and as highly specialized as the mammals we find that digestion is entirely extracellular. Indeed, the only remnant of the phagocytic systems which are so important among invertebrates is that found in the wandering scavenger cells of the reticulo-endothelial system. Relatively little is known about the digestive processes of invertebrates, but the very large literature of the subject indi- cates, in a general manner, that animals as a whole are equipped with enzymes competent to break down fats, proteins and carbo- hydrates into their simple constituents. Proteinoses, lipases and carbohydrates have been detected, either in the extracellular digestive juices or in the cells of the digestive glands themselves, in a very largo number of cases. It is not always possible to bo sure that, because a protein-splitting enzyme is demonstrably present in the cells of a digestive gland, it necessarily has a diges- tivo function. Intracellular proteinases have nevertheless been described having pH optima in rather strongly acid or weakly alkaline media, and thus resembling pepsin and trypsin. In such cases it is possible that the enzymes in question are concerned with digestion. However, other proteinases have been extracted which have optimal proteolytio activity in the region of neu- trality, and, in cases like this, it is at least equally likely that their function is akin to that of the kathepsins. The nature of the chemical operations involved in digestion appears to bo substantially the same in all kinds of animals, whether digestion is intra- or extracellular. Most is known about these processes and the enzymes which catalyse them in the mam mala, and the ensuing description of digestion relates mainly to these animals. It is not unusual to think of digestion as a process which is divisible into a series of nicely defined steps, each of which leads to equally nicely defined products. We find in the text-books an abundance of statements to the effect that pepsin digests pro- teins thus far and no farther, that trypsin digests them farther to another definite point, and so on. While it is perfectly true that each enzyme, taken by itself, will carry out certain perfectly definite operations and cease acting when these have been ac- complished, it must be remembered that digestion is not carried 237 DIGESTION out in this manner. Food that has passed into the small intestine of a mammal, for example, is exposed to the simultaneous activities of all the pancreatic and intestinal enzymes, and its digestion is not separable into a series of discrete steps and stages but is, rather, a continuous process. The fact that the digestive secretions of animals have been resolved into a number of indi- vidual catalysts, each of which can be studied separately, has tended somewhat to encourage the step-by-step outlook on digestion as a whole. Perhaps the best way to check this ten- dency is to think of the enzymes involved in digestion, not as a mere collection of catalysts, but rather as an organized system of catalysts, so ordered and regulated as to carry out a long arid complex but nevertheless continuous chain of processes. The products of digestion form the raw materials for the pro- cesses of metabolism, a general term used to coverall the chemical changes going on in the cells and tissues of living organisms. These changes may result in a chemical simplification of the starting material, in which case wo speak of katabolism, or in an increase of chemical complexity, when we speak of anabolism (cf. Chap. m). Katabolic changes are usually associated with the liberation of a larger or smaller part of the free energy of the starting material and are therefore said to bo excrgonic. A larger or smaller part of this energy can bo harnessed by the organism and used for the performance of work of some kind, e.g, in locomotion or chemical synthesis. Anabolic pro- cesses, on the other hand, are usually endergonic, i.e. they are attended by an uptake of free energy. This energy is drawn from concomitant katabolism or, in autotrophes, from the external world. Living organisms of every kind appear to be able to accom- plish anabolism at the expense of katabolism, but the ability to carry out anabolic changes at the expense of external energy is the prerogative of autotrophic organisms. Thus, when we are studying processes of katabolism we should have constantly in mind the question, how much energy becomes available to the organism? We may also ask in what form it becomes available, and how it is converted into chemical, mechanical, electrical, thermal or osmotic work as the case may be. Similarly, when anabolic changes are being considered, we must inquire whence DIGESTION OF PROTEINS and in what form the necessary energy is forthcoming, and how it is transferred from its source to ita intramolecular destination. These are important questions; questions which, moreover, have remained practically unanswered and unanswerable until very recent times, but at last we have a few clear indications on which we may hope to found a new knowledge of biological energetics. Before going on to consider metabolism in detail it is desirable to examino the phenomena of digestion, taking our information mainly from the mammals. This task we shall attempt in the rest of this chapter. DIOESTION AND ABSORPTION OP PROTEINS Saliva contains no proteolytic enzyme, and the first phase of digestion takes placo in the stomach under the influence of pepsin. Pepsin, it will be remembered, is secreted in the form of an enzymatically inactive precursor, pepsinogen. This is activated by the hydrochloric acid of the gastric juice, which provides at the same time an acid medium in which the pH is about optimal for the action of pepsin. The latter, which acts more rapidly upon denatured than upon native proteins, opens up certain particular peptide links in its substrates, hut whether or not it is able to complete its work depends a good deal on the consistency of the gastric contents. As soon as these have become liquid they are forced through tho pyloric sphincter, whether peptic digestion has been completed or not. After its passage through the pylorus the partially digested food mass, or chyme, is mixed with the pancreatic juice and the bile. Taken together, these secretions contain about enough free alkali to neutralize the acid that has come through from the stomach, and the pH of the intestinal contents is brought nearly to neutrality. It was formerly believed that the pH is about 8‘G at this stage, but more recent measurements show that it usually lies between pH 0-5 and 7. Trypsinogen and chymotrypsinogen, activated by entero- krnase and by trypsin respectively, yield trypsin and chymo- trypsin. These enzymes continue the process of hydrolytic disintegration begun by pepsin and open up more, but different, peptide links, to produce peptide fragments much smaller than 23D ABSORPTION OP PROTEINS the original food proteins. Few free amino-acid molecules arc produced at this stage. Carboxypeptidases, contributed by the pancreatic juice, and aminopeptidase3, secreted by tho intestine, take up tho task of degrading the polypeptide fragments inwards from the cnd3, liberating amino-acid molecules one at a time until, when the dipeptide stage is reached, tho substrates pass out of their range of specificity and into that of the dipeptidases of tho intestinal secretions, and these complete the digestion. Even- tually therefore, the amino -acids which enter into the composi- tion of the food proteins aro set free, absorbed into the portal blood stream, and carried away into the general circulation by way of the liver. In the past there has been considerable discussion as to whether protein foodstuffs are, in fact, completely broken down into their constituent amino-acids before being absorbed. Some favoured this view, while others believed that so long os tho protein has been reduced to some soluble form such, for example, as a mix- ture of peptones, the function of the digestive enzymes has been satisfactorily discharged. There is now evidence in plenty to show that the latter view is erroneous. In the first place it is unlikely that peptones are absorbed as such because, if peptones are injected into the blood stream of mammals, a condition known a3 ‘peptone shock’ results, but nothing comparable fol- lows the consumption of a protein meal. Abel, using an ingenious technique known as vividiffusion, showed that amino-acids, but no protein fragments of larger size, can be detected in the blood leaving those regions of the gut from which absorption takes place. This he did by leading off tho emergent blood through a series of collodion tubes immersed in warm physiological saline, and returning it then to the circulation. After this performance he was able to isolate several amino-acids from the saline medium and to detect the presence of a number of others by chemical tests, but no trace of products more complex than the amino- acids could be detected. There is, moreover, a largo increase in the concentration of free amino-acid nitrogen in the blood while absorption is taking place in a normal animal. It may also be argued, though perhaps the argument savours alittle of teleology, that animals do in fact possess a series of enzymes capable of carrying digestion right through to the free amino-acid stage, 240 DIGESTION OP CARBOHYDRATES and that these enzymes would hardly have been perpetuated in the course of evolution unless they were of some use, i.e. survival value, to the organism. Finally, there is the telling fact that certain students have acquired considerable fame for themselves by consenting over considerable periods to the replacement of their dietary protein by mixtures of purified amino-acids without, however, appearing any the worse for the experience. DIGESTION AND ABSORPTION Or CARBOHYDRATES Few animals are equipped with enzymes capable of attacking cellulose, although this polysaccharide plays a very large part in the nutrition of herbivorous animals. In these creatures the task of digesting cellulose is usually delegated to vast hordes of symbiotic micro-organisms (p. 234), and the useful products of their activity consist in the main of short-chain fatty acids. The mechanisms of this degradation are complex, if only because many different kinds of micro-organisms are involved. Like cellulose, the so-called hemicelluloses (xylans, arabans, mannans, galactans, etc.) and fructofuranosans (such as the Jevans of grasses and the inulins of the Jerusalem artichoke and other Compositae) are not digestible by the enzymes of mo3t animals, although they can be handled by symbiotic micro- organisms and probably yield products similar to those formed from cellulose. In the insoluble granular form in which it occurs in nature, starch is very resistant to digestion and one of the functions of cooking is the disruption and partial solubilization of the starch granules. The digestion of starch and glycogen is initiated by Balivary amylase but, unless the eater follows the precept of Mr Gladstone and chews each mouthful of food quite an un- believable number of times, little digestion takes place in the mouth. The food, more or less intimately mixed with saliva, is swallowed and passes on into the stomach. Although the optimal pH for salivary digestion lies very near to neutrality, the secre- tion of the strongly acid gastric juice does not put a sudden end to salivary digestion because it takes time for the acid to penetrate into the food bolus. The consistency of the food mas3 is therefore an important factor. Eventually, however, the free acid of the 16 241 DIGESTIOK OF CARBOHYDRATES gastric contents reduces the pH to a value at which the salivary amylase is inactive and is actually destroyed, hut in the mean- time starch and glycogen alike have been at least partly broken down to yield maltose, together with some maltotriose and, if digestion is not yet complete, some doxtrins. The gastric juice itself contains no carbohydraso, but a notable concentration of free hydrochlorio acid is present and contri- butes something to the digestion of carbohydrates containing fructofuranoso units. Fructofuranoside3 such os sucrose and inulin are hydrolysed with great ease and rapidity by warm, diluto mineral acids, and it is probable, therefore, that substances such as these undergo at any rate a partial hydrolysis during their stay in the stomach. The hydrolytio activities of the hydro- chloric acid are cut short when the chyme passes through the pyloric sphincter and into the duodenum, where it encounters the Btrongly alkaline pancreatic juice and bile. Here the pH rises nearly to neutrality, and under these conditions the amylase of the pancreatic juice has almost its optimal activity. This enzyme finishes the work begun by the salivary amylase, and the conversion of starch and glycogen into maltose is completed. Some free glucose is produced at the same time. Maltose, however, is only a transitory product, for it is rapidly hydrolysed under the influence of an a-glucosidase, tho so-called ‘maltose’ of the intestinal juice. This secretion also contains a powerful gluco3accharase, which completes the hydrolysis of sucrose, and a /?-galactosklase, ‘lactase’, that deals with lactose. Ultimately , therefore, the digestible carbohydrates of the food are resolved into their constituent monosaccharides and in this form they are absorbed from tho gut. It is improbablo that appreciable quantities of di- or higher saccharides are absorbed because, os is known from injection experiments, disaccharides present in the blood Btream are largely excreted unchanged, and it is only in exceptional and probably abnormal cases that disaccharides appear in the urine, though lactosuria is common during preg- nancy and lactation. The rates of absorption of different monosaccharides vary much more widely than might have been expected in view of the fact that all liexoses have the samo molecular weight, while that of the pentoses is not very different. It follows, therefore, that 2 42 ABSORPTION OF 0 ABB OH YD BATES the absorption of sugars from the gut cannot be explained in terms simply of diffusion. The same conclusion follows from the fact that glucose, for example, can be absorbed from very strong solutions, and therefore against large osmotic gradients. Many experiments have been made to discover what mechanisms are involved in the absorption. The rate of absorption can bo determined by opening up an experimental animal, such as a rat, and introducing a known amount of the sugar to be studied into a loop of intestine, pre- viously tied off at both end9. The animal is kept for a known length of time, and the contents of the intestinal loop are removed and analysed. The amount of sugar absorbed is then found by difference. While experiments carried out on these lines show that galactose and glucose are nbsorbed much more rapidly than other sugars, they are open to criticism. There is, in the first place, a definite possibility that direct damage may be done to the gut, and it is also possible that the anaesthetic that must necessarily be used may interfere with the normal processes of absorption. But other methods of investigation are possible and yield substantially the same results. Cori, working on un- anaesthetized rats which he fed by stomach-tube, obtained the results shown in Table 17. Other workers, including Verz&r and his co-workers, obtained substantially the same figures, though rather different ratios have been found in different animal species. Table 17. Absorption op monosaccharides from THE SMALL INTESTINE OF RATS (After Cori) Relative rate Sugar of absorption D-GaUctoee 110 d-GIucoso 100 D-Fnictose 43 B'Mannose 19 l-Xj lose 15 L-Arabinoso 9 Pentoses are absorbed at the same rate as indifferent sub- stances such as sodium sulphate. Galactose and glucose are absorbed so much more rapidly than the rest that a special mechanism of some kind must bo deemed to be involved in their 243 ABSORPTION OP CARBOHYDRATES case. Verz&r believed that this consists in the phosphorylation of the sugars in the gut mucosa under the influence of a phosphat- ase -which is demonstrably present in the cells. He showed that the selective absorption of glucose and galactose can be abolished by adding iodoacetate or phlorrhizin to the contents of tied-off intestinal loops, and that absorption in the intact animal is much delayed by previous injection of these drugs. Both phlorrhizin and iodoacetate are known to be powerful inhibitors of fermenta- tion and glycolysis, and in both these processes phosphorylation is known to play a fundamental part. Verzdr therefore regarded his results as evidence that phosphorylation is involved in the absorption of sugars from the intestine. Verzdr’s observations might be regarded as strong evidence in favour of Iris contention if it were definitely known that iodoacetate inhibits phosphatases specifically, but there is no indication that this is the case. Iodoacetate is known to inhibit several of the enzymes concerned in glycolysis and in fermenta- tion, but there is no reason at present to believe that it directly inhibits enzymes concerned only with phosphorylation. The most probable explanation of the action of iodoacetate lies in the fact, that, for energetic reasons, the phosphorylation of glucose requires the participation of ATP and hexokinase. The provision of ATP depends in turn upon glycolysis and oxidation, which are powerfully inhibited by iodoacetate. The interference of iodoacetate in the normal absorption of glucose is probably due, therefore, to inhibition of ATP formation rather than to any inhibitory effect it might have upon the gut phosphatase. This argument assumes that the experimental results ob- tained with iodoacetate are valid, and this is very doubtful because the concentrations of iodoacetate used were high enough to cause sloughing of the mucosa from the gut wall in some of the experiments. The only verdict wo can give on the phosphorylation theory is one of not proven and, indeed, it is clear today that absorption is a more complex affair than it formerly appeared. Some recent work on intestinal loops has shown that absorbed glucose does not appear as such, nor in any known phosphorylated form, in the portal blood. A point of interest may be added in passing. It is well known 244 DIGESTION OF FATS that phlorrhizin also abolishes the reabsorption of glucose from the urine by the cells of the kidney tubule, as well as its specific absorption from the small intestine. Phlorrhizin, like iodoacetate, is a powerful inhibitor of glycolysis and oxidation, and the facts suggest that the intestine and the renal tubule alike carry out the work of absorption by essentially similar mechanisms. DIGESTION AND ABSORPTION OF FATS Saliva contains no lipase and, while the presence of a lipase in the gastric juice has been reported in a number of cases, the activity of the alleged gastric lipase at tho pH of the gastric contents is such that it can be of little importance in digestion. Many authors believe that it must be regarded as pancreatic lipase that has regurgitated from the Bmall intestine. But although no appreciable digestion takes place in the stomach, the fats of tho food are wanned and softened, if not actually liquefied. When presently the chyme is somewhat forcibly squirted into the duodenum there is a marked tendency for the fat to become emulsified, a tendency which is emphasized by the presence of bile salts. Tho commonest representatives of this important group of substances are conjugated derivatives of cholic acid with glycine and taurine. They are remarkable for their property of very greatly reducing the surface tension of fat/water inter- faces, and for this reason not only facilitate emulsification but tend to stabilize an emulsion once it has been formed. Until the beginning of the century it was generally believed that finely emulsified fat can be absorbed without previous digestion, but it appears that the bile salts alone cannot produce a sufficiently fine dispersion. Finer emulsions can be prepared With the aid of sodium cetyl sulphate, a synthetic wetting agent, and if emulsions made in tliis way are introduced into the duo- denum of rats, the dispersed fat particles are absorbed. Sodium cetyl sulphate inhibits the action of the digestive lipases, and we have therefore to conclude that direct absorption of unhydro- lysed fats can, indeed, take place. Even paraffin can bo similarly absorbed, provided only that it is sufficiently finely dispersed. The essential conditions for absorption are, according to Frazer, that the particles shall bo less than 0-6 /z in diameter and that 245 ' • ABSORPTION OF FATS they shall be negatively charged. Sodium cetyl sulphate gives emulsions in which these conditions are fulfilled, but does not occur naturally, while the bile salts alone do not yield particles less than about 2/* in diameter. Frazer and hi3 colleagues attempted to find natural emulsi- fying agents which could produce the degree of emulsification, required for direct absorption under physico-chemical conditions similar to those which prevail in the Hmall intestine. The sub- stances studied included bile salts, cholesterol, a free fatty acid (oleic) and a monoglyceride (glyceryl monostenrato), separately and in various combinations. Only with bile salts -f fatty acid + monoglyceride was it found possible to obtain the neces- sary degree of dispersion. But not all the food fat is absorbed in the emulsified form. Animals possess powerful lipolytic enzymes, which probably would not have survived unless they were useful to their 24fl ABSORPTION OP FATS possessors. Moreover, the action of the pancreatic and intestinal lipases upon ordinary neutral fats disengages the fatty acids from their combination with glycerol one at a time, giving, at first, a mixture of free fatty acids with di- and monoglycerides — • precisely the materials required for the emulsification of the remaining unhydrolysed fat. It may therefore be concluded that a part of the fat of the food undergoes digestion before being absorbed, and that the products of its digestion, together with the bile 6al ts, facilitate the emulsification of the remainder, which i3 then absorbed without previous digestive hydrolysis. Digestion is carried out in the small intestine under the in- fluence of pancreatic and intestinal lipaseB. At the pH prevailing in the small gut, the eventual products of hydrolysis are glycerol on the one hand and free fatty acids on the other. It was formerly supposed that the gut contents are alkaline (pH 8-9) in this region, and that the fatty acids are accordingly neutralised to form soaps. These substances are appreciably soluble in water, and their absorption seemed to offer no problems. More recent estimates put the pH at G-5-7-G however, and in tliis range soaps do not exist necessarily as such, but givo rise to free fatty acids. Soaps, moreover, are powerful haemolytic agents, while ulceration of the colon following the use of soap enemata is not an unknown occurrence. Soaps, then, are probably not formed. Free fatty acids, by contrast with the soaps, are characteristically insoluble in water, and the manner of their absorption is therefore more problematical. Bile Balts probably aid the digestion of lipid materials by facilitating their emulsification and so presenting the digestive lipases with a larger surface upon which to attack their sub- strates. But bile salts are not essential for digestion. In experi- mental animals in which the bile duct has been ligated, or in human subjects in whom the bile duct is occluded, e.g. by the presence of gall stones, fat is still digested, as is attested by the presence of free fatty acids in the faeces. Again, if a fat such as olive oil is introduced into a tied-off loop of intestine, with or without the addition of bile salts, no absorption takes place, but if a small amount oflipase is also added, the contents of the loop undergo digestion. But the oleic acid liberated is only absorbed if bilo salts were introduced into the loop at the outset. 247 AB80SFT10N OF FATS Thus bile salts play some part in the absorption of free fatty acids, as "well as in that of unhydrolysed fat. This is attributed to the so-called hydrotropic action of the bile salts, i.e. their ability to form water-soluble complexes with fatty acids. TTm effect can be demonstrated readily enough by adding a solution of 6odium glyco- or taurocholate to an aqueous emulsion of a fatty acid. If enough bile salt is added, the emul- sion becomes water-clear. The simplest and smallest molecular complexes formed in this way are believed to contain one mole- cule of fatty acid and four of bile salt, but larger aggregates can be formed with fatty acid :bile salt ratios of 2 : 7, 3 ; 8, 4 : 9, and eo on. The ability of these complexes to pass through a membrane will therefore be determined by the relative proportions of fatty acids and bile salts. If the fatty acid '.bile salt ratio is low the particles will be small, and their ability to pass through the intestinal barrier will bo proportionately greater. This hydro- tropic effect on the part of the bile salts is exerted upon other lipid materials such as cholesterol, but does not extend to unhydrolysed fats. Tahoe; 18. ABSonrTiON of fatty acids (Ajttr VerzAr) Oleic »eld absorbed Contents of intestinal loop in 6 hr. Oleic acid + b3e salt 29 3 Olcio acid + glycerol + bile salt 24-9 Oleic acid + phosphate + bile salt 10 3 Oleic acid + phosphate 4 glycerol 4 bile salt 48 0 Oleic acid + glycerol phosphate 4 bilo salt 72 7 Oleic acid 4 glycerol phosphate + bile aalt 4 iod cm estate 0 Many experiments were carried out by VerzAr and his col- leagues on the absorption of fatty acids, hut in discussing them it is well to remember that, at the time, Verz&r himself was of the opinion that fats must be fully hydrolysed before they can be absorbed. In his experiments, which were carried out earlier than those of Frazer, use was made of tied-off intestinal loops. Oleio acid and bUe salts were introduced into the loops in all the experiments, together with the other substances indicated in Table 18, which is taken from his work. Neither glycerol nor phosphate alone leads to any acceleration of absorption, but if both are present together the rate increases two or three times. 248 ABSORPTION OP PATS Tliia suggests the possible formation of some compound of fatty acids with glycerol and phosphate, i.e. of a leci thin-like substance. Compounds of this type are soluble in water and might therefore bo freely absorbed. In lecithin itself, it will bo remembered, glycerol is present in combination with phosphoric acid, and the fact that fatty acids are absorbed nearly twice as fast in the presence of glycerol phosphate as when its components are present separately adds considerably to the probability that a phospholipid of some kind is indeed formed from some larger or smaller proportion of the free fatty acids. Like the absorption of sugars, that of fatty acids is inhibited by plilorrhizin and by iodoacetate, probably for the same reasons os in the absorption of glucose. Phosphorylation is perhaps again involved, for the phospholipid content of the blood is higher during the absorption of a fatty meal than it is at any other time, while the phospholipid content of the intestinal lymph rises from a resting level of about 2-2 to about 7-5 mg. % while fat absorption is taking place. Furthermore, if an animal Is fed with fat that has been 'labelled ’ with iodine or with heavy hydrogen, iodized or deuterated phospholipids can be recovered from the gut mucosa while absorption is in progress. It seems likely, therefore, that fatty acids are transported into the cells of the mucosa, presumably in the form of water-soluble com- plexes with bile salts, and then condensed with phosphate and glycerol to form substances resembling lecithin. Phospholipids of the lecithin group typically contain a nitro- genous base, usually choline or ethanolamine, in addition to , glycerol, phosphoric and fatty acids, but since some at least of the glycerol and phosphate required for the synthesis of the presumptive phospholipid must probably be provided by the epithelial cells during normal absorption, it is not inconceivable that, if a nitrogenous base is required, it too can be furnished by the cells. It appears, then, that a part of the food fat is digested by the pancreatic and intestinal Upases, and that tho products of partial digestion, aided by bile salts, serve to emulsify the remainder so finely that it can pass through the intestinal wall without pre- vious hydrolysis. How much of the total fat undergoes digestion and how much is absorbed directly is not certainly known. 249 ABSOBPTION OF FATS Probably it is reasonable to estimate that from one-quarter to one-third undergoes hydrolysis and that the rest, after emulsifi- cation, is directly absorbed. Fat which is absorbed in the emulsified condition passes into the cells of the gut wall, in which it can be observed in the form of minute droplets which stain with dyes such as Sudan IH and have in fact all the histological characteristics of neutral fat. From the cells these droplets, the chylomicrons, make their way into the lacteals and hence, through the Jymphatio system and the thoracio duct, into the blood stream, where they are respon- sible for the condition of post-absorptive lipaemia. That part of the fat which undergoes hydrolysis is ultimately resolved into glycerol and free fatty acids. The latter pass into the intestinal mucosa, apparently in tho form of water-soluble complexes with bile salts, and here they appear, at any rate in part, to be built up into phospholipids of some kind, though some may be resyn* thesized to ordinary neutral fat. These too pass into the lymph, foT in experiments in which isotopically labelled pentadecanoio acid was administered to experimental animals, the isotope was almost quantitatively recovered in the effluent from a cannula tied into the thoracic duct. Even so, other experiments with isotopically labelled acids have shown that the shorter chain acids — up to 8 carbon atoms in length — are largely absorbed by way of the blood stream. 250 CHAPTER X GENERAL METABOLISM OF PROTEINS * AND AMINO-ACIDS FUNCTIONS AND FATE OF PROTEINS AND AMINO-ACIDS Proteins constitute an indispensable article of food for all animals. Wo have abundant direct evidence from feeding experi- ments that the usual laboratory animals require supplies of certain amino-acids, notably tryptophan, lysine and histidine, not only for growth while the animal is young, but also for the maintenance of normal physiological condition during adult life. These essential amino-acids are only to bo found in proteins, and protein foodstuffs are therefore indispensable. In most and pro- bably all herbivores an indirect, secondary source of essential amino-acids is found in the tissue proteins of symbiotic micro- organisms inhabiting the gut but, although the value of this supplement may be considerable in some cases, we do not know if it is over sufficient alone. Evidence regarding the amino-acid requirements of invertebrate animals, unfortunately, is scanty. What information we have relates mostly to insects, but does not give grounds for supposing that their ingenuity in amino- acid synthesis is any greater than our own. Tryptophan, lysine and histidine seem to be essential for animals of erery kind. The naturally occurring amino-acid3 can bo classified as in Table 19. Under the heading of essential amino-acids are some that can bo replaced by other members of the essential group; thus tyrosine can be formed from phenylalanino, wliile cysteine can be produced if methionine fa available. But the reverse fa not possible. Provided that enough phenylalanine fa available to discharge the essential and characteristic functions of phenyl- alanine itself, and to produce at the same time enough tyrosine to fulfil those of tyrosine, tyrosine itself need not be provided. Tyrosine, however, cannot discharge the functions of phenyl- alanine. Glycine, aspartic and glutamio acids, by contrast, need 251 X8BENT1A.Ii AMINO-ACIDS not be prodded at all ; these the organism can mate for itself from non -protein materials, and of all the amino-acids that enter into the composition of the tissue and other proteins, there are less than ten -which the animal organism can produce by its own resources. Table Iff. Nctaitionai. status of Aanwo-Aqins Essential Non-essential Irreplaceable Replaceable Glycine Threonine AlatJino Valine Leucine Aspartic acid uo Leucine Glutamic acid Methionine Cysteine : cyutino Pronin o Phenylalanine Tyrosine flydroxyproline Histidine Arginine Tryptophan Lysine Arginine Tiie position of arginine, which figures as essential and as non-essential alike, calls for special comment. Adult rats remain alive and healthy, and young rats grow, on diets wholly devoid of arginine. But the growth of young rats on an arginine-defi- cient diet can be accelerated by the administration of arginine. A similar effect has been observed in chicks, which also require and cannot synthesize glycine. It is therefore probable that, while tho growing animal can evidently synthesize arginine to some extent, it cannot do bo fast enough to keep pace with the requirements of optimal growth. It may be, indeed, that impor- tant substances other than arginine are synthesized slowly enough to limit the growth-rate of young organisms. Amino-acids, essential and non-essential alike, are required for numerous purposes. Quite apart from tho special products to which particular individual amino-acid3 give rise, new tissue proteins must be synthesized, damaged or wasted tissues must be repaired or replaced, and normal supplies of enzymes and hormones must constantly bo maintained. The formation of adrenaline and thyroxine, for example, makes essential the pro- vision of tyrosine (or phenylalanine), to which they are closely related and from which they are in all probability produced, as witness their respective formulae on p. 253. Hie production of another hormono, insulin, makes particularly heavy demands 252 FUNCTIONS OF AMINO-ACIDS upon tile supplies of essential amino-acids, for it is a polypeptide containing about 16% leucines, 8% phenylalanine, 12% tyro- sine, 4% histidine, 3% threonine, 2% lysine and 12% cystine. tyronnt Provided that the food proteins contain enough of all the amino-acids that aro essential, the organism can probably make good any deficiency of the rest, although, in the ordinary way, it is not likely to be called upon to do so. The essential amino- acids are, on the wholo, the least common, so that an adequate intake of these in the form of protein is necessarily attended by an adequate intake of the rest. It is necessary, however, that the amino-acids required should be presented to the organism all at one time, as they are if an adequate meal of protein is taken. Amino-acids that are not more or less immediately in- corporated into tissue or other proteins undergo deamination, bo they essential or non-essential, and are therefore lost for purposes of protein synthesis. This lias been demonstrated by feeding a variety of mixtures of purified amino-acids simul- taneously and at different times. The elaboration of hormones, enzymes and other special pro- ducts still goes on even during starvation, when it can only be dono at tho expense of the tissue proteins. Prolonged deprivation of protein therefore leads to emaciation and eventually to death . For some time before death ensues there is a small, fairly con- stant, daily excretion of nitrogen, the magnitude of which may be taken as an index to the amount of protein being broken down 253 PROTEIN REQUIREMENTS for processes essential to the functioning of the body machine. Death Itself is heralded by a sudden extreme rise in the rate of nitrogenous excretion, known as the ‘ pre-mortal rise', and this begins when, the available carbohydrate and fat reserves of the tissues having been exhausted, the organism is left with only its tissue proteins as a source of energy production, so that a large- scale degradation of protein begins. On a diet that contains very littlo protein it is possible for the daily intake of protein nitrogen to lie below the output of urinary nitrogen. So long as output exceeds intake the organism, on balance, is the loser, and the deficit of nitrogen is withdrawn from the tissues. If, however, the protein allowance is gradually in- creased, a point is eventually reached at which intake just suffices to balance output. The organism is then said to be in a state of nitrogenous equilibrium. The amount of protein required just to attain this equilibrium condition in a given individual is therefore ft measure of the minimum protein requirement of that Individual, and since proteins are among the most expensive articles of food, this is a matter of economic as well as academic interest. Many workers have accordingly investigated tho minimum protein requirements of the human organism, and the results obtained have been very variable indeed. Rubner and his colleagues put it at about 100-120 g. protein per diem for an average man, whereas Chittenden, using himself as the experimental animal, found that he could satisfy his personal requirements with only some 30-35 g. per diem, Ins health improving as a result of the experiment. These large differences do not, os might at first appear, merely reflect differences in individual requirements, but differences rather in the chemical nature of the food proteins chosen. These proteins must supply enough of the essential amino-acids, and no amount of protein, however great, that fails to accomplish this can suffice to establish nitrogenous equilibrium. Animal proteins are, on the whole, much richer than plant proteins in terms of their content of essential amino- acida, and it follows that smaller amounts of protein arc required when meat, fish, eggs, cheese, milk and the like are chosen than when the food selected consists largely of cereals and pulses. The maize protein, zein, is notoriously deficient in tryptophan and in lysine, and if zein is taken as the sole protein of a diet. 251 PEOTEIN EEQT7IEESIENTS nitrogenous equilibrium can never be established, no matter how much of it is consumed. Gelatin is similarly deficient in trypto- phan and in phenylalanine, and, like zein, is a protein of 'poor biological value’. The primary function of protein food is to supply the amino-acids needed for tke growth, repair and general maintenance of the struc- tural and catalytic machinery of living cells. If, as is commonly the case, the proteins of the food provide more amino-acid units than are required for the discharge of these primary and very specifio functions, the excess can be degraded and made to subserve the secondary and less specific function of providing fuel for the machine. If excess protein is taken, the excess of nitrogen is eliminated, mostly in the form either of ammonia, urea or urio acid, within 24 hr. Proteins and amino-acids are not normally stored to any appreciable extent in the normal adult organism: nitrogen retention on a significant scale is only observed during periods of tissue growth, during childhood and pregnancy, for example, or during periods of protein replacement, as during con- valesence after a wasting disease or after protein starvation. The non-nitrogenous residues of surplus amino-acids are retained and servo to contribute to the stores of ‘energy-producing’ materials, i.e. carbohydrates and fats. Table 20. Fates or auino-aoids administered TO A DIABETIC OH PHLORBRIZINIZED DOO Ketogeoio Leucine (4) woLeucine (4) Phenylalanine (4) Tyrosine (4) Fate uncertain Lysine Histidine Tryptophan Methionine Glucogenic Glycine (2) Alanine (3) Serine {3) Threonine (3) Cysteine (3) Valine (3) wo Leucine (3) Aspartic acid (3) Glutamic acid (3) Arginine (3) Ornithine (3) Proline (3) Hydroiyproline (3) Note. The numbers in brackets indicate the number of carbon atoms undergoing conversion in each case. If a meal of protein is administered to a phlorrhizinized or diabetic animal an increased output of glucose and of acetono bodit’3 is observed. Part of the protein must therefore be 265 PATE OPCC-AMINO-ACIDS considered ns convertible into carbohydrate derivatives and part into fatty metabolites. If the amino-acids are administered individually it is found that some are glucogenic, i.o. give rise to glucose, while others are ketogmic, giving rise to ketone or acetone bodies. The known fates of the amino-acids are summarized in Table 20. It will be noticed that certain amino-acids, including some of the essential group, give rise neither to glucose nor to ketone bodies. It may be that they are incorporated into some sort of protein or peptide which serves as a temporary store, but there is little evidence for the existence of such a store. PATE OF a- AMINO -NITROGEN Neither glucose nor the ketone bodies contain nitrogen. It follows, therefore, that, at an early stage in their metabolism, the amino-acids suffer the removal of their characteristic a-amino- group. In a typical mammal such as a dog, this a-amino-nitrogen ultimately appears in the urine in the form of urea. In birds, snakes and lizards, by contrast, the final end-product ia uric acid, while in most aquatic animals ammonia is excreted instead. The urine of a dog Btarved of protein contains very little urea, but if a protein meal is taken, urea production soon begins and the protein-nitrogen of the food is almost quantitatively eliminated in the form of urea within 24 hr. or thereabouts. Now we are already aware that the food proteins are broken down by digestive peptidases to yield the component amino-acids, and it is in this form that the food proteins are actually absorbed into the blood stream. We have therefore to discover how, where, and in what form the a-amino-nitrogen is detached from the amino-acid molecules, and how urea is elaborated from the primary nitro- genous product. A partial answer to these questions 13 obtained by studying a hepatectomized animal or, alternatively, an animal with an Eck's fistula. An animal of this kind will survive for 6ome days, but dies quickly if it is allowed to eat protein. At death, un- usually largo amounts of amino-acids ore found in the blood, but neither the blood nor the urino contains any urea in the case of a dog, or uric acid in that of a bird. Instead, ammonia is present, and ammonia poisoning is one of the causes of death. 256 DEAMINATION These observations show (a) that the amino-groups of the amino- acids are split off in the form of ammonia, and (6) that the conversion of this ammonia into urea or uric acid, as the case may be, normally takes place only in the liver. The latter con- clusion is confirmed by liver-perfusion experiments and by experiments on liver slices. If small concentrations of ammonia are perfused through a surviving liver or shaken with liver slices, urea is formed In the case of dog liver, while if a bird’s liver is used, the addition of ammonia leads to the production of uric acid. We shall discuss these processes separately, dealing first with the removal of the amino-groups, a process which is known as deamination. DEAMINATION The deamination of amino-acids with production of ammonia might be accomplished in either of two ways, both of which have been considered. It might be a hydrolytic (equation (1)) Or an oxidative process {equation (2)) : Nil, OH (1) R.dH + H,0 - R.dH + NH,. ''boon ''boon NH, O (2) R.dH + JO, - R. / + NH,. ''booH ''boon Very little experimental evidence has ever been adduced in favour of hydrolysis os the mode of deamination in animal tissues, though it is known that hydrolytic deamination takes place in some bacteria. The vast bulk of evidence relating to animal metabolism is in favour of oxidative deamination. The most convincing work on this problem was that carried out by Krebs, who made use of the tissue-slice technique. Slices of various rat tissues were shaken under physiological conditions of temperature, pH, etc., in the presence of various amino-acids. After an hour or two the reaction mixture was deprotein ized and the corresponding a-keto-aeids were sought and found by taking advantage of the fact that they form very insoluble, 257 17 8»A DEA3IIJ4ATI0.N characteristic 2;4-dinitrophenylhydrazones. Among mammalian tissues only liver and kidney deaminateamino-acids at all rapidly, and these tissues use more oxygen when they are deaminating than when they are not. Surviving liver and kidney slices deaminate both the common, naturally occurring L-series and the much rarer 'non-natural’ D-series of amino-acids, but not all amino-acids are attacked at the same rate. Table 21, which is taken from Krebs’s original paper, illustrates the relative differences in the rate of deamina- tion of a number of amino-acids in terms of the extra oxygen uptake resulting from the addition of the amino-acids to kidney tissue slices. Tahle 21. Rates or deamination or amino-acids BV SLICED RAT SIQKEI TISSUE (After Krebs) Qo, Without With m/I 00 Amino-acid added amino-acid amino-acid Clycine -21-1 rl- A lanine -23 0 DL-nor Leucine -17-4 DL- Aspartic acid -23 5 L-Cluumic acid -23 5 DL-Prolmo -18 5 L-Iiydroxyproline -18 5 L-Iaudc -21-1 DL- Vatina -2M L-Tryptopban -IB 9 L-Hutidioo -18 9 DL- Phenylalanine -21-1 t-Tynwine -18 7 dl- L eucine -23-5 L- wo Leucine -23 5 -21-4 -410 -232 -37 0 -43-2 -37-3 - 10-8 -25 4 -250 -135 -210 -233 -24-4 -291 -2L-7 To obtain strictly quantitative evidence in favour of equa- tion (2) is more difficult. If liver tissue is used, the ammonia set free by deamination is converted more or less completely into urea, but this difficulty can be obviated by the use of kidney slices, which do not form urea. But in liver and kidney alike, the other product of deamination, the a-keto-acid, is liable to he further metabolized, by oxidative decarboxylation in the first instance. This process, Krebs found, can be prevented by the addition of arsenious oxide. Working therefore with kidney slices and in the presence of arsemte, he was able to demonstrate DEAMINATION that, for every molecule of ammonia produced, an extra atom of oxygen was consumed and a molecule of the corresponding a-keto-acid formed. Essentially the same results were obtained with extracts prepared from acetone powders of kidney tissue (Table 22). Table 22. Oxidative deamination of amino-acids BY KIDNEY EXTRACT (After Krebs) Amino-acid added MoL O f : NH, : keto-acid dl- A lanine 1 : 1 04 : 1-83 DL- Valine 1 : 2 08 : 2 20 Dh-rwr Leucine 1 ; 1-85 : 1-83 Dl.- Leucine 1 : 2 42 : 2 28 DL- Phenylalanine 1 ; 2-17 : 1-85 Kreb3 went on to seek information regarding the mechanism of the process by studying tissue extracts. Pulp preparations of liver and kidney alike act upon both the l- and the D-series of amino-acids, again in an oxidative manner, but as soon as the pulp is appreciably diluted its ability to attack the naturally occurring L-acids disappears. The enzyme responsible for the deamination of the D-series, however, is resistant to dilution, and powerful preparations of the D-amino-acid oxidase can be made by extracting fresh, finely divided kidney tissue with water or buffer, centrifuging to remove the tissue debris, and treating the clear extract with 10 vol. of ice-cold acetone under ice-cold conditions. By filtering off the resulting acetone powder and drying it carefully, a stable preparation can be had which retains its activity for some weeks, and from which active enzyme solu- tions can be made by extraction with water or with phosphate buffer. Preparations made in this way deaminate all the amino- acids of the D-series with three exceptions: glycine, D-glutamic acid and D-lysine. A specifio oxidaso was later discovered which deals with glycine, but thero is reason to tliink that lysine is never deaminated at all (p. 203). The D-amino-acid oxidase has been extensively concentrated and finally isolated, and a more detailed description of its nature and properties will be found on p. 153. So far, however, no evidence had been obtained about the enzyme or enzyme systems involved in the deamination of the L-series of amino-acids. Numerous attempts were made to DEAMINATION obtain enzyme preparations which would act upon the naturally occurring amino-acids, but for a number of years only one such enzyme was known. This enzyme, which occurs in liver and kidney, is completely specific with respect to L-glutamio acid. It requires either DPH or TPN, and is, in fact, a typical, coenzyme-speciGo dehydro- genase, now known as b-glutamic dehydrogenase. Unlike that of D -amino-acid oxidase its action is freely reversible. With tho more recent discovery and eventual isolation of an L-amino-acid oxidase from mammalian liver and kidney, we are able to account for the deamination of the majority of L-amino- acids, for the L-oxidase resembles the p-enzyme in being a true oxidase. It is a group-specific enzyme and attacks all tho mono- amino-znono-carboxylic amino-acids except glycine and those that contain a hydroxyl radical, but has no action upon the diamino- or dicarboxylic acids. How important this enzyme may be seems uncertain, for even in the rat it acts relatively feebly. Whether it is catalysed by the d- or by the L-amino-acid oxidase, or by L-glutamic dehydrogenase, deamination is always oxidative and takes place in two stages. In the first a pair of hydrogen atoms is transferred to the appropriate hydrogen acceptor and the corresponding a-imino-acid is formed: NH, NH / / (I) R.CH — R.C + 2H (to acceptor], \ooh \oon The imino-acid then reacts, apparently spontaneously, with water to yield the a-keto-acid, together with ammonia: NH S (2) R.C Nxjoh 0 + H.O - R.C + NH„ '''coon Special non -oxidative mechanisms are involved in the deamina- tion of the hydroxy-acida, serine and threonine (p. 103), but imino-acids are again formed as intermediates. 260 TRANS DEAMINATION TRANSDEAMINATION In addition to deaminating enzymes, animal and plant tissues possess catalytic mechanisms which catalyse the transference of amino-groups from amino-acids to certain a-keto-acids. If L-glu- tamic and pyruvic acids are added together to chopped liver or muscle tissue, the a-amino-radical of the glutamic acid is in part transferred to the pyruvic acid, bo that a-ketoglutaric acid and alanine are formed. The system tends towards an equilibrium which can be approached equally from either side, and the pro- cess, which for obvious reasons is referred to as ‘ transamination was attributed by its discoverers to an enzyme which they named ‘aminophorase’. This name has now been generally abandoned in favour of 'transaminase': COOH transaminase Enzymes of this kind seem to be very widely distributed in plant and animal tissues alike, and it is worth while to notice that, unlike the deamin&ting enzymes, they ere not confined to liver and kidney among animal tissues, but are present also in brain, kidney, muscle, and heart, for example. They are specific, more- over, towards amino-acids of the natural n-series. One of these transaminases is specific towards aspartic acid and the corre- sponding a-ketosuccinic (oxaloacetic) acid, and another towards glutamic and the corresponding a-ketoglutario acids. The 261 TRA5TSOEASIJNATION glutamic enzyme seems to predominate in animal tissues and also in many plants. Braunstein suggested that the deamination of L-amino-acids in general might involve transaminase. It was already known that a-ketoglutaric acid i3 a common metabolite, arising as it does from carbohydrate as well as from protein sources. Under the influence of glutamic transaminase the et-amino- groups of any incoming amino-acid could, it was suggested, be transferred to a-ketoglutaric acid to yield glutamic acid which then, under the influence of then-glutamic dehydrogenase of liver or kidney, would undergo deamination, a-ketoglutario acid being regenerated and ammonia set free: E.CH(NHJCOOH— II --v. a-fcetogmtanc acid R.CO.CO OH-*^ glotamic acid — ' L-gliUamic z^glutamic CH CHOU ioon iooii iooH propUmie acrylic lactic add and acid CH, 0 glycogen pyrtitK “ A 208 OLYCO0EHESIS: KETOGENESIS In this chapter wo shall content ourselves with tracing, as far as we can, the routes of conversion of amino-acids into sub' stances which, like propionic, lactic and pyruvic acids, are known carbohydrate-formers. The rest of the stages are common to the metabolism of carbohydrates and carbohydrate derivatives, and will be considered in later chapters. The amino-acids are classified as essential or non-essential according as they can or cannot be synthesized in the animal body. If the reader will refer again to Tables 19 and 20 (pp. 252 and 255) several interesting points will be noticed. First, all the non-essential amino-acids are glycogenic , which probably indicates that their conversion into carbohydrate is a reversible operation. Of the essential amino-acids only a few are glycogenic , and the fact that they are essential presumably indicates that the reaction chains through which they are transformed into carbohydrate include some irreversible step or steps. Thus carbohydrate can contribute to the synthesis of some amino-acids, but not of all. All the ketogenic amino-acids are essential. They give rise to fatty metabolites, but the fact that they are essential, i.e. cannot be synthesized by the animal organism, shows that their con- version into ketone bodies is not a reversible performance. Thus fat, unlike carbohydrate, contributes little or nothing towards the synthesis of amino-acids or of protein. These general points may be summarized as follows: The first step in the transformation of the amino-acids consists in the deamination or transdeamination of the amino-acids, the mechanisms of which we have already discussed. For con- venience of reference the known modes of deamination of the naturally occurring L-acids are summarized in Table 23. It may be noticed that there is serious doubt whether lysine, ornithine and arginine undergo biological deamination (p. 263). It is known that serine and threonine too can be atypically 209 ACTION or BACTERIA deaminatcd by non -oxidative enzymes present in rat liver (p. 103), which, in the absence of any other name, we may call 'serine deaminase’. Table 23. Deamination of amino-acids of the naturally OOCUBRINO L-SEBIES L-Amino-aeid IN ANIMAL TISSUES Amino-acid oxidase Specific enzyme Glycine - Glycine oxidase Alanine + ‘Serine deaminase’ Threonine — ‘Threonine deaminase’ Selhionine + + Valint + Leucine + f*j Leucine + Aspartic acid _ Glutamic acid - L-Glntamio dehydrogenaso Arginine - Ornithine — Lysine - Phenylalanine + Tyrosine + Tryptophan Histidine + + Moat and probably all can bo tronsdeaminated except arginine, ornithine and lysine. Note. The we of a negative sign indicate* only that the amino-acid U not at present known to be deaminatcd by L-amino-acid oxidase. BACTERIAL ATTACK AND DETOXICATION Bacteria of many kinds possess powerful and specific amino- acid decarboxylases, and although these do not fall within the scope of thi3 book they must be mentioned here because they have considerable interest for animal metabolism. The alimen- tary canal of animals is always densely populated with bacteria at some region or other, and the activities of the members of this population lead to the production of materials which may bo later absorbed by the animal itself. The extent of this absorption depends upon the region in which bacterial activity takes place. In herbivorous animals, of course, micro-organisms play a very large and important part in the digestion of the food, but even in animals that do not delegate tlicir digestive operations to symbiotio bacteria, the metabolism of the micro-organisms has to be taken into account. 270 DETOXICATION The bacterial inhabitants of the intestine have access to the food, or to the products of digestion of the food, among -which amino-acids are included; and certain bacteria decarboxyiate one or more of these to produce the corresponding amines: r.ch(Kh i )!coo.‘h - r.ch.nh, + co,. Some of these products are intensely poisonous and, when they are absorbed into the animal's blood stream, undergo what is known as detoxication. This is accomplished by oxidation, reduc- tion, acetylation, methylation.or in Borne other manner. Further- more, by progressive bacterial degradation of the side-chains of acids containing aromatic rings, poisonous phenolic substances aro formed, and these, like the amines, undergo detoxication in the animal body. We shall deal individually with individual cases, and we shall also comment upon the parts played by the amino-acids themselves, since several of them act as important detoxicating agents. In addition to amino-acids, many other substances play apart in detoxication, and these may be briefly mentioned at tliis point. Acetylation (p. 432) is a common fate among aromatic substances containing — NH 2 groups, and the administration of aniline, sub- stituted anilines, sulphonamide drugs and the like, is followed by their excretion, partly or wholly in the acetylatcd form. Amines are commonly oxidized, yielding harmless aldehydes or acids, by the amine and diamine oxidases of the tissues (pp. 155-0). Phenolic substances are frequently excreted in conjugation with glucuronio acid or with sulphuric acid, the latter being probably derived from sulphur-containing amino-acids. Plus book does not contain a special chapter on detoxication or, as it is often termed, protective synthesis ; it has seemed more suitable to the author to dispense with such a chapter, since a largo part of the subject can be dealt with under the special functions vfindcridiial antia• glycogen Aooh Aooh cooh The desulphurose reaction is anaerobic: under aerobic conditions the HjS can be oxidized to sulphate. It is also known that cysteine suiphintc acid can undergo a transamination reaction 27ft cysteine: cystine with a-ketoglotaric acid yielding glutamate and pyruvic add. The latter can then be split enzymatically into pyruvate and sulphate: CH.SH CUfif 0 ch-s / 0 CH, I I H)H I CH.NH, f CH.NHj *■ C=0 + H,0 B*SO* + CO — > glycogon iooH ioon iooH ” iooH cysteine cysleine-fl- fi-tulphinyl tutphinic acid pyruvic acid That dietary cysteine and cystine can be replaced bymclktonine argues that they can be derived from it, and this is indeed the case. If methionine containing radioactive sulphur is admin* istered to animals, radioactive sulphur can be recovered in cysteine and in cystine isolated from the tissues. Cysteino does not arise directly from methionine but from its demcthylatcd product, homocysteine. If sliced rat liver is incubated with homo- cysteine in the presence of serine, cysteine is formed by a trans* thiolation reaction in which an — SH group changes place with an — OH. An intermediate reaction complex, cystathionine, is formed: CH£H CH.OH CII r S— — C5i, CH.OH ClhSII in, + (m.NB, -1^? iH, Ah.nh, liS? in, + in.NH, in.NH, iooH iii.NH, iooB in.NH, iooa iooH iooH iooB homocysteine ferine cystathionine homoserine cysteine There is some evidence that two enzymes are involved, one in the synthesis and the second in the breakdown of cystathionine. Pyridoxal phosphate acts as a coenzyme for one or both of these enzymes. Cysteine and cystine are particularly important because of the ease with which a pair of — SH groups of cysteine can be oxidized to give the — S — S — bond of cystine, and vice versa. This property extends to many compounds into the composition of which cyBtcine enters. Thus it is present in glutathione, which we may represent as G . SH. This compound is very readily oxidized, e.g. by molecular oxygen in the presence of traces of heavy metals, to give the oxidized form, G . S — S .G, and it is upon this cysteine: cystine behaviour that the functional importance of glutathione appears to depend. Linkages of the — S — S — type also play an important part in the intramolecular structure of hair keratin and other sclero- proteins. Hair contains about 7-3 % of cystine and it is believed that — S — S — bonds are formed between adjacent molecular fibres, and that the tensile strength and other mechanical pro- perties of the hair fibre as a whole are largely due to these linkages. The — SH group is important also among enzymes, many of which are active only so long as their — SH groups are in the free state. If these are oxidized to give — S — S — bonds, catalytic activity disappears, but can be recovered by the addi- tion of reduced glutathione. Although this is not by any means a universal property of enzymes, certain dehydrogenases in particular are reversibly inhibited by mild oxidation, and irreversibly by iodoacetate, which reacts with and blocks — SH groups in the following manner: — SH + I.Cff.COOH - HI + _S.CH.CO0H. A considerable number of war gases (cf. p. 181), including lachry- mators and vesicants, act by blocking the — SH groups of enzymes which can, however, be ‘protected’ or reactivated by certain dithiols, in particular by British anti-lewisite (‘BAL’; 2:3-dimercaptopropanol) : CH..CH..CH.OH iff iff These act by reducing — S — S — bonds to — SH. Cysteine is the mother substance of taurine, a compound which is Very widely distributed, often in remarkably large amounts (p. 340). It is found in the bile of many vertebrates as a conjugant in the bile salts (p. 246). Taurine is known to arise from cysteic acid through the agency of a specific cysteic acid decarboxylase, one of the few ‘straight* decarboxylases known to occur in animal tissues (liver and kidney). Cysteic acid itself is believed to be formed from cysteine by oxidation, most probably by way of cysteine sulphinic acid. That the reaction takes place cannot be seriously doubted, for the administration of metliionine con- taining radioactive sulphur can be followed by the isolation, not 281 cysteine: cystine only of radioactive cysteine and cystine from the tissues, but by that of radioactive taurine also. The formation of taurine is therefore believed to take the following course: CHJ5H CJI r s/° CHJ50.H CH^O.H I I N>n i - co, I CH.NH, CH.NH, CH.NH, ► CH.NH, ioon iooH i:ooH cj/sttin* fy.itfinj cytUic taurine tuljihinie acid By decarboxylation of cysteine itself fl-mercaplo-eihylamhie is formed; this is a constituent of coenzyme a (p. 356). The oxidation of cysteine to cysteic acid probably represents a step in the oxidative degradation of cysteine and cystine, the sulphur of which ultimately appears in the urine of mammals in tUo form of inorganic sulphates. Perhaps cysteine is also the source of the so-called ethereal sulphates which appear in the urine following the absorption of phenolic substances into the body: their conjugation with sulphuric acid is one of several devices involved in the detoxication of phenols. Examples of ethereal sulphates are the following: phenoltvlpAvnc indory'.tvtphuric acid otnl (vnnnry iruiican) Unlike cysteio acid and taurine, these ethereal sulphates aro true sulphates and not sulphonio acids: the direct carbon-to-sulpliur linkage characteristio of the sulphur-containing amino-acids is absent from the ethereal sulphates. Cysteine also contributes to the formation of mercapturic acids, which are the products of detoxication of certain aromatic sub- stances. A classical example of this is seen in the fate of bromo- benzene administered to dogs. The substance is eliminated in conjugation with A T -acetylcysteine in the form of/j-bromopbenyl- mercapturic acid, a remarkable achievement on the part of an METHIONINE animal that is never likely to meet bromobenzene except through the medium of the laboratory. Naphthalene abo is converted in part into a mercapturic acid: p-bromopKenyLmereapluric acid napMhyUmcrcapluric acid In addition to the inorganic and etheral sulphates, mam- malian urine contains a third fraction, the so-called ‘ neutral sulphur’. This comprises a mixed bag of sulphur compounds, including traces of thio-alcohols (mercaptans) and mercapturic acids. Not much is known about this fraction, but there is no doubt that much of it arises from the sulphur-containing amino- acids. There exists, however, a rare condition known as cystinuria , in which the neutral sulphur fraction is very large and consists mainly of cystine itself. The administration of cystine to a cysti- nuric does not, however, increase the output of urinary cystino, showing that the cystine excreted is not directly derived from that of the food. METHIONINE, S.CH, Ah.nh, Aoon (essential), is not known to give rise either to glucose or to ketone bodies. Its main known function, which it discharges in the form of S-adenosyl-methionine (p. 130), is that of a biological methylating agent. Thus it completes the biological synthesis of creatine by the transfer of a methyl group to glycocyamine (p. 228). Very probably it is the methylating agent involved in the detoxication of pyridine and certain pyridine derivatives, including nicotinio acid, for pyridine itself is excreted in the form of N-methylpyridino, and nicotinio acid partly as trigonelline, as shown on p. 284. There is evidence that methio- nine supplies a methyl radical for the synthesis of adrenaline (p. 202) and sarcosine (p. 276). By undergoing demethylation. 283 methionine: valine methionine is converted into homocysteine, the — SH group of •which can be transferred to serine in the synthesis of cysteine by way of cystathionine (p. 2S0). On account of the ease with which homocysteine can be remethylated at the expense of the methyl radicals of choline, methionine perhaps plays a part of some importance in the metabolism of phospholipids by con- verting ethanolamine into cholino and vice versa (p. 326). Homocysteine can be remethylated at the expense of choline, glycine betaine and possibly of sarcosine, but not at that of creatine. I OH" I OU- CH, CH , H-rntPiylptfriHtM IrigontlliM It has been Bhown in recent times that formaldehyde can Berve as a source of methyl groups in pigeon liver: CH^H CB,S-CH,On cn^.cu, in, + u.cho — ► bt, — *■ ^h, in.NH, is.Nii, ia.Nn, cooh ioou iooH Little is known at present about the intimate details of these reactions. V ALIKE, cn.cn, in. mi, ioOH (essential: glycogenic), was formerly believed to bo ketogcnic, in common with the other branched-chain amino-acids. Veiy little is known about the metabolism of this and the other amino-acids containing branched chains. Its conversion into glycogen has been studied extensively in the last few years largely with the aid of isotopes and it now appears that, contrary to expectation, thero is not a removal of one of the methyl groups. The first two steps follow the usual pattern — deamination and oxidative 284 LEHCINE.* ZSO-LT3UGINJS decarboxylation, leading to wo-butyrio acid. One of the methyl groups is then oxidized and the product decarboxylated to give propionic acid and carbon dioxide : CH, CH, CH,CH, Ya Ih.nh, to * COOH Aooh CH.CHO Y H AoOH CH, CH, Yl -2H toolT"* CH, Ah, Aho CH, CH, CH, CH,OH Y +H,0 Yr toOH * to OH tn, glycogen toon LEUCINE, CH, CH, Y tn, Ah.nh. (essential: ketogenic). Its conversion into ketone bodies can be explained in terms of the following reactions, but the evidence is less secure than that in the case of valine: CH, CH, CH, CH, Yh _ Yh -c Ah, * Ah, Ao Aooh AoO] CH, CH, , Y "As, ' Aooh ch, CH, Yo 'Y-*betone bodies + ' CH.COOH iso-LEUCINE, CH, Ah,ch, Y Ah.nh, Aooh (essential: glycogenic and ketogenic), might be converted into glycogen by mechanisms similar to those postulated for valine. ASPAltTIC ACID ASPARTIC ACID, COOH iir, iii.NH, ioon (non essential : glycogenic), yields oxaloacetic acid on deamina- tion or transdeamination. This product is somewhat unstable and undergoes slow, spontaneous ^-decarboxylation under physio- logical conditions of temperature and pH. The liver contains an enzyme, ^-carboxylase or oxaloacetic decarboxylase , which catalyses this reaction, of which the product, pyruvic acid, is known to give rise freely to glucose and glycogen: COO H + CO, iii, cn, io io >■ gljcogtn (bon iooa This rather rare process of /^-decarboxylation contrasts sharply with the oxidative decarboxylation that is characteristic of a-keto-acid3 in general. Aspartic acid can react with ammonia in the presence of ATP and Coxand under the influenceof an enzyme to form asparagine, which is itself non-essential and glycogenic. This system plays an important part in the storage of amino-groups in the tissues of certain plants (p. 266). Aspartic acid also plays a centra! part in transamination and transdeamination in some animal and plant tissues and plays a part also in ureogenesis by donating its amino- group to citrulline to yield arginine (p. 315). Finally, by a reaction with carbamyl phosphate, aspartic acid gives rise to carbamyl -aspartic acid in tho presence of the appropriate enzyme: COOH Nil, coon + (in, — ► io in, +HO.® n,x.iir Li— iir ioon ioon The product, also known as ureidosuccinic acid, is the starting KH, io.o© GLUTAMIC ACID material for pyrimidine synthesis (p. 347) and also plays a part in purine formation (p. 320). GLUTAMIC ACID. COOH in, in, Ah.nh, ioon (non-essential: glycogenic), yields a-ketoglutaric acid on deami- nation. The product, an a-keto-acid, undergoes oxidative derarb- oxylation in the usual way, giving rise to Buccinic acid. Succinic is converted into fumaric acid by dehydrogenation, catalysed by succinic dehydrogenase, and fumaric acid, under the influence of fumaraso, takes on water to give malic acid. Malio acid is dehydrogenated in its turn by the action of malic dehydrogenase and DPN, to give oxaloacetic acid. The latter, on /7-decarboxyl- ation, yields pyruvic acid and hence gives rise to glycogen: coon coou cooh coon coo n + co f in. Ah, -211A11 + h,o in, -211 ch, ch, I * ► I 1 ► n 1* I ► ) — *■ I — ► glycogen ch, £h, ch CHon co co io ioon Aooh Aooh A oon Aooh ioon + co. Glutamic acid can also give rise to and be formed from ‘prolint (p. 207). The reaction involves glutamic-y-semialdehyde and , reduced DPN or TPN : cn,— CH, +2H An, Ah .coon ch, — ch, ch,— ch, ioon in. cooh -*Aho Ah.i cn,— ch, .♦Ah Ah. cooh (DPN) \ N / The existence of a glutamic-a-decarborylase in animal tissues accounts for the formation of y-o.minobu.tync acid , which occurs in brain tissue and elsewhere: H,N .CH,CH,Cn,COOn. Like /7-alanine this is a somewhat uncommon and thoroughly atypical amino-acid and perhaps it is worth noticing that 287 OI/DTAM1C ACID /J-alanino might conceivably arise by the action of an analogous enzyme upon aspartic acid. Glutamic acid together with glutamic dehydrogenase plays a central part in transamination and transdeamination, and is a constituent of glutathione, while its amide, glutamine, occurs in considerable quantities in many plant and animal tissues, in which it represents a store of amino groups. Glutamine, like glutamic acid, is non-essential and glycogenic, giving rise to glutamic acid and free ammonia under the influence of glutaminase (p. 101). It is formed from glutamic acid and ammonia by an endergonio process involving ATP (p. 124). There is evidence too that it can lose ita amido-group and simultaneously transfer its a-amino-group to certain a-keto- acids by a special kind of transamination process. In addition, glutamine plays a special part in ammonia storage (p. 205) and also in the detoxication of aromatio acids, though only among Primates, according to present information, Fhenylacetic acid is excreted in the form of a conjugate with glycine by most animals, but in man and the chimpanzee it gives rise to phenyl- acetylglutamine: CO. Nil, l'II.KII — OC. ioon pkenylacetylgl ulamine Glutamine is also involved in uricogenesis (p- 320); acetyl glutamate and carbamyl glutamate can act catalytically in ureogenests (p. 314). JBBt A HOI XI XE, HN=CC >XH til. Nil, coou (‘half-essential’, see p. 252: glycogenio), can lose ita amidine group under the hydrolytic influence of arginase to yield 283 ARGININE: OITRITLLINE ornithine, which is also glycogenic. There are indications that arginine does not normally undergo deamination (p. 263). Arginine can also part with its amidine radical by participating in group-transfer reactions, i.e. by transamidination. It plays a vital part in the synthesis of urea by the so-called ornithine cycle of Krebs ; the mechanisms leading to its resynthesis from omitliine are considered elsewhere (pp. 314-15). Arginine occurs much more extensively in invertebrates than among vertebrates, for it forms the guanidine base of the commonest of the in- vertebrate phosphagena (p. 334): in addition, arginine is probably the parent substance of some at least of the peculiar guanidine bases found in invertebrate tissues such, for example, as octopine and agmatine (Chap. xm). yNH, C1TRULLINE. 0=C< \s T u (in,)* CH.NH, iooH (non-essential: glycogenic), does not, so far as is known, enter into the composition of proteins, but occurs as an intermediary between ornithine and arginine in the ‘ornithine cycle ’ (p. 314). It can transfer its carbarayl radical to aspartic acid, yielding carbamyl aspartic acid (p. 322). ORNITHINE, NH, (Aim. CH.NHj Aooh (non-essential: glycogenic), has not been isolated from protein hydrolysates, but arises from the action of arginase upon arginine and participates in the * ornithine cycle *. It yields citrulline by ac- cepting a carbamyl radical (— CO.NH*) apparently from carbamyl phosphate (see p. 314). There is some doubt whether it under- goes biological deamination (cf. lysine, p. 263) but it has been shown in feeding experiments that deuterium-labelled ornithine gives rise to glutamic acid and hence, presumably by way of the y*semialdehyde, to proline. Both of these products arc glycogenic. >9 ornithine: lysine Among birds, ornithine discharges a special function in therf<- toxication of aromatic acids such as benzoic: the latter is excreted La the urine of birds in the form of dibeazoyloraithine, or orat- thuric add (p. 125): pO (CHJ, in.Nii— oc^^) COOK onutAvne acid NH, (in,), nh, jwtrctcine By bacterial decarboxylation in the intestine and elsewhere, ornithine can givo rise to the toxic diamine putresdne. Small amounts of this substance are absorbed by animals and are deioxicaJed , probably undergoing oxidation at the hands of diamine oxidase (p. 156). LTSIS'E, Nil, (iiij. CH.NH, ioon (essential: not known to be glycogenic or ketogenic), is the next higher homologue of ornithine. It is believed not to undergo deamination in the body {p. 203) but is mctabolically degraded in other ways. The details are still far from clear but the fol- lowing sequence of reactions has been suggested and bos a cer- tain amount of experimental evidence to support it: nh. in. + Nil, T COOK coon COOII in, in, in, iii. u in. i„. ~iu. — .in, — . I 1 Ju. at. cn. iooH ■h t crusty itt.XK* In ioon coon ioon CO, a-Omino- a-letu. glutaric adipic acid adipic acid acid The fate of glutaric acid is unknown. 290 PHENYL alanine: TYBOBINE Very little is known about the specific functions which, as an essential amino-acid, it must be presumed to discharge in the organism. Under the influence of bacterial enzymes it can yield cadaverine. H 1 N'(CH 1 ),NH„ which, like putrescine, is highly toxic and can be oxidatively detoxicated by diamine oxidase. PHENYLALANINE, ^cn,cH{Nn,)C00H (essential: kctogenic), and TYROSINE, CH,CH(NH,)COOH (replaceable by phenylalanine: ketogenic). It may be presumed that phenylalanine is irreversibly convertible into tyrosine (p. 251), but we have no certain knowledge of the mechanisms in- volved. These important amino-acids are known to be ketogenic and it is usually considered that the ring must be opened in the process because the side-chain contains only three carbon atoms whereas four are required for the production of acetoacetic acid. Work with isotopically labelled phenylalanine and tyrosine indicates that the a- and /?-carbon atoms of the side-chain and two more from the ring furnish the four carbon atoms of acetoacetate. Recent experiments suggest that the following reactions are involved, though the intermediate stages have not yet been completely worked out: ^^cn.ciUNii.jcoon — » no^n.cHtNH^coon phenylalanine tyrosine 03 CII — ci \ no^^ai.co.coon p- hydroxy phenyl pymne acid -cii cn.crr.cooH cn co.cn.coon — ra.coon N b=*dn ''boirfH, ch.co.ch.cooh ill hrmojentitie arid fumaryl acetoaeelic acid 291 Ip*3 PHENYL ALANINE: TYROSINE Enzymes capable of catalysing these postulated reactions have been found in liver preparations of various kinds. The metabolism of these aromatic acids goes astray in a group of interesting hereditary ' inborn errors of metabolism ' presumably because of the deficiency or absence of one or more of the numerous enzymes concerned in the normal metabolism of these aromatic amino-acids. A peculiar form of mental deficiency known as imbecillilas (oligophrenia) phenylpyruvtca owes its name to the curious fact that the urine of the afflicted regularly contains small amounts of phenylpyruvic acid. In albinism the enzyme tyrosinase is completely lacking, and the dark-coloured melanic pigments (p. 161) are characteristically absent from the skin, hair, eyes and other usual situations. A single case of tyrosinosis has been reported, the urine in this disorder con- taining traces of tyrosine as a regular feature. Alcaptonuria is another disorder in which the metabolism of the aromatio amino-acids appears to be blocked, and hamogenti&ic acid is found in the urine (p. 21C). Phenylalanine and tyrosine are believed to bo of particular importance in animal metabolism as the parent substances of two hormones, adrenaline and thyroxine. This belief has been confirmed by the use of isotopes. Tyramine can bo formed from tyrosine by the action of a weak, specific tyrosine decarboxylase that occurs in the kidney and liver of mammals, and may possibly be an intermediate in tbo elaboration of adrenalino. If so, it must presumably be attacked by the monophenol oxidase component of tyrosinase to yield the amino corresponding to dihydroxyphenyl- alanine dopa’). It is known, however, that dopa can be formed from tyrosine by the action of tyrosinase and, moreover, that there exists a specific dopa decarboxylase which, acting upon dopa itself, could yield the same amine once more. Little ia known about the remaining stages on the route to adrenaline, but the possible stages just described may be summarized in tho manner shown on p. 293. The tf-methyl group of adrenalino is probably transferred from methionine, for administration of methyl -labelled p'C) methionine leads to tho production of iY-m ethyl -labelled adrenaline in tho adrenal medulla. Thyroxine is a heavily iodinated derivative of tyrosine but very littlo is known about its biological formation. It occurs, 292 PHENYL ALANINE: TYROSINE together -with di-iodotyrosinc , in tho thyroid tissue of vertebrates. Di-iodotyrosine, together with the corresponding dibromotyro - sine, has also beezi described as a constituent amino-acid of the skeletal protein material of a coral, Gorgonia, for which reason these kalogcnated tyrosines are sometimes called iodogorgoio and bromogorgoic acids respectively. CH,CH(NH,)COOH di-iodolyrotin* cn,cn(Nn,)cooK dilromotyroaint CH 1 CH(NH,)COOH V thyroxxw Phenylalanine and tyrosine give rise to a series of toxic pro- ducts when submitted to bacterial attack. These include phenol, p-cresol, tyramino and phenylethylamine. The amines are pro- bably oxidatively detoxicated by amine oxidase, and the phenols by conjugation, usually with sulphuric acid. UISTJDINE, j===jOT»CH(NH,)COOir V/ (essential: neither glycogenic nor ketogenic), occurs in combina- tion with /7-alanine in the dipeptido camosine (p. 338), which is nxsTtDisE: TRYPTornAK present in the muscles of vertebrates of most kinds, though invariably absent from invertebrate tissues. The analogous com- pound anserine (p. 330) ia similarly distributed and also contains /J-alanine, but histidine is here replaced by ita 1 -tf -methyl deriva- tive. The methyl group arises by transference from methionine. Comparatively little is known about the metabolism of histi- dine, though it was at one time thought to be concerned in the synthesis of purines. Animal tissues, especially the liver, are known to contain enzymes winch open the imidazole ring and yield, through a series of reactions, glutamic and formic acids, together with ammonia. The first step leads to the formation of urocanic acid and i3 catalysed by a specific hislidase, p=*ecn 1 CH(NH t )cooH t p=pr=cn.cooH K NH — Nil, N Nil V" V/ urocanic acid Histidine is the mother substance of histamine , being attacked by a specific histidine decarboxylase, traces of wliich are present in liver and kidney. Histamine can also be produced by bacterial activity in the intestine and elsewhere, and is detoxicated by a histamine oxidase, wliich may bo identical with the diamine oxidase of animal tissues, though its distribution is a little peculiar. TRYPTOPHAN. (essential: not known to bo cither glycogenio or ketogenic). This amino-acid gives rise, when administered in fairly largo doses, to the excretion of bynurenic acid. The product ia formed by way of kynurtnine, apparently through the following reactions: tryplopka* 2-tyJroxytryptojJmn 204 TBYPTOFHA.S :o.cn»cn(NHocooH I.N'U, kynurenine • 0 r o-aminobemoylpyruvie OH oeid lynurenic acid There is evidence that kynurenine may also be metabolized to yield nicotinic acid in the animal ; this is the only known case in which a vitamin can be produced from an essential amino-acid. 3-Hydroxyanthranilic acid, which can be formed from kyn- ureninc, i 3 known to be a precursor of nicotinic acid and the intermediate stages are thought to include the following: :.CH,Cn(NH,)COOH V kynurtnine Qr- 3 -hjdroxy- anlhramlic acut a NU, COOH [uiutahU intermediate) lo.jCH.cnpiHjcoon Lvn, Z-KydroxyhynureniM -a COOH -a -Cr N ** norm nicotinic acid c°,/\ • (Jbo. picolinic Bacterial decarboxylation of tryptophan leads to the forma- tion of a poisonous amine, tryptamine, which is pro a y e atroyed by amine oxidase, like other amines. Further a of the side- chain by bacteria is also possible and leads to the formation of a pair of foul-smelling compounds, indole and .forlok; these are Bald to be largely responsible for thB odour of 295 tryptophan: pbolinr faeces. Indole undergoes biological conversion into the corre- sponding alcohol, indoxyl, which, if absorbed, is detoxicated by conjugation with sulphuric acid and excreted in the urine in the form of the corresponding ethereal sulphate: tjuWe indoxyl indoxyltvlphurit acid (urinary indican) Finally, mention may be made of two natural pigments that are of some historical interest and are related to tryptophan, viz. natural indigo, from the woad and indigo plants, and the Royal or Tyrian purple of tho ancients, which can be prepared from a variety of marine gastropod molluscs, the classical source being Mur ex spp. Natural indigo arises from a ^-glycoside of indoxyl that is present in tho plant juices and undergoes decom- position when the tissues are bruised ; free indoxyl is thus formed and undergoes oxidativo coupling in tho presence of oxygen to yield indigo. Tyrian purple is similarly formed from a derivative, thought to bo a mercaptan, of 4-brom -indoxyl : natural indigo Tyrian pvrpU (i-i'dUnminJigo) PH0L1NE, H,C CH, H.C dll COOK Jl (non-essential-glycogenic) js, strictly speaking, an imino-rather than an amino-acid. Little is known about its metabolism, apart from the fact that proline oxidase, which is present in kidney tissue, can open the ring and give riso to glutamic acid. This may perhaps bo a preliminary to the deamination of proline. The ring opening is not a hydrolytic but essentially an oxidative process. 29C PROLINE: HYDROXYPBOLINE Since glutamic acid is glycogenic, the formation of glucose and glycogen from proline can be understood: H,c CH, t | proline oxidise J I CH.COOH -coon CH.COOH Xjf/ H CH, CH, Ah.c I™, There is evidence that this process can in Borne way be reversed since glutamic acid can act as a precursor of proline itself (p. 287). There is also evidence that proline can arise from ornithine by way of gl u tamic-y-semialdehyde (p. 289). BYDROXYPROUNE , HO.CH — CH, H.t ilH.COOH \ N / H (non-essential : glycogenic), like proline, is an imino- acid. How it is converted into carbohydrate we do not know: conceivably it might first be reduced to proline and then attacked by proline oxidase. Alternatively, it might perhaps be attacked directly by proline oxidase or some similar enzyme to give y-hydroxy- glutamic acid, which occurs in nature and is known to be glycogenic. CHAPTER XII EXCRETORY METABOLISM OF PROTEINS AND AMINO -ACIDS NATURE OF THE NITBOOENOUS END-PRODUCTS Tiie great bulk of all the nitrogen entering a typical animal arises from the a-amino-nitrogcn of its food proteins. In mam- mals, at any rate, this is split off by deamination or transdeami- nation in the form of ammonia. Small quantities of ammonia also arise from other sources, such as the deamination of amino- purines and ominopyrimidines, but the great mass originates in tho food proteins. The tissues have little capacity to store proteins or amino-acid3 as such, but considerable amounts of ammonia can undoubtedly be stored in animal tissues in the form of the amide-N of glutamine. Tho storago capacity with respect to ammonia-N is, however, small compared with the average daily turnover of protein and amino-acid nitrogen. In plants, which have no excretory apparatus, larger quantities of nitrogen can bo stored, either in the form of asparagino or glutamine or both, according to the species, but in animals, which possess efficient excretory machinery, the superfluous nitrogen is excreted and tho excreta of animals contain a variety of nitrogenous substances of varying degrees of complexity. Only among invertebrates do wo find significant amounts of amino-acid nitrogen being excreted as such, and in certain In- vertebrate groups os much as 20-30% of the total nitrogen ingested may be excreted in the form of unchanged amino-acids. Whether this is due simply to leakage of amino-acid3 from the body fluids of these animals, or whether it indicates some BOrt of metabolic disability we do not know. In marine invertebrates, at any rate, it is known that the surface membranes aro perme- able to water, to ions and to small molecules, and it is therefore possible that amino-acid molecules might be lost by diffusion to some extent. Up to the present we have dealt mainly with the nitrogenous TOXICITY OF AMMONIA metabolism of mammals, chiefly because so much more is known about them than about any other group of animals. But there is reason to think that animals of every kind possess digestive enzymes capable of dismantling their food proteins completely to yield the component amino-acids. Although an excretion of unchanged amino-acids is observed among many invertebrates, the greater part of the ingested nitrogen is excreted in the form of ammonia, even among these animals. It is probable that all animals deaminate at any rate the greater part of their incoming amino-acids with production of ammonia. Whether the deami- nating machinery is always the same or even of the same general kind we do not at present know, but the evidence points to a large-scale production of ammonia by transdeamination in all animals. Now ammonia is a very toxic substance. Just how toxic it is can bo appreciated if wo consider some experiments carried out by Sumner, who injected crystalline urease into rabbits. The blood of the rabbit contains a small amount of urea and, from this urea, ammonia was formed by the hydrolytic action of the enzyme. The animals died as soon as the concentration of ammonia in the blood rose to about 6 mg. per 100 ml., i.e. about X part in 20,000, a very high order of toxicity indeed. Death occurred before any change in the pH of the blood could be detected, and it is therefore probable that the toxicity of ammonia is due to some specific property of the ammonium ion rather than to the basicity of ammonium hydroxide. It is extremely improbable that death was due to any toxic properties of the enzyme itself, for urease was also injected into birds, the blood of which does not normally contain urea, and in this case tho animals were unharmed ; but fatal results followed the in- jection of urease together with urea. If we examine the excreta of many different kinds of animals, representing as many different phyla and classes as possible, we find that among the nitrogenous substances present, some one compound always predominates. Over and above the traces of assorted odds and ends such as creatine, purines, betaines and the like, we find either ammonia, urea or uric acid accounting as a rule for two-thirds or more of the total nitrogen excreted. In a few special cases, some compound other than these 209 EXCRETION OF NITROGEN predominates, but such cases are rare and, in fact, animals as a whole may be divided rather sharply into three groups according as their main nitrogenous excretory product is ammonia, urea or urio acid. These three groups are respectively said to be ammonioletic, wreoteftcand uricotelic. Tins discovery raises several important problems. First we must inquire why some animals are content to excrete their waste ammonia unchanged, and why others convert ammonia, the primary product of deamination, into secondary products in the form of urea and urio acid. Then we must ask why it is that, among animals that do elaborate these secondary products, some produce urea and others uric acid. Finally, we have to inquire into tho mechanisms whereby these ultimate end-products are synthesized. The nature of the predominant end-product in any particular case seems to he conditioned by the nature of the habitual environment of the particular organism, and the known facts are best explained on the supposition that the conversion of ammonia to other products is an indispensable adaptation to limita- tion of the availability of water. If we consider the invertebrates first of all it may he said at once that they fall into a very large group of ammonioteles on tho one hand, and a much smaller group of uricotcles on the other. Aquatic invertebrates, almost without exception, are ammonio- tolic. Ureotelism seems not to have been developed by members of the invertebrate phyla, while uricotelism is found only among terrestrial representatives of groups which, like the insects and the gastropod snails, have succeeded in colonizing the dry land. Animals living in water have at their disposal a relatively vast reservoir into which they can discharge waste ammonia, a rela- tively diffusible substance, without running any grave risk of being poisoned by their own excrement. Terrestrial invertebrates, on the other hand, are often hard put to it to find enough water for their essential needs, and tho impossibility of disposing of ammonia fast enough to avoid toxaemia is overcome by the biological conversion of ammonia, a very soluble and highly poisonous material, into the insoluble and relatively innocuous nric acid. Terrestrial woodlice, however, are ammoniotclie, and in their case adaptation to terrestrial existence has been achieved, not by uricotelism, but by on over-all reduction in protein 300 COMPARATIVE ASPECTS metabolism. The daily turnover of nitrogen here is of the order of oniy 10% of that of related marine and fresh -water species, and thi3 suppression of protein metabolism is certainly the simplest and probably the most primitive device for combating the dangers of ammonaemia. The general picture appears particularly clearly among the vertebrates. Taking the fishes first, it must be remembered that they fall into two main classes, the teleosfcs, or bony fishes, and the elasmohranchs, or cartilaginous fishes. Each of these classes is well represented in fresh and in salt water alike. To appreciate their position in the matter of water supply must involve a short digression. The lives of aquatic animals are complicated by a factor which terrestrial creatures like ourselves have little reason to appre- ciate. The fact that an animal is aquatic does not necessarily mean that it enjoys an unlimited supply of water. Among marine invertebrates the membranes bounding the body surface are, as a rule, more or less permeable to water and to small molecules. Ammonia formed in the body of such an organism can therefore escape comparatively readily by diffusion into the external en- vironment. In fresh-water invertebrates, however, the boundary membranes are much less permeable: indeed, the main part of the body surface is impermeable to salt9, and not very permeable even to water. This impermeability is important because the cells and tissues can only survive in the presence of considerably higher concentrations of salts thin are present in the surrounding water, and surface impermeability is a device that serves to prevent leakage of salts out of the animal. But even so, the animal not only lives in water, it breathes in water, and this means that, in certain organs specialized for the purposes of respiration, the animal’s blood comes into very close proximity to the sur- rounding water. In these respiratory organs, oxygen is taken into the blood and carbon dioxide eliminated, and the membranes of these organs have necessarily to be freely permeable to dis- solved gases. It seems that the necessary degree of permeability to dissolved gases is inseparable from an appreciable degree of permeability to water. In the respiratory organs of a fresh- water animal, therefore, we find membranes that are perme- able to water, though impermeable to Balts; they are, in fact, 301 COttPAEATlVE ASPECTS approximately semiperraeable. On tho outer side of these mem- branes we have fresh water, which is virtually free of dissolved salts, and on the other lies the animal’s blood, which contains on the average about 1 % of dissolved salts. For this reason there is a considerable osmotic force tending to drive water into the animal from outside. The entry of this water leads to dilution of the salts of the blood, but aquatic animals possess elaborate Balt-absorbing and excretory organs which enable them to turn out the unwanted water, while maintaining at tile same time a constant internal salinity. A fresh-water invertebrate may therefore be pictured as having a constant, osraotically-driven current of water passing through its body. Any ammonia formed in the cells and tissues can diffuse into the blood of such an animal and be carried away with the water in the form of a copious but very dilute urine. The position is substantially the same for fresh-iraler tdeosts. Ammonia formed in the tissues escapes rapidly and readily by way of the urine, and hero, as in aquatic invertebrates, there is no danger of toxaemia duo to the accumulation of ammonia. For marine teleosta x however, tho position is considerably more difficult. The gill membranes and the mucous membranes of the mouth are apprcciftbty permeable to water, as they are in the fresh-water forms. But sea water contains about 3% of salts as against the 1 % or thereabouts present in the blood, and the osmotic flow of water tn this case is therefore away from the fish, instead of towards it. Marine tcleosts, therefore, although they inhabit a watery medium, are nevertheless poorly supplied with water. They lose water constantly to their environment and arc liable to die of desiccation, unlike their fresh-water relatives, whose lives are constantly imperilled by the imminent threat of flooding.* The nitrogenous excretion of marine tcleosts has been investi- gated in a very ingenious experiment devised by Homer Smith. A wooden box is divided into two compartments by a watertight rubber dam pierced by a hole large enough to fit closely round tho belly of a fish. The animal is placed in the apparatus, which • For further inforraition rrjj»Ttiiiijt tbr osmotic regulation of aquatic animat*, •eo Baldwin, An Inlradueium lo Comj^rntut fiuxhtmUlry ; also Krogk Rrp&sho* in Ajvai le Animal*, for detailed Information. 302 COMPABATIVE ASPECTS is filled with sea water, in such a way that the head and the gills are accommodated in one compartment and the tail and the excretory aperture in the other. After a suitable time, samples of water from either compartment are withdrawn and analysed for nitrogenous compounds, and it then appears that 80-90% of the total nitrogen excreted is found in the forward compart- ment and must therefore have been excreted by way of the gills, only a small part of the whole being evacuated by way of the kidneys About two-thirds of all the nitrogenous material ex- creted consists of ammonia, indicating that, although the fish has a comparatively poor water supply, it can, nevertheless, dispose of most of its ammonia by diffusion across the gill mem- branes, without previously converting it into any less noxious nitrogenous compound. The remaining third is not present in the form of urea, nor yet as uric acid; indeed, it defied identifi- cation for some time, but turned out in the end to be IrimelkyU amine oxide (CHjJjN - -*■ 0. This is a practically neutral, soluble and innocuous material. There is no record of the occurrence of significant amounts of trimethylamino oxide in the tissues or in the excreta of fresh- water teleosts, though its presence in the tissues and excreta of marine forms has been abundantly confirmed. The possibility that it represents a detoxicated form of ammonia has therefore to be considered (but see p. 322). The elasmobranch fishes present a slightly more difficult problem. Marine elasmobranchs produco and retain within their bodies large amounts of urea. Retention of urea in the blood and tissue fluids of these fishes is possible because the gills are impermeable to urea, while the kidney possesses a specialized mechanism that can control the loss of urea from the body. Enough urea is always retained to keep op a concentration of 2-2-5 % of urea in the blood ; over and above this concentration, Urea is excreted, and the elasmobranch fishes are, in fact, ureo- telic. The presence of so much urea in the blood raises the total osmotic pressure of the blood to a level slightly higher than that of the surrounding sea water, and these fishes therefore escape the constant loss of water which threatens the existence of the marine teleosts. Instead of losing water to their environment, they constantly receive an osmotically driven stream of water 303 COMPARATIVE ASPECTS from the sea. By resorting to ureotelism, therefore, the marine elasmobnmchs are not only protected against toxaemia due to ammonia but, by retaining some of the urea they produce, now find themselves in a very favourable position as regards water supply. Like marine teleosts, the marine elasmobranclis excrete a part of their waste nitrogen in the form of trimethylamine oxide. This suggests that they have at some time in the past experienced the same osmotic difficulties os confront the marine teleosts of the present day, and that they faced them by making urea, which is even less toxic than trimethylamine oxide, and, by retaining enough of it in their tissues, managed in the end to turn the osmotic gradient to their advantage instead of their detriment. The fresh-water dasmobranchs are believed to be descended from their marine cousins, which they resemble in being urco- telic, although in their cose the amount of urea retained is only of the order of 1 %. Thus, oven among the fishes, an essentially aquatic group, we find ureotelism already well developed. But no discussion of the fishes would be complete without some mention of the Dipnoi, or lung-fishes. These creatures inhabit swamps and rivers in tropical regions. During the hot season the water dries up, and the lung-fishes shut themselves up in cocoon-Iiko structures in the mud to wait until the rains come. As long as water is available, theso fishes behave lika fresh-water teleosts and are essentially ammoniotelio. But during the period while they lie dormant and cut off from the water, they switch over to ureotelism and, when the rains come and the rivers fill again, almost their first act on emerging is to excrete a mass of urea that has accumulated during their aestivation. Thi3 cose is a particularly interesting one, since it constitutes a test case of the validity of our general hypothesis — that the detoxication of ammonia is essentially an adaptation to restriction of the water supply. Going on now to tho Amphibia, the frogs, toads, newts and the rest, wo arc in the company of animals which arc able to spend longer or shorter periods away from tho water. We should expect, in tho terms of our hypothesis, that no animal could live long away from water without exposing itself to the hazards of ammonia -poisoning and, indeed, that the colonization of dry 304 COMPARATIVE ASPECTS land could hardly have been begun until some mechanism had been evolved by means of which ammonia could be detoxicated. The Amphibia would be expected, then, to be either ureotelic or uricotelic . Weget some very interesting evidence here by studying the humble tadpole. Tadpoles are aquatic and ammoniotclic. Later, the tadpole undergoes the metamorphosis that changes it from a wholly aquatic animal into a true amphibian and, at the same time precisely, it also undergoes a chemical metamorphosis and abandons ammoniotelism in favour of ureotelism. The adult frog, in common with the adult forms of other Amphibia, is ureotelio, and it seems as though, in the course of its develop- ment, it recapitulates some of the essential features of its evolu- tionary past. Table 24. Nitrogen excretion of some ohelonian reptiles {Averagos, after V. Moyle) Habitat % of total N as Amtnoma Urea Uric acid Wholly aquatio Serai-aquatic Wholly terrestrial: lly graph lloua Xerophilous 20-25 20-25 5 6 40-00 5 . 6 30 7 5 10-20 50-60 The rest of the vertebrates are generally considered as having evolved from Borne primitive kind of amphibian stock which, wo may reasonably suppose, must have been ureotelic. Leading from the Amphibia we find two main, diverging lines of evolu- tion, one leading to the mammals and the other to the reptiles and the birds. There exists among the reptiles one group, the Chelonia (tortoises and turtles) which are of rather particular evolutionary interest as a transitional group, at any rate from the chemical point of view. Probably the original chelonian stock was terrestrial or amphibious, but to-day we find wholly aquatic, semi-aquatic and wholly terrestrial species. Table 24 presents some data relating to the nitrogen excretion of some of these animals. Those which are terrestrial but favour marshy surroundings are essentially ureotelic; little ammonia or uric acid is produced. In wholly aquatic species a significant degree of ureotelism is still retained, but the ratio of urca-N to ammonia -N 305 COMPARATIVE ASPECTS is much lower, suggesting that theso forms tend to revert towards an ammoniutelic habit. But in the dry-living and wholly terrestrial forms wo find that, although urea formation still persists, the hulk of the total N is now excreted in the form of uric acid. Other dry-living reptiles, tho Sauria (snakes and lizards), together with the birds, have altogether abandoned urcotelism in favour of uricotelism whereas the mammals have continued in the amphibian manner and clung to the more primitive ureotclic habit. Joseph Needham considers that tho choico between urco- and uricotelism is determined by tho conditions undor wluch em- bryonic development takes place. Embryos developing in eggs that are laid in water can probably dis]>oso of ammonia by Birapio diffusion and have no need to detoxicate it. Things are different for the embryos of terrestrial animals however. The case of tho Chclonia has not been very thoroughly studied, but tho position is much clearer in the other great ureotclic group, the mammals. There still remain a few egg-laying mam- mals, c.g. Echidna, and their eggs are incubated always in wet situations. Not much is known about the water relationships of theso eggs, but it has nevertheless been established that the adults are ureotclic. The rest of the mammals undergo embryonic development in intimate contact with the maternal circulation. Food materials diffuse from the maternal blood stream across tho placenta to tho embryo, and waste products can likewise diffuse back across the placental barrier to bo excreted by tho maternal kidneys. Tho niammaban embryo, with tho entire water re- sources and the excretory apparatus of tho maternal organism, at it* disposal, has no need to do otherwise than remain ureotclic and excrete its waste nitrogen by proxy. Conditions are very different in the eggs of certain tortoises and all malts, lizards and birds. These eggs are laid with a supply of water just sufficient to see them through development, and no more hi to he had, apart from, metabolic water formed by the oxidation of food reserves as development proceeds, because tho eggs are surrounded by tough membranes or hard shells which aro practically impermeable to water. In such a system the production of ammonia could be nothing short of disastrous. 30 « COMPARATIVE ASPECTS Urea would be a more suitable end-product if only because it is relatively harmless, but apart from the elasmobranch fishes, no organisms are known that can stand up to more than a very jnild uraemia without more or less serious disturbances of normal physiological function. Needham has calculated that, if the waste nitrogen actually turned out during the embryonic de- velopment of the chick were converted into urea, the resulting uraemia, by human standards, would be sufficient to give the embryo a bad headache at the very least. ‘In which case’, as Needham says, ‘natural selection would hardly have preserved it for our entertainment.’ Embryos which develop in these closed-box, or ‘cleidoic’, eggs solve their problems by the con- version of waste ammonia, not into urea, but into uric acid, and the habit of uricotelism which they acquire during embryonic existence persists into, and throughout, their adult life. Whereas urea is a very soluble compound, the excretion of which requires a comparatively liberal supply of water, uric acid, which is almost equally innocuous, is exceedingly insoluble and can simply be dumped in the solid form. It is carried away from tho embryo proper and deposited in a little membranous bag, the allantois, the contents of which include solid nodules of uric acid at the end of development. Finally, mention may bo made of a case of chemical recapitu- lation comparable to that already mentioned in the case of the frog. Needham has studied the nitrogenous excretory products of chick embryos at different stages throughout development. His results, which are shown graphically in Fig. 25, show that at the very beginning of development the chick produces am- monia, like an aquatio animal. This is quickly switched off in favour of urea, the embryo behaving for a time like an amphibious animal. Finally, tho chick appears in its true colours, as a truly terrestrial, uricotelic organism, developing inside a cleidoic egg. To summarize, we may make the following statements. Among invertebrates, the ammoniotelic type of metabolism is found in aquatic animals; ureotelism appears not to be employed, while Uricotelism is confined to organisms that have become adapted to life under terrestrial conditions. Among vertebrates, ammonio- telism is confined to animals that are entirely aquatic and, even 307 COMPARATIVE A8FECTS among these, ureotelism and perhaps trimethylamine oxide for- mation have been exploited by animals whioh, though aquatic, experience considerable shortage of water. With the conquest of the land, ureotelism appears to have been generally adopted and is still found to-day among the Amphibia. It has been retained by the mammals, whose embryos have the water of the maternal blood-stream at their disposal. The dry-living reptiles, together Fig. 25. Nitrogenous excretion of developing chick embryo (tiler Needham). Maximum of | Days Ammonia I 4 Urea 0 Uric acid ] II Sole difference* in the scale*. with the birds, have abandoned ureotelism in favour of urico- telism, a change wliich is associated with embryonic develop- ment under the conditions of acute water shortage implied by the clcidoicity of the egg. Tlius the detoxication of ammonia by conversion to urea or uric acid appears in every case to bo intimately associated with limitations of water supply. A more pictorial form of summary is given in Tablo 25. SYNTHESIS OF THE END-PRODUCTS: UREA Most of the early work on the biological synthesis of urea was, not unnaturally, carried out on mammalian materials. It will be recalled that hepatectomy in the dog leads to cessation of urea production, an observation that points to the liver as the solo seat of urea synthesis in the mammalian organism. Work with 308 TJREOGBNESIS perfused mammalian liver confirmed this conclusion, for a syn- thetic formation of urea from added ammonia was readily demonstrated. Little was known about the mechanisms of the synthesis for many years, although several alternative theories were expounded. Table 25. Nixboqen excretion of vertebrates IN RELATION TO WATER SUPPLY Environ- Water Uric Group ment Bupply (CH.hN—O NH, Urea acid Piscea: Telcos tei FW Abundant _ + _ — SW Poor + + - - Elaamobranchii FW Abundant _ + _ sw Good + - + - Dipnoi FW Abundant _ + - _ T* None _ _ Amphibia: Urodela FW Abundant _ + w Anura: Tadpole FW Abundant _ + - - Frog Reptilia; Chelonia FW/T Good/poor " " + - FW Abundant _ + + - FW/T Good/poor - - + - T Poor - - + + Sauna T Poor - - - ■b Avea T Poor _ - - + Mammalia T Poor - - + - FW ■•fresh water; 8W—sea water; T — terrestrial. * During aestivation. The existence of a urea-producing enzyme in mammalian liver was suspected at the end of the last century, following the dis- covery that, if liver tissue is allowed to autolyse, urea is produced. ICossel and Dakin showed that this urea originates in arginine (set free by autolysis of the tissue proteins) under the influence of a hydrolytic enzyme which they called arginase, and which cata- lyses the following reaction : JfH, ,NH, HN=e< + 11,0 - II,N~C/ XjfH ^0 + (in.). cn. nii, iooH arginine ti rta NH, (An,), Ah.nh, COOH ornithine 309 DBEOOENE8IS It vaa Clementi who first drew attention to the striking fact that, while arginase occurs in high concentrations in the liver oj ureoldic animals, it is present in traces at most in the liver of those which are uricotelic (Table 26). It was already clear that arginase could be held responsible for the production of somo of the urea excreted by mammals, inasmuch as arginine occurs in considerable amounts in most proteins. Arginine arising from the food proteins could therefore account for some, though not by any means for all, of the urea formed. Table 26 . Distribution or arqinase in liver AND KIDNEY OF VERTEBRATES (Data from Clementi) Class Species Liver Kldne Mammalia Dog Ox r and glutamine, hypoxantlune f°™arion ha, now been traced through the somewhat ^hy aenes of reactions shown on p. 3=0. Env.ymcs capable of ca talymng al these reactions have been found in pigeon iver on , products isolated, though information aofaC *™ “cd possible intermediates is not yet complete. Al the postulated Intermediates are convertible into uno acid in the pigeon. URICOGENESIS The initial reaction (1) appears to take place between PItPP and the amide N of glutamine, thus: r*. m r Tho product, represented below as ©K-NH S , now reacts first with glycine (2) and then with formate (3) to givo glycinamidc- ribosyl-S-phosphale. and Jormylgbjcinamide-ribosyl-S-pho&phale: <4 2 ) NIT, + glycine J,TI It© It© NH efr, '''cno to (3) \ •f formate Nil (4) {Co r) It© + NII, NH c(i \no l vjf \ t© In the next step (4) an amino-group is transferred, again from glutamine, followed by ring closure (5) : G-amino-iminazoli-ribotyl' 5'-f\a»phatt Now a carbamyl radical is transferred from carbamyl aspar- tate (C) toyic]dG- - xanthine. By oxidation of the latter at the an o oxidase xanthine is produced and further oxidized I by the same enzyme to uric acid. The further metabolism of uric acid is discussed later (pp. 359-61). 0 _ rnf i lpH : B In the meantime one interesting aspec o p 5™ ^ may be mentioned. Uric acid arises from ypoxan of xanthine through the action of .“tarn uricotelic animals, replaces the urea produce y ■ ureoteUc. No matter which end-product amyl groups play an important part m the^arlier^ineoteUsm 3 te Steibm w^lccompShed, not so ™ of entirelymew enzjMt^achmery^as that^arbamyl phosphate ^ar^^oiua.eariumdioxideandAWin^P^e of certain glutamic acid derivatives m ureotehe same may well be true of uricoteles. In radicals are transferred to ornithine, yie g __ ’ rtate uricotelio auimals carbamyl groups are transferred to aspartate BDA TTRICOOENESIS and then am utilized in the synthesis of the pyrimidine ring of the purines. Citrulline, which can transfer its carbamyl group to aspartate, is perhaps a common intermediate to both: NH,+CO, +ATP glutamic dcriratire arginine urea -v- carbamyl phosphate Little work has been done on uricogcnesis in tho reptiles. As far as the uricotelic invertebrates are concerned, a good deal of work has been done, but the results arc, at best, inconclusive. The outlook in this field has been much prejudiced by the view, now abandoned in so far as it affects uricotelic vertebrates, that urea is an intermediary in the synthesis. The best attitude to adopt at the present time is ono of ignorance. BYNTHE8I8 OF OTHER END-PRODUCTS Trimelhylamine oxide. Feeding experiments have been carried out with young salmon to elucidate tho origin of this compound. These fishes are curyhaline and were kept at first in fresh water oa & diet of fresh, ground ox Uxor, when no trimothylamino oxide was produced. They were then transferred to sea water, still on tho same diet, but still no trimethylamino oxido ap- peared until food containing preformed trimethylamino oxide was given in place of tho ox liver. Tliis would seem to indicate 322 TBIMETBYLAMINE OXIDE that this compound is wholly exogenous in origin, but the excretion in some cases of nearly 50 % of the total nitrogen in the form of trimethylamine oxide by marine fishes suggests that this substance must have n synthetic origin in these fishes, to some extent at least. Probably a part originates os such in tho food, for many marine organisms, including tho small crusta- ceans which form an important article of diet for many fishes, contain substantial amounts of trimethylamine oxide, which is taken over by the feeder and excreted unchanged. Another possible source is glycine betaine. This substance occurs abun- dantly in some animals and in many plants. It Is known that if cows are fed on sugar-beet residues, which are a rather rich source of glycine betaine, trimethylamine oxide appears in the milk. But although betaine gives rise to trimethylamine oxido when administered to cows, it is generally believed that the conversion is not due to the tissues of the cow itself, but rather to the activities of the symbiotic micro-organisms which inhabit its rumen. Animal tissues do not, in general, appear to be capable of converting betaine into trimethylamine oxide, though trimethylamine itself is oxidized by mammalian tissues to yield the oxide, and a trimethylamine oxidase baa been discovered in fish muscle. One can do little more at present than guess at the extent to which such a conversion can take place in fishes, and in any case there remains to be explained the excretion of tri- methylamine oxide by marine yet not by fresh-water fishes, for there is no obvious reason to think that the food of fresh- water species contains less precursors than that of marine forms. It is conceivable that trimethylamine itself might first be formed by the biological methylation of ammonia, and subse- quently oxidized to the oxide in animal tissues; mechanisms for methylation are known (p. 130), and it is known too that tri- methylamine can be oxidized to its oxide in the tissues of some animals. Guanine (2-amino-6-oxypurine). Special mention must be made of guanine, for this purine replaces uric acid in the excreta of spiders. In spite of the popular belief to the contrary, the spiders constitute a group that is morphologically quite dis- tinct from the insects: they demonstrate their independence 31-3 323 GUANINE chemically, too, by excreting guanino in place of nric acid. Guanine, if anything, is even less soluble than uric acid, and contains ono amino-group per molecule over and abovo the four ring-bound nitrogen atoms of the purine ring. Evidently, there- fore, guanine is well qualified to take over the excretory functions of uric acid. The manner of its synthesis is uncertain: probably it arises from xanthine by animation (see p. 347). CHAPTER XIII SOME SPECIAL ASPECTS OF NITROGEN METABOLISM Nitrogenous substances of many different kinds have been isolated from time to time from various animal materials, and although their origins, functions and metabolic fates are known in only a few cases, some mention of these compounds and their distribution seems desirable here. DISTRIBUTION OF CHOLINE, TETRAMETHYL AMMONIUM HYDROXIDE, TRIMETHYL AMINE AND TRIMETI1YL AMINE OXIDE Many different animal tissues have been found to contain larger or smaller amounts of highly methylated nitrogenous com- pounds such as trimethylamine oxide and various betaines, and the suggestion has often been made that they must arise from choline. At the present time, however, it seems less probable that they arise directly from choline than that they are formed by the methylation of other substances, choline entering into the picture only as a source of methyl groups. CHOLINE, (CH,),N + .CH 1 CH,OH, bH" is probably universally distributed as the basic constituent of phospholipids of the lecithin type. Traces of free choline have been isolated from animal tissues of many kinds, but it is doubtful whether it occurs in the free state to any great extent. Probably it arises by autolysis, for, to take only one example, dog liver worked up as rapidly a a possible after the death of the animal contains only 0-43 mg. free choline per kg., rising to 136-164 mg. per kg. if the tissue is allowed to stand for 6 hr. before being worked up. In addition to its importance as a constituent of the lecithins, 326 CROLIKF. choline is important as the raw material from which acetyfcAoh'ne, the neuro-hormone of the parasympathetic nervous system, 13 synthesized by acetylation (see p. 131). The methyl groups of choline can be transferred to homocysteine to yield methionine which, in its turn, acts as a biological methylating agent (see p. 130): u (cn t ), | * in +3 9 H * in, oh Ah.k ioOD chotint homo- cy sttiru b.ch, U sin, in-NU, ioon tlhrtnol- mtthumiiu XII, .Ah, • in,OH The other product, ethanolaminz (cholaminc), it will bo remem- bered, replaces choline in phospholipids of the kephalin type and can also bo formed by the decarboxylation of serine (p. 278). The metabolic importance of choline may be judged from the fact that it appears to bo an essential dietary constituent, usually classified with the B a group of vitamins. Little is known about its metabolism however. The discovery of a choline dehydro- genase in mammalian liver indicates that choline can be oxidized, the product being betaine aldehydo, and there is a possibility that the latter may then be further oxidized to glycine betaino itself, e.g. by the group-specific aldehyde oxidase of the liver: - 211 - 211 (CH,),X + .CD 1 Cn 1 OH . (CHJ,N*.CU,CnO (Cn,),N*.CH,COO- : : + H.o oir oir cKdino Uiaint aUthydo fflynns btlain* TETEA 3IETI1 YLA ItllOXIUll UT DEOXIDE f letrmmine *). (CH,t,N r ...0ir, lias been isolated from only one animal source, the coclcntcrnto Actinia equina, from which it was obtained in remarkably high yield, some 12 g, of the chloride being obtained from 33 kg, of the fresh material. Like most quaternary ammonium bases it has a powerful paralysant action and may, it is thought, bo responsible for the paralysing ‘sting’ which many coelcntcmtcs are capable of inflicting. 320 TRIMETHYLAMINE OXIDE TlilMETH YLAMINE, (CH,),N, and TRIMETHYLAMINE OXIDE, (CU,),N — 0, have been obtained from many animal sources, vertebrate and invertebrate alike. As a rule the oxide occurs in quantities that completely overshadow those of the free base and, in general, it seems that trimethylamine arises from its oxide through bacterial action. The characteristic odour of dead marine fishes, which incidentally is not observable in fresh-water species, is largely due to free trimethylamine formed by the action of putrefactive bacteria upon trimethylamine oxide present in the tissue. Perfectly fresh fish muscle contains traces at most of the free base. Free trimethylamine has long been known as a trace con- stituent of mammalian urine, and it has also been detected in human menstrual blood. Little is known about its metabolic origin and fate except that it is almost quantitatively converted into its oxide when fed to mammals (cow, man). Perhaps the most striking fact that has emerged in connexion with trimethylamine oxide is that, while it is present in a great variety of marine animals, it seem3 never to occur on a sub- stantial scale among fresh-water organisms. Its function in the fishes has been studied a good deal. That it occurs only in marine species suggests that it might play a part, analogous to that of urea in marine elasmobranchs, in tho regulation of the osmotic pressure of tho blood, for it resembles urea in being nearly neutral and relatively innocuous. Elasmobranch bloods contain about 100-120 mu per litre, as compared with about 330-440 mil of urea. It accounts therefore for 20-25 % of that part of the total osmotic pressure not due to salts. Further, the concentra- tion of trimethylamine oxide in the urine of marine elasmo- branchs is only about one-tenth as great as that present in the blood, so that this substance, like urea, is actively retained by tho elasmobranch kidney. Probably, therefore, it plays a signi- ficant part in osmotic regulation in these fishes. Among teleo3ta, however, matters are different. The trimethyl- amine oxide content of fresh cod muscle, for example, has been estimated at about 20 mu per kg., i.e. only about one-fifth of the amount present in elasmobranch muscle. Furthermore, there is 327 BETAINES no reason to believe that the substance is retained, for it is one of the major nitrogenous constituents of the excreta of marine tcleosts. It is therefore unlikely that trimethylamine oxide plays any significant osmotic role in marino teleosts; it is far more probable that it is essentially an excretory' product and may possibly represent a detoxicated form of ammonia (p. 303). Its origin has been discussed in an earlier section (p. 322). DISTRIBUTION OF BETAINES Betaines of various kinds are widely distributed among animals and plants aliko, but their mode of formation is still somewhat obscure. It has usually been supposed that they are formed by the methylation of simpler substances, glycine betaino arising from glycine for example: n,N\cn,coo~ — ► (cn t ),N\cn,coo' glycine (rtnUm Phylum Protozoa Porifera Coelenterata PUtyhelminthea Nemerte* Stoll OSCA Annelida rboronldcn Arthropod* Echinodennata Frotochordata Vertebra ta Class Arginine Creatine Scyphotoa + Actmoroa + Ctcnopbora + Amphineora LameUibranehlaU Gastropoda Cephalopoda Polychaeta Oligochaeta Gepbyrea Crustacea Insects Arachnids Crinoidca Aaterolde* Ophiuroidea Holothuroidea Echinoidca Tnnlcot* EnteropneusU OphalocLorda Piscca Amphibia ReptiUa Mammalia No oomparable cases have been found in the Vertcbrata proper but among the Protochordata, a group of creatures which rescmblo the true vertebrates in their possession of a primitive 334 DISTRIBUTION OF PHOSPHAQ ENS notochord, one similar case has been recorded. The Proto- chordata comprise three groups, the Tunicata (sea-squirts), the Enteropneusta (a small group of worm-like animals) and the Cephalochorda (tancelets). These Protochordata are of special interest since they are regarded on morphological grounds as lying on the border-line between the true Vertebrata on the one hand and the invertebrate phyla on the other. Of the Cephalochorda it may bo said that they resemble the vertebrates rather than the invertebrates, for not only do they possess a well-developed notochord but, chemically speaking, the relationship is clear from the fact that they contain creatine but not arginine. Superficially, the adult Tunicata resemble the invertebrates rather than the vertebrates in their general appear- ance and mode of life, but their tadpole-like larvae possess a well developed notochord. Recent work has shown that the adults, which possess no notochord after metamorphosis, nevertheless contain creatino phosphate and that, contrary to previous opinion, arginine phosphate is absent. In the Enteropneusta, however, we find one case* in which there is evidence for the presence of arginine and creatine side bj T side in the musculature. The Enteropneusta thus appear to be related to the Vertebrata in that they contain creatine and, at the same time, to the invertebrate phyla in that they contain arginine. In particular, they show an affinity with the Echinodermata, for only in these two groups have arginine and creatine been found to co-cxist in significant quantities. Similar relationships were established on purely morphological grounds by the classical investigations of Miiller, Metschnikow and Bateson. As Bateson showed, in addi- tion to their gill-slits the Enteropneusta possess a short but well- defined notochord, features which establish their relationship to the Vertebrata. But in their larval forms the Enteropneusta so closely resemble the echinoderms that their larvae were classi- fied with those of the Echinodermata before the adul t forms were discovered. It was Metschnikow who, some time later, showed that the Tomaria larva, far from being an echinoderm larva as was previously believed, gives rise in the adult form to Balanoglossua, an enteropneust. * In Halanoglosnt/i salmoneui. The closely related B. davtgema and two Specie* of Sauoglosrus contain creatine bat no arginine, 335 pnoscitAGEN's: aqxayzke From the phylogenetic standpoint the natural distribution of these two bases, arginine and creatine, is clearly of considerable interest. The chemical evidence available favours Bateson’s views on the origin of vertebrates by pointing to the echinodenns and the enteropneusts as links between the invertebrates on the one hand and the vertebrates on the other. Other theories of vertebrate descent have been put forward by morphologists in the past, but for nono of them has any substantial chemical support yet been found. U coon « nsmac N XII ,x -<; XU, L. hii£o,u in. COOK Aii.xh, iu, a-r— o.iii, iu lotnirictn* Passing reference may hero be made to the annelid worms. These arc most versatile animals. Nono of them contains either arginine or arginine phosphate, but taurocyamint, glycocyaminc and even creatine phosphates have all been found among the marine species. Sometimes more than one of these has been found in a single spccie3. The earthworms (oligochaetes), while they contain appreciable amounts of free arginino, contain no arginino phosphate; instead they have what appears to bo a unique phosphagen, lombricine phosphate. yXU, AO it ATI SE, IIX=C( 'XU (jaw. is a strong base which can be prepared chemically by the decarb- oxylation of arginino. It has been isolated from a few inverte- brate sources, notably from the sponge, Oeodia gigas, and from several cephalopoda. Its modo of formation is not known: perhaps it is formed by the action of an arginine decarboxylase, but there is no evidence 03 yet for tho occurrence of such an enzyme in animal tissues, although bacterial arginino decarb- oxylases are known. 330 OOTOfclNE: AfiCAltf OCTOPINE, HN=C< (in,), ch, I:h.nh-Ah ioOH ioOH This interesting substance was first isolated from the muscles of Octopus, but has since been obtained from several other cephalopod species and a few lamellibranchs. It is not present in perfectly fresh muscle but arises as a post-mortem product, arginine disappearing as octopine is formed. Its constitution has been established by synthesis, which can be accomplished by the reductive condensation of arginine with pyruvic acid. It Is possible that octopine arises in this manner in Octopus muscle, for it has been known for some time that this, unlike most muscular tissues, contains very little lactic acid in fatigue or at death. Lactic acid is formed as a general rule by the reduction of pyruvic acid, but in the case of Octopus it seems possible that pyruvic acid may condense with arginine to yield an intermediate complex, which then undergoes reduction in place of pyruvic acid itself. Alternatively, octopine might arise directly by the condensation of arginine with lactic acid, but present evidence is insufficient to allow a definite decision on this point. ,NH, ARC AIN, HN=Gf X NH (iH.h Jm HN=C< X NH, (tetramethylene diguanidino), has been isolated only from Area noae. It was again isolated from a second batch of the same organism, but none could be found in another lamellibranch, Mytilus edulis. Structurally it is nearly related to the synthetic substance synthalin (decamethylene diguanidine), winch has a profound effect in lowering the level of the blood sugar in mammals, and was at one time used experimentally for that purpose in the treatment of diabetes mellitus. 337 BP* ASTERTTBIN*. CARNOSINE How areain is formed we do not know. Several suggestions have been made, but there is no evidence to favour one rather than another. Probably it must arise in some way from arginine. yS(CBJ, ASTERVBIS. UN=CC >KH in, in,so,H is not, as its name might suggest, a pigment, but a sulphur* containing guanidine derivative which, up to the present, has been obtained only from two species of star-fishes. Its mode of synthesis w unknown. It may bo considered as being derived by methylation from the amidinated taurine, taurocyamine (p.33G)> taurine itself being widely distributed in nature. Asterubm, like taurine and taurocyamine, belongs to the class of sulphonio acids, a somewhat rare group in nature. DISTRIBUTION OF ISUNAZOLE RASES In addition to the amino-acid histidine, in which the iminozolo ring is present, several other iminazolo compounds have been found in animal tissues. Of these the most widely distributed are carnosine and anserine, which appear to bo peculiar to the muscular tissues of vertebrates, for neither has ever been found among tho extractives of invertebrate materials. i— jcn.dn Nxjoii N Nil .nr— oc.cii.cn, nu, (/?-alanylhistidine), is something of a biological curiosity, being derived from tho rare /1-amino-acid, /7-alanine. It is, therefore, not a typical dipeptido, sine© all ouch are derived wholly from a-amino-acids. Carnosine has been known since 1000, when it was isolated from Liebig's meat extract, and ainco that time it has been obtained from tho muscles of representatives of all classes of vertebrate animals. 338 ANSERINE -N H— OC . CH.CH.NH, ANSERINE, \x>OK N N.CH, \/ a methylated camosine, is also a derivative of ^-alanine. It was first isolated from the muscle of the goose, Atiser, from which it received its name. Camosine and anserine often occur side by side in one and the same tissue. The function of these peculiar bases is quite unknown. Attempts have been made to show that the amount of these iminazole bases is correlated with the activity of the muscle, but without success. Indeed, the only hint we have about their possible function is the fact that both are strong buffers. How camosine and anserine are formed in animal tissues is still unknown. Anserine can arise by the transmethylation of camosine. Camosine itself can replace the essential amino-acid histidine in the diet of rats, from which we may conclude that camosine can be hydrolysed in the tissues and a specific camo- sinase is in fact known to occur in the rat and a corresponding nnserinase has been found in fish muscle. It is possible, there- fore, that it may arise by condensation between ^-alanine, or some forerunner of /halanine, and histidine. If this is so, the origin of /?-alanine remains to be accounted for. Certain bacteria can produce it by the a-decarboxylation of aspartic acid, . and it may be that a similar process can take place in plants: HOOC.Cn.CJIfNH,) COO.H — * HOOC.CH.CH.NH, + CO,. / There is no indication at present that this reaction takes place in animal tissues, but, if it occurs in plants, /i-alanino is presumably available to animals in their food. DISTRIBUTION OP OTHER NJTBOOENOUS COMPOUNDS Many nitrogenous substances have been isolated at one time or another from animal tissues of various kinds, including free amino-acids (notably glycine) and purine bases, in addition to those already discussed. Of these, taurine deserves special men- tion, if only because it occurs in Ter}' large amounts in certain tissues. 339 TAUBINE TAURINE, CU,SO,n in.KH, has long been known os a constituent of the bile of some though not of all vertebrates, in winch it is present in conjugation with cholic and other bile acids (p. 246). Its metabolio relationships with cysteine have already been discussed (p. SSI) and wo have noted its presence in taurocyamine and in osterubin. That free taurino often occurs in very notable quantities among invertebrates has been known for many years. Thus the adductor muscle of the edible mussel, Mytilus edulis, contains 4-5%, while the muscles of on annelid worm, Audomnia spira- brcmcAua, contain 3 % of taurine. Muscles of vertebra to organisms also contain free taurino, though in much smaller amounts. Its function in muscle, however, is unknown. 340 CHAPTER XIV METABOLISM OF PURINE DERIVATIVES NUCLEOPROTEINS In thi3 chapter we have to deal with a group of natural com- pounds called nucleoproteins and with certain products arising by their breakdown. Nucleoproteins contribute only a small proportion to the total nitrogen of any average diet and account for only a small fraction of the total nitrogen of most cells and tissues but, as their name implies, they occur especially in cell nuclei, and are very important substances. Any cell that con- tains a high proportion of nuclear material will form a good source of nucleoproteins, and glandular materials such as pan- creas and thymus have been used extensively for their prepara- tion. The richest source of all seems to be the heads of ripe spermatozoa: indeed, it has been estimated that the nucleo- protein content of fresh spermatozoa is from 50-80% of the total solid matter. Ripe fish milt (soft roes) therefore provides a valuable source of nucleoproteins and their derivatives. It is only during the last decade that the nucleoproteins have attracted very much interest. The early preparations were rather crude, insoluble substances unattractive to the pure chemist, and the present phase of interest began when it was discovered that certain plant viruses can be isolated in crystalline form and are, in fact, nucleoproteins. Unlike the products for- merly obtained, these virus proteins give beautiful, water-soluble crystals. A number of plant viruses have now been shown to consist of crysfcallizahle nucleoproteins, and among animal viruses there are many, including vaccinia, the virus of cowpox, which contain, even if they do not consist entirely of, nucleo- protein material. It h ad hitherto been supposed that viruses are living organisms, small enough to pass through the pores of bacterial filters and to be invisible under the microscope. Yet virus diseases can be transmitted to healthy plants cither by inoculation with sap 341 >'T7CLEOPROT£IK8 from an infected plant or with the crystalline virus protein. If healthy plants are infected by means of a pure virus nuclco- protein and allowed to develop the disease, the virus protein can later be isolated in quantities very much in excess of those used for the original inoculation. These discoveries produced some shocks among biologists who, as a wbolo, had always supposed that there exists some sharp line of demarcation between things that are living and tilings that are not. Yet, in these virus proteins, wo have something which can bo crystallised and can at the same time transmit disease from a sick to a healthy plant and, moreover, reduplicates or 'reproduces* as the disease develops. It has been shown too that chromosomes also consist largely of nucleoprotein material, so that wc must look on the nucleo- protcins not only as the causative agents of a large number of infectious diseases, but also as the vehicles whereby hereditary characteristics are transmitted from parents to offspring. Virus proteins and chromosomes have several important features in common. Both exist as long, fibrous or filamentous structures, and both, given the interior of the right kind of cell as environ- ment, can reduplicate. Further, both show tho phenomenon of mutation, either in the course of nature or under tho artificial influence of X-rays or y-radiation. Just os new strains of organisms can suddenly appear as a result of genetic mutations, so, too, new virus strains can suddenly appear os a result of mutations in the old. This process of mutation among viruses is quite probably the reason for the hitherto inexplicable variability in tho virulence of many virus-borne diseases, e.g. influenza. Finally, there is reason to believe that nucleopro tains play an important if still poorly defined part in tho all-important matter of protein synthesis. It will bo clear to tho reader that tho nucleopro terns are at present a subject of manifold interest. A full knowledge of their chemical constitution must necessarily await further develop- ments in protein chemistry os far as their protein components are concerned, but, in the meantime, considerable strides are being made in the chemistry of their non-protein, prosthetic components, tho nucleic acids. 342 NUCLEIC ACIDS NUCLEIC ACIDS Nucleoproteins are conjugated proteins, formed by the essen- tially salt-liko union of a nucleic acid with a basic protein such as a protamine or a histone. If thymus gland material is macerated with large volumes of water the thymus nucleo- protein is extracted, and the protein component, in this case a histone, can be precipitated by saturation of the extract with sodium chloride. On the further addition of ethyl alcohol, nucleic acid 13 precipitated as a fibrous mass. Two chief types of nucleic acids have so far been recognized, one of which can be obtained from thymus gland and the other from yeast. These acids, the structure of which is extremely complex and still incompletely understood, are built up from smaller units known as nucleotides, which yield on hydrolysis one molecule each of a nitrogenous base, a pentose sugar and phosphoric acid. These nucleotides are, in fact, phosphate esters of the iV-glyeosides of certain nitrogenous bases. According to the older work, four of these nucleotide units go to make up one molecule of nucleic acid, but recent determinations of the par- ticle weight, carried out by the method of ultracentrifugation, give values ranging from about 200,000 to several millions. Physical studies show that the particles are long, rod-like or thread-like objects, each of which appears to consist of aggre- gates of smaller units. These aggregates can be made to depoly- merize in various ways, and the smallest units so far obtained by disaggregation have particle weights of the order of 15,000. If, as seems possible, these are really molecular units, it follows that each molecule of nucleic acid must contain many more than the four nucleotide radicals formerly postulated. At present the tendency is to think of nucleic acids as being built up by the union of large numbers of nucleotide units, much in the way that proteins are built up by the union of large numbers of amino- acid units. The possible number of nucleio acids, like that of proteins, is exceedingly large. The most striking differences between nucleio acids of the yeast and thymus types lie in the nature of the sugar radicals involved in the nucleotide units. Nucleic acid prepared from 343 NUCLEIC ACIDS yeast contains /7-D-ribofuranose, wliilo that from the thymus gland of animals contains /J-D-S-desoxyribofuranose: fi-Tj-ribofanmut p-X^Z-duoxyribofuranOH It was at one time believed that plant and animal cells always and only contain ribonucleic and desoxyribonucleic acids respec- tively. This view has turned out to bo entirely erroneous. Probably all cells contain nucleic acids of both types, the desoxy- ribose compounds preponderating in the nucleus and the ribose compounds in the cytoplasm generally. Both types of nucleic acids contain phosphate radicals, and both contain bases belonging to the purine and pyrimidino groups, i.e. bases containing the following ring systems: i \.s-/ /v\ i N / pyrimidine ring pinM riny {—pyrimidine 4 imiwuoM Hydrolysis of the nucleic acids by means of diluto acids or tho appropriate enzymes yields the following recognized products: Pt PynmtdlMs: Punntt ' CYTOPLASM Rjboxccxtjc Ano ('yowl nucleic acid’) D-ritxmo f j-loaioo; uracil adenine; guanine NUCLEUS Pmoxtriboscclho Acid I’MymonvcUic add’) D-deeOtyrib c*o cyloaine; thymine adenine j guanine Small amounts of other bases arc sometimes found, e.g. 5-methyl- cytosine in thymus nucleic acid. Tho chief differences lie, therefore, in the nature of tho pentose radicals, and in the presence of thymine and of uracil in desoxy- ribonucleic {‘DNA ’J and ribonucleic acids (‘RNA ’) respectively. Since at least these four different bases are found in whichever nucleic acid is taken, it follows that at least four nucleotide 344 DIGESTION OF NtJCBEOPBOTEINS radicals enter into the composition of the nucleic acid concerned ; but, as has been pointed out already, there is reason to believe that many more than four nucleotides, perhaps some fairly high multiple of four, are present. DIGESTION OF NU0LE0PB0TEIN8 The salt-like union between the nucleic acid and the basic protein component of a typical nucleoprotein is disrupted by the acidic contents of the stomach, the protein fragment being digested along with the other food proteins. Nucleic acids are further split by enzymes contributed by the pancreatic and intestinal j uices. Our knowledge of these enzymes is still far from complete, but they appear to comprise (1) nucleates, which liberate the component nucleotides (p. 99), (ii) nucleotidases, which catalyse the dephosphorylation of the nucleotides, yielding nucleosides, which are -glycosides of the nitrogenous bases (p. 99), and (iii) nucleosidases, which hydrolyse the nucleosides to liberate the basio and glycoaidio components. Relatively little is known about the fates of the pyrimidine bases and we shall not consider them here. Their structures, together with that of pyrimidine itself, are appended as a matter of interest, and in passing it should be noticed that a large and important group of drugs, the barbiturates, are structurally related to the pyrimidine group, and that vitamin B x also is a pyrimidine derivative. Similarly, it may be pointed out, caffein H NH. 1 0 0 Nai («>CH Ah Hkx«x!a o'c n h a a z uric acid (2-6- S-trihydrozyp urine) PUEINE METABOLISM to yield xanthine (2:6-dihydroxypurine). Hypoxanthine and xanthine then undergo serial oxidation tinder the influence of xanthine oxidase to give uric acid. These metabolio relation- ships may be summarized as shown on p. 358. In passing, it should be noticed that all the hydroxypurines (often known as ‘oxypurines’) are tautomeric substances which readily undergo transformation at the — N=€(OH)— * — * — NH-CO— groupings. In the scheme above, only the enol forms are given for the sake of clarity, except in tho case of uric acid, in which the keto form is believed to predominate. The distribution of adenose, guanase and xanthine oxidase among animal tissues is very erratic. It is said, for example, that man and the rat possess no adenase, though the enzyme is common elsewhere, while tho tissues of the embryonic pig are stated to contain guanaso, in contradistinction to those of the adult, which do not. Again, xanthine oxidase is present in the liver of most birds, e.g. goose and domestio fowl, but is absent from that of the pigeon (p. 317). METABOLISM OF UBIO ACID Uric acid may arise from purine bases in animals of any kind, whether they are ammoniotelic, ureotelic or uricotelic. The bio- synthesis of the purine ring system has already been discussed (pp. 3 1 6-2 1 ). U ricotelic animals, as we have Been , convert tho bulk of their waste nitrogen into uric acid, but the amounts of uric acid that arise from purine metabolism are relatively very small, accounting perhaps for about 5 % of all the nitrogen excreted. Uric acid is excreted without further chemical manipulation by uricotelio animals, but in mo3t other forms it is more or less extensively degraded before being excreted (see Table 30). The hrstf stage m the process of un'coiysis consists m the oxidation of urio acid itself to the more soluble substance allantoin, under the influence of urico-oxidaso. This takes place in all mammals apart from man and the higher apes (Primates), while the Dalmatian coach-hound is peculiar among dogs in that it excretes only a small part of its total purines in the form of allantoin. It is a strange fact that the liver of this dog is nevertheless fairly rich 369 TFBIGOt* Y SIS Table 30. End-products of fubtne metabolism ( After Florkin & Duchatcau) ■fffX ■U/" MI I Kl y 4y V : i ct Ml, tio 'V io L > Nn, COOIINH, io CH CO VY H It allanto te tttd | »lbntolc**« V"' coon SCO 4 I 1 CIIO Excreted by Primates Eirds Uricotelic rtptllr* Insect* (other than Diptera) (other thin Primates) Insect* (Dipten only) Gastropods Fishes (uoo tcieosU) lube* (la general) Amphibia Lameliibranehs (fresh water) Cephytcan worm* Lameilibrani-hs (marine) 360 URIC0I*Y8IS in urico-oxidase ; one possible explanation of this paradox is that the renal threshold for uric acid may he abnormally low in this animal, so that uric acid escapes very rapidly into the urine before the enzyme has had time to oxidize all of it. In other mammals, however, allantoin is excreted in place of uric acid, but uricolysis stops at this stage. In mo3t other non-uricotelic animals, allantoin is further degraded to yield allantoic acid, thence to urea, and finally even to ammonia, though in many animal groups the complete set of uricolytic enzymes is lacking. The stages involved in the complete process are summarized in Table 30, together with the names of the enzymes concerned and some indications of their distribution. It is worthy of note that, like adenase, guanase and xanthine oxidase, the uricolytic enzymes are very erratically distributed among animals. In particular, it is interesting to notice that, with the evolution of more complex forms of life, the tendency, as far as purine metabolism is concerned, has been to lose old enzymes rather than to acquire new ones, a fact which is amply illustrated by Table 30. 36) CHAPTER XV CARBOHYDRATE PRODUCTION IN THE GREEN PLANT INTRODUCTION The green plants possess specialized machinery which does not depend mainly , as most animal tissues do, upon ATP as prin- cipal energy -source ; instead they draw energy for their synthetic operations from the sun’s radiations. The photosynthetio formation of carbohydrates from CO, can be classified as an anaerobic process; indeed, while it is going on oxygen is actually evolved. In the presence of light, carbon dioxide, water and a handful of simple inorganic salts the green plant can produce an astonishing variety of organic products. Everything an animal can produce a plant can produce, and animals depend upon the synthetic powers of the plants for their own supplies of essential amino-acids, vitamins and other compounds which the plants, but not they themselves, can make. In thi3 section we shall consider only the photosynthetic formation of carbohydrates, especially of starch and sucrose, probably the most abundant carbohydrates in the world, apart from cellulose. We bavo, in point of fact, already reviewed the enzymes and processes involved in the production of amylose and amylopectin from glucose and the glucose phosphates (pp. 120-3), and wo shall shortly see that any substance lying on the glycolytic reaction chain, or giving rise to any substance lying on that chain, is convertible into one or other of the glucose and fructose phosphates. The overall equation usually written for photosynthesis is the precise reverse of that for the oxidative metabolism of carbo- hydrate. pholotyrJketU 6CO, + 011,0 t ^ - ■ 60, + C,tf„0,. oxidation Now, it is possible to consider the processes involved in photosynthesis in two parts, one concerned with the photolytic PHOTOLYSIS OF WATER splitting of water, which requires light for its accomplishment, and the fixation of carbon dioxide, which depends in its earliest stages upon the photolysis of water and is therefore light- dependentbut which, once the earliest Btages have been achieved, can continue in the dark. PHOTOLYSIS OF WATEB The origin of the oxygen evolved by an actively photoayn- thesizing plant has been much discussed and much studied, and the general consensus of opinion is that it arises, not from carbon dioxide or some other metabolite, but from water. The most striking demonstration of this was provided by experi- ments in which plants were kept in water enriched with heavy oxygen ( 18 0 2 ), when the oxygen evolved proved to be iso- topically labelled. Moreover, as Hill has shown, oxygen evolu- tion can bo demonstrated in isolated chloroplasts with a simul- taneous reduction of ferric oxalate for example, but without any concomitant synthesis of carbohydrate material, showing that the photolysis of water is separable from photosynthesis itself. The splitting of water involves light, chlorophyll and a number of other reactants. Certainly it is a complex process and, equally certainly, it is still far from being understood, despite its impor- tance and fascination. Suffice it for our purposes to say that the photolytio process, in which water is oxidized to oxygen, possibly by way of hydrogen peroxide, must necessarily be associated with the reduction of some other substance. If we represent the Bubstance undergoing reduction as X we can write: 1211,0 + 12X * 60, + 12X.H,. The fixation of carbon dixoide is a reductive process for which we can write: 6C0, + I2XH, *■ C 4 n„0, + I2X + 6H,0. The resultant of the two equations is of course, 6C0, + 611,0 ► C,H„0, + 60,. leaving X as an indeterminate compound. Now it is known that the reductive stage in C0 2 -fixation is catalysed by a TPN-specific triosephosphate dehydrogenase, 363 FIXATION OF CARBON DIOXIDE working *in reverse’ and requiring a continuous supply of reduced TPN (TPN.Hj) to continue in operation. Since this reaction ceases immediately illumination is cut off, it seems reasonable to think that the reduction of TPN must be achieved directly or indirectly by the photolytio splitting of water. It may well he that there are a number of intermediate steps between photolysis and the reduction of TPN, but that the two are intimately associated each with the other can hardly bo seriously doubted. If, for the sake of argument, we suppose that there arc three intermediate carriers, X, Y and 2, it is probably permissible to write: 11,0—*. oxidized X redace4~. oxidized-*. oxiAatA^r '^■icductd T JL ■reduced — -oxidutd or, more simply: 2TPN •*-2TPN.H, FIXATION OF OARBON DIOXIDE Over a great number of years attempts hnvo been mado to discover the first-formed produot of CO s -Gxation and many theories have been propounded, but it is only in tho Inst few years that any convincing evidence has been forthcoming. Tho newer work has been mado possible largely through tho avail- abUity of isotopic carbon and by the introduction of the tech- niques of paper chromatography. If suspensions of green algae such as Chlorella or Scencde&mus are illuminated in tho presenco of 1, G in the form of carbon dioxide or bicarbonate, the isotope is rapidly incorporated into a large number of different substances, including sugars, poly- saccharides and amino-acids. But if tho period of illumination is very short, say 0- 1 sec., one substance and one substance only contains the isotope, viz. glyceric acid-3-phosphate. Tor each molecule of carbon dioxide fixed, 2 molecules of this product 304 FIXATION OF CARBON DIOXIDE are formed. With slightly longer periods of illumination, e.g. 1-5 sec., it is possible to observe the passage of isotope into other substances, notably triosephosphates, fructose mono- and diphosphates and certain other sugar derivatives, and it has indeed been possible to trace out the early stages of the photo- synthetic pathway in this manner. Unfortunately however there are many interlinking metabolic pathways and the labelled carbon follows them all, so that the solution of the problem of picking out the paths that lead to carbohydrate formation has had to depend a great deal upon the separation of enzymes from plant materials and piecing together the reactions they catalyse. Since one molecule of carbon dioxide gives rise to two mole- cules of glyceric acid phosphate, each with 3 carbon atoms, it follows that a 6-carbon acceptor of some kind must probably be involved. Such a precursor has been detected and identified as the l:6-diphosphate of the pentose sugar, ribulose. An enzyme, variously known as carboxydismutase, ribulose diphosphate carb- oxylase and ‘carboxylation enzyme’ has been purified from spinach leaves and from extracts of Chlorella and shown to catalyso the formation of 2 molecules of glyceric acid-3-phos- phate from C0 2 and ribulose-l:5-diphosphate. The reaction is a curious one and is thought to proceed in the following manner: CO, cn,o® CH,0© CH,0® J +H,0 CH.O© (Ihoh io C.OII + HO— 0=0 — ► C(OH)COOH +H,0 J — ► C00H CHOH *~=sl!>.ON L ""to Ahoh Ah.o® C00H dnoH ufose-l:5 (enol-) diphosphate (intermediate) glyceric acid- Z-phosphate This is the first of a network of reactions that lead back eventu- ally to tho regeneration of the ribulose diphosphate. From glyceric acid phosphate tho isotope passes on to trioso- phosphate. Green plants contain several iriosephosphate deJiy- drogenases at least one of which resembles that found in animals 305 CARBON PATHWAYS IN PHOTOSYNTHESIS (p. 1 86). In particular, however, there is a TPN -specific enzyme that is apparently confined to the green parts of plants and catalyses the reduction of glyceric acid phosphate to glycer- aldehyde phosphate at the expense of reduced TPN. This reac- tion stops immediately illumination is cut off, indicating that the production of the reduced TPN is light-dependent and is, in fact, coupled in some way to the photo lytic splitting of water: cn,o® cn,o® B. AlIOH + TPN.H, — ► AlIOH + TPN Aooh Aho Then, under the influence of triosephoapliale isomerase, equi- librium is set up between the two forms of trioso phosphate; CH,0® CH.O® c. Ahoh Aho Ah, on The triosephosphates lie on the direct linos of fermentation, glycolysis and carbohydrate synthesis and their fates in these reaction sequences will be discussed in later chapters. Their immediate fate in photosynthesis is at first the same as in the synthesis of carbohydrate in animal and other tissues, for fructose-1: 6-diphosphate and fructose-O-phosphate are formed, but from this point on the processes become more involved. They can he followed by means of the ' map ’ shown in Fig. 27. AU the Table 31. Enzymes concerned in photos yntretic CARBON - FIXATION Reaction Enzyme A Ribnloee diphoepbate carboxylase B Trioaephaephate debydrogenaao C Tnoeephoephate iso top rasa 1 Aldolase 2 Specific phoepbat&ae 3 Traoaketolaae 4 Ph oephokrtopenloee epimenuw 5 Fbcephoribulokinaae 6 Aldolase 7 Specific phosphatase 8 Transketoisae Q Pboephokelopefllnae epimeraae 10 Fboaphorifcw iaomeraae 11 Fboephoribulok&ue CARBON PATHWAYS IN PHOTOSYNTHESIS Fig. 27. Reaction network in CO, -fixation. The formation of trioeepboaphnte is described in the text. See Table 31 for summary of enzymes involved. 367 OABBON PATHWAYS 15? PHOTOSYNTHESIS enzymes concerned in catalysing these reactions, and all the intermediate substances, are detectable in plants and all the intermediates can be isotopically labelled. Several of the en- zymes have been isolated in pure, crystalline form. Molecules of each of the two forms of triosepbosphate react together under the influence of a typical aldolase (reaction 1) to form fructose-l;6-diphosphate. This is a veil -known reaction in all kinds of colls. The next step consists in the removal of one phosphate radical (2) under the influence of a specific phos- phatase, giving fructose-G-phosphate. This reaction too is veil known and constitutes another step in the direction of carbo- hydrate synthesis, but now the pathways diverge. The next reaction (3) is catalysed by transketolase. The two terminal carbon atoms of fructose-6-phosphate are transferred to a molecule of glyceraldehyde phosphate to give xyluIose-6- phosphate, which is transformed by phosphokelopentose epi- tnerase into ribulose-5-phospbate (reaction 4), and the latter, under the influence of a specific phosphoribulokinase, reacts with ATP (5) to regenerate ribulose-l:5-diphosphato. But this is not all. The second product of reaction (3) is the tetrose sugar erythrose, and further supplies of ribulose diphos- phate arise from tlii3 source. The tetrose phosphate reacts with a molecule of dihydroxyacetone phosphate in a typical aldolase reaction and gives rise to sedoheptuloso-l:7-diphosphate (0), which is attacked by a specific phosphatase and yields sedohep- tulose-7-phosphate (reaction 7). The product now reacts with another molecule of glyceraldehyde phosphate, with transkelo- last acting as catalyst (reaction 8). The products are: xylulose- 6-phosphate, which epimerises (reaction 9) to give ribuIoso-6- phosphate: and riboso-6-phosphate, which is converted into ribulose-5-phosphate by phosphoriboisomtrast (reaction 10), and the two molecules of ribuloso-6-phosphato thus produced are further phosphorylated by phosphorilmlokinase and ATP to form two molecules of ribulose-l-:5-diphosphate (reaction II). In all, therefore, three molecules of rihulosc-l:&-dipliosphate are formed by the complete reaction network. PHOTOSYNTHESIS OF STAKOH PRODUCTION OF STABQH For the production of these 3 molecules of pentose diphos- phate 5 molecules of triosephosphate are consumed, 3 of the aldose and 2 of the ketose, the production of which would require the fixation of 2\ molecules of C0 2 by 2£ molecules of ribulose diphosphate in reaction A. Putting this into terms of whole molecules: 6 pentose diphosphate + SCO, ► 6 pentose diphosphato. (25 carbon) (30 carbon) In other words, for each pair of complete operations, 6 molecules of carbon are fixed. Presumably, however, the 6 additional carbon atoms, although they could pass on and give ribulose diphosphate, do not do so, since after a long enough period of illumination the plant would consist almost entirely of this substance. One may suppose that enough ribulose diphosphate accumulates to allow of the optimal rate of carbon dioxide fixation under the conditions prevailing, and that some at least of the carbon so fixed passes out of the reaction network through triosephosphate, fructose diphosphate or fructose monophos- phate, all of which lie on the direct route for synthesis or kata- bolism of carbohydrate, whether as simple monosaccharides, di- or higher or even polysaccharides, particularly starch. In the case of synthesis we know that the following chain of reactions can take place under the influence of enzymes known to occur in plants (pp. 120-3): all these enzymes are described in Part I of this book. J Trioxphoaphile PHOTOSYNTHESIS-. FructoM 1 Mipho«pl)»i» | tpttijie pAoppfatM \ Frurto»e-9-pbo*pIi»U Claocw-S-p barpbafe Amploptctui | Q-m frw AmploM jl P-tHtynt phwpbUa fkatpkofliumuUM 24 369 PHOTOSYNTHESIS OF SUOBOSE PRODUCTION OF SUCROSE If photosynthesis is allowed to proceed in the presence of ”C0 t , sucrose appears fairly early and contains isotopio carbon. It has been established that sucrose can bo formed in plant material (wheat germ) through a reaction in which uridine diphosphate glucose is involved. Fructose-G-phosphate, an intermediate in the photosynthetio network, enters into the synthesis; (а) Froetoae-C-© + UDPG »ucrose-© + UDP, (б) Sucroso-© + 11,0 -* sucrose + HO.©- Sucrose phosphorylase (p. 118) appears not to be present in plant materials. It will be noticed that if thi3 synthesis i3 to bo a continuous process, UDP must be reconverted to UDPG. This takes place by two reactions, in the first of which. UDP reacts with ATP: UDP + ATP -v UTF + ADP. This is followed by a reaction between UTF and a-glucose-l- phosphate, which can be formed from fructose- 6- phosphate by way of glucoso-G-phosphate: UTP + glucose- 1 -phosphite s=» UDPG + pyrophoipbnte. PRO DUCT 10H OF OTHER CARBOHYDRATES Plants are known to contain a variety of polysaccharides besides amylose nnd arajiopectin. Members of the Compositao store inulin, a pulyfructofuranoside, rather than starch. Other poly- saccharides contain other sugars and sugar derivatives, c.g. galactose, tf-acctylglucosarmne, and, although wo know that UDPG plays a part in the production of a number of the mono- saccharide units, little is yot known about the details of the mechanisms concerned. In the case of galactose, however, more is known. It is formed by an enzyme that occurs in animalsamlgalactoso-trained yeasts os well as in plants. The overall reaction can bo written: UDP -f glucoae-l-© UDP-glueoec UDP-galoelosa gaUctwio-l-© + UDP. It now seems certain that the Walden inversion which takes place in the UDP-glucose ?=* UDP-galactoso transformation 370 PHOTOSYNTHESIS involves DPN and is acliieved by dehydrogenation followed by rehydrogenation, probably thus: UDPG plays a part in a great many processes in which carbohydrates are involved and is known to form compounds not only with glucose and galactose but with iV-acctylgluco3- amine and with glucuronic acid, with N -acetylgalactosamine, galactonio acid and even with glycerol, all of which enter into one or another of the numerous known plant polysaccharides. Some workers believe that UDP may play a part in transglyco- sylation reactions generally, while UDP and UMP appear to play some part in the interconversion of phosphorylascs a and b (p. 121) and, indeed, even in the action of the a enzyme itself, probably in the role of a prosthetic group. The many new discoveries concerning UDP and its functions suggest that a long new chapter in carbohydrate biochemistry is about to be written. FORMATION OF AMINO-ACIDS In addition to undergoing synthetic reactions to produce poly- saccharides, sucrose and other substances, triosephosphate can follow the katabolic pathways of glycolysis to produce pyruvate. Under oxidative conditions pyruvate can enter into the citric acid cycle, which we shall deal with later, in the course of which a-ketoglutario acid and oxaloacetic acids are formed. Like pyruvate these are a-keto-acids which can be aminated or transaminated to yield the corresponding a-atnino-acids, alanine, glutamic and aspartic, respectively. Aspartic and glutamic acids can act as transaminating agents for other a-keto-acids produced in other line3 of metabolism. The green plants can, apparently, produce a-keto-acids corre- sponding to all the naturally-occurring amino-acids and can transform these into the amino-acids themselves by transamination. 371 CHAPTER XVI ANAEROBIC METABOLISM OF CARBOHYDRATES: ALCOHOLIC FERMENTATION INTRODUCTION Relatively little has been learned about the aerobic break- down of carbohydrates until recent years, though a great deal was known about their anaerobio metabolism in yeast and in muscle. It may strike the reader as curious that these two kinds of cells, so different in their organization and function, should have been selected for examination rather than any others- Yeast, however, has long been a matter of great commercial importance for the production of alcoholic beverages and for the manufacture of industrial alcohol. Furthermore, various im- portant by-products of fermentation, such as the components of fusel oil, find many important applications in chemical tech- nology. No wonder, then, that alcoholic fermentation has been extensively studied. In the ease of muscle, interest has been aroused by more academic considerations. Muscle does mechani- cal work. Many muscles can be isolated and made to cpntract outside the body, and in theso, beyond all other tissues, wo have an opportunity of measuring the amount of work done by a bio- logical system and attempting to correlate it with the amount of chemical change simultaneously taking place. It was rather late in tlio history of the subject before it was realized that, in spite of their many apparent differences, yeast and muscle both derive the energy they expend through very similar chemical manipula- tions of their carbohydrate starting-materials. The enzymes concerned in the anaerobic degradation of carbo- hydrates can readily bo obtained in particle-free cell extracts, whereas those concerned with their oxidative metabolism are intimately bound up with the structural elements of the cell, especially with the mitochondria. Anaerobio metabolism can therefore proceed and can be studied independently by the use 372 BIOLOGICAL EFFICIENCY of simple aqueous extracts of cells and tissues after the solid matter has been removed by filtration or centrifugation. We know now that the aerobic metabolism of Btarch, glycogen and glucose is, so to speak, a continuation of their anaerobic meta- bolism and, moreover, that it is much more complicated. Here, therefore, we shall deal first with anaerobic and later with aerobic metabolism. Starch, glycogen and glucose provide major sources of energy for plants and animals, to make no mention of the innumerable micro-organisms which likewise derive energy and employment from the breakdown of these substances. Their breakdown is attended by the liberation of some at least of the intrinsic energy of the carbohydrate molecule, but how much of the free, or available energy becomes biologically accessible to any given organism depends upon the nature of the chemical changes the organism is able to accomplish. When glucose is burned in a bomb calorimeter the heat set free (—AH) amounts to about 674,000 cal. perg.mol. The change in the entropy term ( T.AS ) corresponds to some 12,000 cal., so that the loss of free energy { - A F) associated with the complete combustion of 1 g.mol. of glucose is approximately 086,000 cal. The synthesis of 1 g.mol. of glucose, in a green plant for ex- ample, therefore requires the provision of about 680,000 cal. of free energy. When an animal oxidizes I g.mol. of glucose to carbon dioxide and water, there is a loss of 686,000 cal. of free energy. The biological efficiency of an organism can be measured in terms of the extent to which these 686,000 cal. can be harnessed by the organism and put to service for its biological purposes. In considering biological efficiency, however, it is necessary to dis- tinguish between the efficiency of energy capture, i.e. the efficiency with which the free energy of the substrates of metabolism can be 'tapVuit'A Vn Vue form of KYf*, and rffitranty wVJn T&atVi energy so captured can be utilized in the performance of mechanical or some other kind of biological work. We may therefore distinguish between the energy -capture efficiency and the overall efficiency of a cell, tissue or organism. Generally speaking, living cells can achieve an energy-capture efficiency of 56-75 %, but their overall efficiency is not much greater than that of the majority of man-made machines. 373 ANAEROBIOSI8 Now glucose can be broken down in other ways than by complete oxidation. Muscle cells, working under anaerobic con* ditions, can convert glucose into lactic acid, a process known os glycolysis : C,H fl O, - 2CH,Cn(OII)COOH. Yeast, again, can cany out an anaerobic fermentation of glucose, yielding ethyl alcohol and carbon dioxide: c,n u o, - 2c,n 4 on + sco,. In neither of these transformations is the change of free energy as large as it is in complete combustion, for a large proportion of the total intrinsic energy of the starting material remains Btored up in the products, and access to this can only bo gained by further degradation of these Bubstancea. The change of free energy associated with complete oxidation of lactic acid amounts to about 325,000 cal. per g.mol., or 050.000 cal. for the 2 g.mol. formed from 1 g.mol. of glucose. Consequently, the loss of free energy associated with glycolysis is less than that associated with complete combustion of glucose by approximately 050,000 cal. per g.mol. of glucose transformed. The loss of free energy in glycolysis therefore amounts to only (686,000 -050,000) = 30,000 cal per g.mol. of glucose approxi- mately. Anaerobically, therefore, muscle gains access to only 36.000 cal. as contrasted with the 680,000 cal. which become available when the same amount of glucoso is completely oxidized under aerobic conditions. To gain access to the same amount of energy, therefore, a muscle will require to glycolyso nearly 20 times os much glucose as it will if it oxidizes glucoso to carbon dioxide and water completely. Aerobic metabolism, in fact, is far more efficient than anaerobic. But in neither case does it necessarily follow that the cell or tissuo can actually harness and utUi 20 all the energy to winch it gains access by oxidizing, glycolysing or fermenting its food materials. How this energy is trapped, and how much of it can bo trapped, we shall see in ensuing chapters, but at the present time wo are only at tho beginning of a knowledge of biochemical energetics. Ono of the tasks confronting biochemistry is the invention and develop* ment of a new thermodynamics. It is convenient to classify organisms as aerobio or anaerobic 03 the case may be. Relatively few living organisms are strictly 374 CELL-FREE FERMENTATION anaerobic. Indeed, as far as we know, strict anaerobiosis is practically restricted to a few groups of bacteria, and these are not merely unable to utilize oxygen but are actually poisoned by it. The vast majority of micro-organisms are facultative anaerobes, i.o. they can utilize oxygen 'when it is available and oxidize their foodstuffs completely, but can still survive under anaerobic conditions by catalysing a partial or ‘fermentative’ breakdown of the same food materials. Animals for the most part might almost be classified as * strict aerobes ’, since few of them can live for long in Complote absence of oxygen. But certain processes can go on in animal tissues under anacrobio conditions, provided that the ‘oxygen debt’ thus incurred can sufficiently soon be repaid. ALCOHOLIC FERMENTATION OF GLUCOSE Alcoholic fermentation has been familiar to the human species since prehistoric times, yet it was not until after 1857 that its cause was discovered. In that year Louis Pasteur was studying the lactic fermentation of milk and trying to discover its cause. The views held at that time look very strange by modem standards, for the great Liebig himself considered that the nitro- genous constituents of the fermenting mixture reacted with air, setting up ‘unstabilizing vibrations’ as they did so, and these vibrations were believed to rupture the fermenting molecules. The fact that a new fermentation could be initiated by inocu- lating the medium with a traco of an already fermenting fluid was attributed to the transference of vibrating material to the new medium. Pasteur began his experiments with media containing very simple substances such as sugars, ammonium tartrate and mineral phosphates, dodo of which could reasonably be expected to develop ‘unstabilizing vibrations’. His results were simple and clear-cut. Fermentation took place only in the presence of certain microscopic organisms, the lactic acid -producing bacteria of the present day. When precautions were taken to exclude these organisms, no fermentation occurred. Extending his studies to alcoholic fermentation in 1860, Pasteur showed that whenever it took place the appropriate micro-organism, in this 375 CELL-FREE FERMENTATION case yeast, grew and multiplied. He therefore concluded that fermentation is a physiological process, intimately hound up with and wholly dependent upon the life of the yeast cell. In 1875, having shown that fermentation can take place in complete absence of oxygen, Pasteur defined fermentation as ‘Life with- out oxygen'. More than 20 years elapsed before the next major step forward, but in 1897 Hans and Eduard Buchner made a key discovery which opened the door not only to the investigation of the mechanisms of fermentation but to the whole of modem en- zyme chemistry. Like many other great discoveries, that of the Buchners had in it an element of chance. They were primarily interested in making cell-freo extracts of yeast for thera- peutic purposes, and this they accomplished by grinding yeast with sand, mixing it with kieselguhr, and squeezing out the juice with a hydraulic press. There then arose the problem of pre- serving their product. Since it was to bo used for experiments on animals the ordinary antiseptics could not bo used as preserva- tives, so they tried the method usual in kitchen -chemistry of adding largo amounts of sucrose. This led to the momentous discovery that sucrose is rapidly fermented by yeast juice. Here, for the first time, fermentation was observed in the complete absence of living cells, and at last it wa3 possible to study the processes of alcoholic fermentation independently of all the other processes — growth, multiplication and excretion — -which accompany fermentation in the living yeast cell. The Buchners’ work was soon followed by intensive studies of fcho properties of yeast juice. It was found capablo of fermenting glucose, fructose, mannose, sucrose and maltose, all of which are fermented by living yeast. The dlsaecha rides, sucrose and maltose, are broken down in some way to yield their constituent monosaccharides before being fermented. Glucose itself is almost quantitatively converted into ethyl alcohol and carbon dioxide according to the equation c,ir tt o 4 - 2 co, + 2cu,cn,oiL Traces of glycerol are always found among the products. Fresh yeast juice is much lcs3 active than living yeast. The rate of fermentation can bo followed by measurements of the INFLUENCE OF EHOSPHATE8 rate of evolution of carbon dioxide, and experiments carried out in this way show that living yeast works 10-20 times as fast as an equivalent quantity of yeast juice. Moreover, the fermenta- tive power of the yeast juice falls off rapidly with time. The juice is not inactivated by drying at 30-35° C. or by the addition of chloroform, but loses its activity if heated to 60° C., suggesting that enzymes must be involved. The first important step towards analysing the mode of action of yeast juice was made by Harden & Young in 1905. If fresh yeast juice is added to a solution of glucose at pH 5-6, fermenta- tion begins almost at once. The rate of carbon dioxide production presently falls off, but the original rate can be restored by the addition of inorganic phosphate. The recovery is only temporary, however; the added phosphate disappears, and the rate of fermentation falls off as the concentration of free phosphate declines. The addition of more phosphate produces another burst of fermentation and so on. The disappearance of added inorganio phosphate from fer- menting mixtures suggested that organio phosphate esters must probably be formed and, as Harden & Young showed, this is indeed the case, for they were able to isolate Buch an ester in the form of fructofuranose-l:6-diphosphate (‘hexose diphosphate’). This substance, like glucose, is fermented if added to an actively fermenting system, and must probably be an intermediate in the process of fermentation. Later Robison isolated another sugar phosphate, this time a monophosphate which, on de- tailed examination, proved to consist of an equilibrium mixture of glucopyranose-O-phosphate and fructofuranose-G-phospliate. Like hexose diphosphate these esters are fermentable. It seemed clear that these substances must arise by the coupling of in- organic phosphate with glucose, the respective esters probably arising in the order shown on p. 378. How these esters are formed, and in what way the fructose diphosphate is eventually converted into alcohol and carbon dioxide, are questions which were only answered over a period of decades and by the efforts of many workers in many different countries. Certain stages were first elucidated by studies of muscle extracts, for it became clear in time that the fermentation of glucose by yeast juice runs closely parallel to the glycolysis of glycogen by suitable muscle extracts. 377 FORMATION OF HEX08E PHOSPHATES Among the names that stand out in connexion with the further analysis of fermentation andglycolysis ore those of Erabden, Neu- berg, Meyerhof, Pamas, D. M. Needham and the Corn, but these are only a few of the distinguished many who have contributed. ch.oh cn.o® a-ghxcoit a-shieopyranote-6-phospfiaU HO H HO II ct-fruciofuranofc-C-pfiotphale a fruciofurancne-1 : Q-Jtph&tpfiaSe The next fundamental step forward was also made by Harden & Young when they discovered that yeast juice loses its activity if dialysed. Activity could bo restored to dialysed juice, cither by adding the dialysate, or by means of small quantities of boiled juice. This allowed that, in addition to enzymes, yeast juice contains dialysable, thermostable substances which function as coenzymes. Yeast juice thus came to bo regarded as consisting of ‘zymase’, a non-dialysablo, thcrmolabile enzyme, plus, ‘co- zymaso’, a dialysable, thermostable fraction. Wo know now, of course, that zymase is in reality a complex mixture of enzymes and that cozymaso consists not of one substance only but of several. It is neither possible nor desirable hero to give an historical account of subsequent work on the problem of fermentation. There were mistakes and gaps in the schemes that replaced one another in quick succession during the ensuing years, but one by ono the mistakes were rectified and the gaps filled in until, at the present time, we have what we believe to be a clear picture of moat of tho details of the process. Before we can study this picture it is necessary to know something more about the composition of cozymase. 37S COENZYMES AND INHIBIT0B8 Cozymase comprises a number of factors. Co-carboxylase, now known to be identical with the pyrophosphate of vitamin Bj (aneurin, thiamine), is the coenzyme of carboxylase, an enzyme which catalyses the ' straight’ decarboxylation of pyruvic acid to form acetaldehyde and carbon dioxide. DPN (also known as cozymase) is identical with the adenino-nicotinic amide dinucleo- tide which wo have discussed already. In fermentation, as in respiration, this substance functions in collaboration with certain dehydrogenases as a hydrogen acceptor, donator and carrier. Adenosine triphosphate, which we have discussed, acts as a phosphate carrier. Magnesium ions , too, are involved. They function as activators for many enzymes concerned with phos- phate metabolism and, in particular, for enolose, an adding enzyme. In addition to these ‘classical’ coenzymea, dialysis removes other substances, including the ions of inorganic phos- phate, calcium and potassium, and evidence is accumulating to show that, for certain reactions at least, even these substances are of great importance and may strictly be classified as coenzymes of fermentation. Since all of these co-substances are essential components of the fermenting system of yeast juice, it follows that the break- down brought about by dialysis is due, not to the abolition of some one particular reaction, but of many. Dialysed juice, with and without the addition of one or more of the known cofactors - has therefore played a large part in unravelling the intricate reaction sequence that underlies fermentation. Much further information has been gained by taking advantage of the fact that certain substances have empirically been found to slow down or stop particular reactions. The addition of these selective inhibitors leads to the accumulation of intermediate products which can be isolated and identified. The reagents most widely used for this purpose have been sodium bisulphite, sodium fluoride and sodium iodoacetate. Tor purposes of discussion the reaction sequence of fermenta- tion can be arbitrarily divided into several stages, each of which involves one or more individual chemical operations, but because we can dissect the whole process into stages and steps in this way, it is not to be supposed that fermentation as such is a fl ^ e P*by-8tep process, catalysed by a mere mixture of enzymes. 379 ALCOHOLIC FEEMENTATIOX The living cell is somctliing more than a mere bag full of enzymes ; fermentation is a highly organized procession of chemical events, the overall result of which is the decomposition of glucose, with production of alcohol and carbon dioxide, together with the provision and capture of free energy which enables the cells to carry out tho synthesis of the new tissue materials required for their maintenance, growth and reproduction. (i) Formation of phosphorylaled sugars. We have already seen that several sugar phosphates can be isolated from fermenting systems to which glucose and inorganic phosphate have been added. If glucose and inorganic phosphate are added to a dialysed juice, however, there is no fermentation and no sugar esters are formed, showing that one or another of the coenzymes must play a part in their synthesis. If ATP is added to tho dialysed juice, however, phosphorylation of the sugar begins again, and £ructofuranose-l:0-diphosphate, fructofuranoso-6- phosphate and glucopyranoso-6-phosphate can be isolated from the system. Work with highly purified enzymes has resolved tins stage into three separate reactions. First of all one phosphate radical is transferred from ATP to glucose, yielding glueopyra- noso-G-phosphato and ADP, aproccss which is catalysed by hexo- kinase (reaction 1 ). Next the glucose ester is reversibly converted into fructofuranose-G-phosphate (reaction 2), tho catalyst being ; phosphohexoisomerase . A phosphato radical is then transferred from a second molecule of ATP to the fructose mono-ester, yielding fructofuranose-l:G-diphosphato. This reaction (reac- tion 3) is catalysed by phosphohexokinase. Tiiis group of reactions may bo summarized as follows, writing only tho forward reactions for tho sake of clarity: (ii) Splitting of the hexost chain. If glucoso or one of the intermediate esters is added to yeast juico in tho presence of iodoacetatc, small amounts of ‘triose phosphate ’ can bo isolated. ALCOHOLIC FERMENTATION showing that hexose diphosphate is split into two 3-carbon frag- ments which, on isolation, prove to consist of an equilibrium mixture of n-glyceraldekyde-3-phosphate and dihydroxy- acetone phosphate. The enzyme concerned, aldolase, has been isolated, and the reaction it catalyses (4) shown to be reversible : CH,0® CO CH a OH dihydroxyactlont phosphate CH.O® CHOH CHO U-glyceraldehyie. Z-phosphate Of the two components of triose phosphate gIyceraldehyde-3- phosphate is the more important from our present point of view, since its derivatives appear lower down in the reaction chain, whereas no direct derivatives of dihydroxyacetone phosphate are found except under special conditions. But dihydroxy- acetone phosphate is not lost to the system, which contains a powerful triosephosphate isomerase. This enzyme catalyses the interconversion of the two triose phosphates (reaction 5) ; cn.o® CH,0® to J2.U. CH a 01I CHO (90%) (4%) The original hexose has now been phosphorylated and quantita- tively split into phosphorylated triose. (iii) Oxidation of glyceraldehyde phosphate. If hexose diphos- phate or ' triose phosphate ’ is added to yeast juice in the presence of fluoride, two further phosphorylated derivatives of glycerol accumulate, viz. glycerol phosphate and glyceric acid phosphate in equimolecular proportions. On isolation and examination the acid proves to be an equilibrium mixture of glyceric acid 2- and 3-phosphates. Of these the primary product must presumably be tho 3-compound since it is formed from glyceraIdehyde-3- phosphate. These products arise by the oxidation of a molecule of glyceraldehyde phosphate at the expense of the reduction of a molecule of dihydroxyacetone phosphate. Yeast juice contains a powerful triosephosphate dehydrogenase, an enzyme which requires DPN”, which is also present. But the 381 ALCOHOLIC FERMENTATION amount of coenzyme is very small, and tlie whole would soon become reduced by acting as hydrogen acceptor for the oxidation of glyceraldehyde phosphate (reacting in its hydrated form): CH,o® I CHOH , CH.O® CHOH + ^DPN.H, I COOH Once all the available coenzymo had been reduced in this way, fermentation would come to an end. But yeast juice contains also a soluble a-glycerophosphale dehydroytnast (see p. 183). This dehydrogenase also co-operates with DPN and, liko dehydro- genases generally, can act reversibly. The reduced coenzyme can therefore become reoxidized by passing on its 2H to a molecule of dihydroxyacetone phosphate, which is thereby reduced to L-gly cerol-3-phosphato : (It should be noted that if n-glyceraldehyde-3 -phosphate was reduced instead of the optically inactive dihydroxyacctono phosphate, D-glyceroI phosphate would bo formed.) Ab a result of tills operation the reduced coenzymo becomes reoxidized and available, therefore, for the oxidation of another batch of molecules of glyceraldehyde phosphate. It may be pointed out in passing that the reduction of the ketone to glycerol phosphate is not normally a large-scale process, but one that only takes place under unusual circumstances such. ALCOHOLIC FERMENTATION for instance, as when the system is poisoned with fluoride, and the reasons for this we Bhall discover presently. In recent years it has been found that tho oxidation of glycer- aldehyde phosphate is a considerably more complex process than was formerly supposed. It was observed by Meyerhof and by D. M. Needham, independently and at the same time, that when glyceraldehyde phosphate is oxidized to glyceric acid phosphate in muscle extracts, one molecule of ATP is synthesized for every molecule of glycerio acid phosphate formed. A similar phenomenon could also be observed in yeast-juice fermentation, and attempts were made to discover its cause, using highly purified (crystalline) triosephosphate dehydrogenase. It wa3 then found that no oxidation of glyceraldehyde phosphate takes place except in the presence of inorganio phosphate. When the latter was added, howover, a brisk oxidation took place, one molecule of inorganic phosphate disappearing for every molecule of glyceraldehyde phosphate oxidized. The product of oxidation now proved to be, not glyceric acid-3-phosphato, but a new compound, glyceric acid-1 :3-diphosphate. It now appears that the dehydrogenase has glutathione as a prosthetic group and that it unites with its substrate through the — SH group. This accounts for the fact that the enzyme is very sensitive to iodoacetate. Dehydrogenation follows with reduction of DPN and the oxidized product is split from the enzyme by a phosphorolytic reaction, and this is why the reaction requires the presence of inorganic phosphate. The intermediate stages are shown on p. 187; for our present pur- poses they may be described as follows. The substrate, an aldehyde, may be considered to be hydrated (reaction 6), and then dehydrogenated under the influence of triosephosphate dehydrogenase and in the presence of DPN and inorganic phosphate, yielding glyceric acid-l:3-dipho3phate. Reaction 7 rs inhibited byiodvacetfc acre?, mV/trn htaaks the — SH groups of the dehydrogenase and by dialysis, which removes inorganio phosphate and the coenzyme. The noxt stage consists in the transference of the phosphate radical at position 1 to a molecule of ADP, so that glyceric acid-3-phosphate and ATP are formed (reaction 8), the process being catalysed by phospho- glyceric ptiosphokinase. The 3-phosphate is then converted into ALCOHOLIC FERMENTATION the 2 -ester (reaction 9) through the agency of phosphoglycero* mutase. The reactions, all of which are reversible, maybe written in the following manner: (iv) Dephosphorylation of glyceric acid phosphate. If one or both of the glyceric acid phosphates is added to whole yeast juice it undergoes fermentation. If, however, a dialysed juice is used there is no fermentation, but a new intermediate accu- mulates, viz. end-pyruvic acid phosphate. This arises by the dehydration of glyceric acid-2-phosphato (reaction 10) at the hands of enolast. It is at this point that fluoride inhibits fermen- tation ; it does so because enolose requires magnesium ions for activity and is, apparently, a magnesium protein. Fluoride forms a complex magnesium fluorophosphato in tho presence of inorganic phosphate. Dialysis, as ordinarily performed, does not stop enolaso activity; it does, however, removo tho coen- zyme required for the next reaction, viz. tho decomposition of pyruvic acid phosphate. If ADP is ndded to a dialysed juice containing pyruvic acid phosphate, the latter begins to break down, and pyruvic acid appears. This reaction ( 1 1) is catalysed by pyru viephosph oki nose , and the phosphate radical is transferred to ADP, yielding ATP once again (eco p. 385). The eno/-pyruvic acid liberated in reaction 11 may pass over into the more stablo Icto-lorm (reaction 12); it is probable, however, that tho less stable and therefore more reactive end-form enters tho next reaction as fast as it is produced. Pyruvic acid accumulates in a dialysed extract provided with ADP -and pyruvic acid phosphate. It does so bccauso the next 384 ALCOHOLIC FERMENTATION reaction, in which the pyruvic acid 13 decarboxylated, requires the presence of co-carboxylase (thiamine diphosphate), together with the enzyme carboxyla-se. The products of this reaction (13) are carbon dioxide and acetaldehyde, and the formation of the latter can be demonstrated by adding sodium bisulphite to a fermenting mixture, when the addition compound, acetalde- hyde-sodium bisulphite, CH 3 CH(0H)S0 3 Na, is formed and can be isolated. The splitting of pyruvic acid is probably the only irreversible process in the whole fermentation sequence, apart from the initial phosphorylations (reactions 1, 3). CH.OH 1 ( 10 ) CHO® — *■ | -HjO COOH ADP ATP (v) Production of alcohol. The final stage of the process con- sists in the reduction of acetaldehyde to ethyl alcohol, and the mechanism of this reaction requires special consideration. It will be remembered that, in the presence of fluoride, glyceric acid phosphate and glycerol phosphate are produced. Glyceric acid phosphate is formed from glyceraldehyde phosphate by reactions which involve the reduction of DPN. The reduced coenzymo passes on its 2H to a molecule of dihydroxyacetone phosphate and, without this, the whole of the available co- enzyme would soon become and remain reduced. As no more glycerio acid phosphate could then be formed, fermentation would speedily come to an end. In a normal as opposed to a fluoride fermentation, dihydroxy- acetone phosphate is not required at this point, for there is available an alternative hydrogen acceptor in the form of acetaldehyde. Under the influence of alcohol dehydrogenase , working ‘in reverse’, the 2H of the reduced coenzyme are trans- ferred instead to acetaldehyde, alcohol is formed, and the oxidized form of DPN is regenerated (reaction 14) and can be „ 386 BOA ALCOHOLIC FERMENTATION used over again (in reaction 7). Tim final operation can be written as follows: CU.OH Reaction 14, like 7, is inhibited by iodoacetate, which blocks the — SH groups of alcohol dehydrogenase. The overall results of this reaction sequence, which is sum- marized in Fig. 28 ore, first, that for each molecule of glucose fermented, iico molecules of alcohol and tico of carbon dioxide are Tig. 2S. Scheme to inmmsriro reaction* of aJcohoHe fermentation of glaooee by yeaat juice. The reaction* are cambered to correspond to the deecription jpeca la the text. For name# of enzyme*, ooenzyrae* and inhibitor*, *ee Table 32. ALCOHOLIC FERMENTATION formed. Secondly, for each molecule of glyceraldehyde phosphate oxidized, one molecule of DPN is reduced, and later reoxidized at the expense of the molecule of acetaldehyde formed from the glyceraldehyde phosphate, so that the coenzyme finishes in the oxi- dized condition in which it began. Thirdly, two molecules of ATP are dephosphorylated in the phosphorylation of each molecule of glucose. Each molecule of the phosphorylated product, fructose-l-.G-diphosphate, yields two molecules of glyceralde- hyde-3-phosphate, and each of these takes up a molecule of inorganic phosphate in the process of oxidation. After oxidation has taken place, the phosphate radicals, two for each molecule of glucose entering the system, are returned in the form of ATP in reaction 8, so that, at this stage in fermentation, the yeast has just recovered the amount of ATP used in the first stages. Presently, however, two more molecules of ADP are taken in and, from these, two fresh molecules of ATP are formed in reaction 11. Thus, as far as the ADP/ ATP system is concerned, two new molecules of ATP are gained for each molecule of glucose fermented. Table 32. Alcoholic fermentation: enzymes, COENZY ME B AND INHIBITORS (See also Fig. 28) Coenzyme Inhibited by ATP Dialysis ATP Dialysis DPN; — PD,H f Dialysis; CH.I.COOH ADP Dialysis Glyceric acid- « 2-3 -diphosphate Mg ions NaF ADP Dialysis Cocarboxylase Dialysis DPN.H. Dialysis; NallSO,; CH.T.COOH Reaction Enzyme 1 Hexokinase 2 Phosphohexoisomerase 3 Phosphohexokinaae 4 Aldolase 8 Phosphotriose Uomerase 6 Spontaneous 7 Triosepboaph a te dehydro- genase B Pbosphoglyceric phospho- kinase 9 Phoephoglyceromutase 10 Enolase 11 Pyruvic phosphokinase 12 t Spontaneous 13 Carboxylase 14 Alcohol dehydrogenase 387 FERMENTATION OP OTHER SUGARS ALCOHOLIC FERMENTATION OP OTHER SHOALS Yeast juico is capable of fermenting glucose, fructose, mannose, sucrose and maltoso, all of whiclx are fermented by living yeast. The fermentation of the first three of these presents no special problems since yeast hexokinase catalyses the phosphorylation of all three to yield the corresponding G-phosphate esters. Of these, glucose-G- and fructose-O-phosphates lio on the normal, direct line of fermentation, while mannose-6-phosphate is con- vertible into fructose-C-phoaphate by pftospAomannotsomerose. Yeast cells, but not yeast juice, can bo ‘trained’ to ferment galactose if it is grown on media in which this sugar is present. Lactose too is fermented by Buch a trained yeast. The training process induces the formation of catalytic systems winch, in effect, convert galactose into glucose. Three reactions are in- volved. First, galactose is phosphorylatcd by a spccifio galacto- kinase to yield galactose- 1-phosphato; secondly, galactose-1- phospbato is transformed into glucose-l-phosphato through the agency of the specific phosphogalactoisomcrase together with its prosthetic group, uridine diphosphate glucose (p. 370) and DPN. The product can then either (n) contribute to the polysaccharide reserves of the cells through the action of a yeast plmphorylase or can be transformed into glucose-G-phospbate by ph osphoylucomu tase and so fermented. The fermentation of the disaccharides, sucrose and maltose raises other problems and possibilities. Sinco yeast juice con- tains a saccharose and a maltose it seems likely that these sugars are first hydrolysed to yield the constituent monosaccharides (glucose and fructose; 2 glucose respectively) which can then be phosphorylnted by hexokinase and fed into the fermentation reaction sequence. There are, however, other possibilities. There is present in another micro-organism, Neisseria perflava, a so- called amylosucrase (p. 113) which acts upon sucrose as follows: introa® -*■ Iru close + - fgljtogen'), while some strains of Each, coli possess an amylomaltaso (p. 114) : nuRoae -*■ elacose + - (‘gljcOgan’V ENERGETICS OF FERMENTATION In each case, one molecule of a fermentable sugar is set free and one monosaccharide unit is contributed to the polysaccharide reserves of the organism. It is not impossible that similar pro- cesses occur in yeast. Yeast cells can, in fact, remove fermentable sugars from, their surroundings more rapidly than they ferment them and can lay down a glycogen-like substance as a reserve of carbohydrate upon which to fall back when no glucose is avail- able in the surrounding medium. ENERGETICS OF FERMENTATION Now let us recall the overall equation of alcoholio fermentation : C f H, t O ( - 2CO, + 2011,011, OH. The loss of free energy in this reaction is roughly 50,000 cal. per g.mol. glucose fermented. Two new are formed at the cost of one molecule of glucose. Now each of these repre- sents some 11,600 cal. of immediately available free energy. It follows that of the 60,000 cal. or thereabouts which become avail- able when a gram-molecule of glucose is fermented, 23,000 cal. are transferred from their source, glucose, to the high-energy phosphate radicals of adenosine triphosphate. About 46% of the total free energy lost when glucose undergoes fermentation is thus rendered immediately accessible to the cell. That part of the energy which is not transferred from glucose to ATP is degraded in the form of heat, in part at any rate, and this, probably, is why the temperature of a fermenting liquor is always rather higher than that of its surroundings. This, how- ever, is not altogether disadvantageous, since, within limits, fermentation , growth and multiplication all proceed more rapidly at higher temperatures. The question is often asked, why is it that yeast does not break up glucose into alcohol and carbon dioxide directly, in- stead of in this rather complicated manner? The total free-energy yield of the process would be the same, no matter how the sugar fermented, but, by working in the way it does, the yeast is able, step-by-step, to transfer a large proportion of the total free energy of the process to the directly utilizable high-energy Phosphate radicals of ATP. If the glucose were directly split, even if enzymes existed that could catalyse this process, the ENERGETICS OF FERMENTATION chances are that the vast balk of the free energy that fermenta- tion renders available would be degraded a a heat, and thus lost to the system. Let us now sec how this important transference of chemical energy from one substance to another is achieved. Living cells seem never to have discovered enzymes capable of catalysing a complete breakdown of the 6-carbon chain of unmodified glucose. The preliminary phosphorylation reactions seem, there- fore, to be devices for getting glucose into a metabolizable form. To accomplish this, chemical work has to bo done, and is carried out at the expense of the terminal high-energy phosphate groups of two molecules of ATP (reactions 1, 3), Then, and only then apparently, the G-carbon chain can be ruptured. In the subse- quent metabolism of the products, tho energy put in is recovered (reaction 8) and tho energetic statu* quo is re-established. Later, still more energy becomes available (reaction 11), and may bo used to start off tho fermentation of fresh molecules of glucose, for example, or for tho synthesis of new and complex tissue materials, bo that the cells may grow and, in due time, divide. The resynthesis of ATP from ADP requires the provision of some 11,500 cal. of free energy per gram-molecule. This is pro- vided by the generation of new ~<£) in tho partial-breakdown products of glucose. Thus, in tho enolaso reaction (10) tho removal of a molecule of water and tho consequent rearrange- ment of molecular architecture is attended by a redistribution of the freo energy of the molecule and tho generation of a now high- cnergy phosphate radical from one that formerly was of tho low energy type. The process can bo written as follows: CH.on CH f ino © cl.o~® + n.o. ioon ioon Another high-energy phosphate is produced in reaction T, in which triosepbosphflte undergoes its phosphate-dependent oxi- dation to glyceric acid-l:3-diphosphato and hero, as in the former case, the now is transferred to ADP to yield a new ATP molecule. FERMENTATION BY LIVING YEAST Yeast-juice fermentation differs from fermentation by live yeast cells in several noteworthy respects. In the first place, the juice is far less active than intact cells. This is probably because the enzymes and coenzymes are not arranged at random in the cell, as presumably they must be in the extract, but in some definite, orderly maimer. It is probably safe to assume that, in the yeast cell, as in a factory, the machinery is arranged in a manner calculated to yield the greatest possible degree of efficiency, and it may not be going too far to suggest that the organization is such that the substance produced by one enzyme in the series is passed immediately on to the next. We know relatively little about the submicroscopic internal organization of this or of any other kind of cell, but that an organization of a high order of complexity exists can hardly be doubted. A second important difference between yeast and yeast juice lies in the effect of inorganic phosphate upon fermentation in the two cases. As Harden & Young first showed, yeast juice can only ferment sugar so long as there is free inorganic phosphate in the medium. The reason for this is clear from what we now know about the mechanisms involved, for inorganic phosphate is required for the phospliorolytic removal of the oxidized sub- strate of triosepho3phate dehydrogenase from the enzyme itself (p. 187). This is a component part of reaction 7, so that this important oxidative process ceases when inorganic phosphate is not available. Any free phosphate that is present is taken up and transferred by way of the diphosphate of glycerio acid to ADP (reaction 8), and the ATP so formed is used to esterify more glucose (reactions 1,3). If inorganic phosphate is added to a juice fermentation, therefore, it disappears and is replaced by the organically bound phosphate of the sugar esters. But the addition of inorganic phosphate has no effect on the rate of fermentation of sugar by intact yeast cells. Once again the notion of intracellular organization has to be invoked: one must suppose that the interior of the cell is so arranged that inorganic phosphate is always available in the cell at the right place and at the right time. In the intact cell, we must believe, ATP synthesized by the 391 ‘live’ fermentation cell’s fermentative activities is utilized for the performance of work of various kinds, especially for tho synthesis of new tissue materials for growth and reproduction, so that the terminal phosphate units of ATP are set free again in one way or another. This inorganic phosphate is caught up by tho fermentation macliine, recharged, so to speak, and again returned to ATP, and so on; a continual cycle of phosphate is built up and used to transfer energy obtained by fermentation to tho places at which it is required and, presently, is actually put to employment. Related to tho effect of phosphate there is an interesting phenomenon known as tho arsenate effect. If arsenate is added to a juice fermentation that has stopped through lack of phos- phate, a long-continuin g but very slow fermentation begins. This is because arsenate is able to replace phosphate in reaction 7, 60 that a glyceric acid arseno-phosphato is formed in reaction 7. The product fails, however, to react with ADP in reaction 8, but phosphoglycerio acid-I-arseno-3'phosphato is rather unstable and breaks down slowly, liberating arsenate, so that glyceric acid-3*phosphate is slowly produced. Tim re-enters the reaction sequence at reaction 0 so that a slow fermentation takes place. An important feature of tills arsenato effect is that reaction 8 is by -passed, so that the high-energy phosphate normally generated at this stage is no longer available to the system. Tho final products of fermentation, by yeast cells or by juico, always includo small quantities o( glycerol and other substances. The formation of glycerol can readily be accounted for. At the very beginning of fermentation, glucose is phosphorylated and split to yield glyceraldehyde phosphate and dihy droxyaceton o phosphate. If fermentation is to proceed, tho glyceraldehyde phosphate must be oxidized to form glyceric acid phosphate, a process in which DPN is reduced. As yet, no acetaldehyde has been formed by the reduction of which reduced DPN can be reoxidized and bo put back into commission. But, os we have learned from experiments on fluoride inhibition, dihydroxy- acctone phosphate can be used instead of acetaldehyde, and this does in fact take place until soma acotaldehydo has been pro- duced. Even when acetaldehyde is being formed, however, small amounts of dihydroxyacetone phosphate continue to bo reduced, for the system contains a soluble a-glj'cerophosphato 302 PRODUCTION OF OLYOEBOL dehydrogenase. The acetaldehyde/alcohol dehydrogenase sys- tem gets the lion’s share of the reduced coenzyme, partly because acetaldehyde is more readily reduced than is dihydroxy- acetone phosphate, and partly because thealcoho! dehydrogenase is more abundant than the glycerophosphate enzyme. A Bmall proportion of the reduced coenzyme nevertheless reacts with dihydroxyacetone phosphate so that a little glycerol phosphate is formed. This is the L-isomer, which proves that it is formed from the optically inactive dihydroxyacetone phosphate (see p. 183) and not, as was formerly supposed, from glyceraldehyde phosphate. The latter arises from hexose diphosphate in the D-form (p. 107) and would yield D-glycerol phosphate on reduc- tion (p. 180). Finally the glycerol phosphate is hydrolysed by a phosphatase that occurs in yeast, and glycerol itself is set free. FERMENTATIVE MANUFACTURE OF GLYCEROL Glycerol is a very important article of commerce, especially in time of war when large a mounts are used in the manufacture of explosives. In ordinary times, the glycerol of commerce is a by- product from the manufacture of soaps by the saponification of fats, and fats are always in short supply in war-time. During the war of 1914-18 the British blockade led to a serious fat shortage in Germany, and the resultant shortage of glycerol meant a shortage also of explosives. The problem was met by making use of the ability of yeast to form glycerol. High yields of glycerol can be obtained from sugar by modi- fying the course of normal fermentation in either of two ways. The two modified forms are known as Neu berg’s ‘second’ and * third ’ forms of fermentation respectively, the ‘ first ’ form being normal alcoholic fermentation. In Neuberg’s second form, sodium bisulphite is introduced into the fermenting liquors. This gives an addition-compound with acetaldehyde, thus depriving the cells of their normal hydrogen acceptor for the reoxidation of reduced DPN. Its place is taken by dihydroxyacetone phos- phate, and one molecule of glycerol phosphate is accordingly formed for each molecule of glyceric acid phosphate. The glycerol phosphate is hydrolysed by the yeast phosphatase, PRODUCTION OF GLYCEROL while the glyceric acid phosphate continues along its usual path until acetaldehyde is formed and reacts -with bisulphite. Each moleculo of glucose therefore yields one molecule of glycerol and one each of carbon dioxide and the nldebydo-bisulpluto CH,0© CU.O® CH» I I I C*H„0, 2CHOH 2CHOH ► 2CO 2CO,+2CH,C«,OH cho coon coon (a) Kcubtrq'i frit form of fermentation CII t O© CH,0© CII.OH CO — - CHOH CHOH * CHjOIt ClfjOH ^H.011 hi<>« \ CH.O© CH.O® CH, ' v (!:hoh - CHOH 1 +CO, CHO CHO coon (trapped) <6) Ntubrry't ttco*i form of fermentation CH.O® CH.O© cir.oif 1 / 2CO — * sillOH — - sinou / in, on in.oii CH.OH CH,0© CH.O© CH, * 2CHOH —* 2^HOH oj +2C0, CHO ioOJl ClIO cn, cir, £ooh cir.on (<■) Stubtrj'* third form of femrntotion Fig. 29. Neu berg's three ‘forms’ of fermentation. addition compound. The process is sketched out in Fig. 296, which may bo compared with Fig. 29 a, which represents norma! fermentation in similar terms. The third form of fermentation sets in if tho fermenting liquors are made and kept alkalino. Under alkaline conditions, acetaldehyde is no longer reduced to alcohol in tho normal 294 PRODUCTION OF OIiYOEEOL manner, but instead undergoes a Cannizzaro reaction. One molecule is oxidized to acetic acid and a second simultaneously reduced to alcohol, and this takes place quite independently of the normal reactions. Again acetaldehyde is no longer available for the reoxidation of reduced DPN, and its place in that reaction is again taken by dihydroxyacetone phosphate. In this case, therefore, each molecule of glucose gives rise to one of glycerol and one of acetaldehyde, one-half of which is further trans- formed into acetic acid and the other into ethyl alcohol (Fig. 29 c). In this third form of fermentation, which takes place only in alkaline solutions, the yeast cell changes its metabolism in such a manner as to produce acetic acid. Unless steps are taken to maintain the alkalinity of the medium, therefore, the pH falls until the medium becomes faintly acid, when the normal form of fermentation reasserts itself and no more acid is produced. We are accustomed to the idea that changes in the environ- ment of living organisms can bring about changes in those organisms, and in the present case the effect of alkalinity in the medium is to change the course of metabolism in the organism. But the organism reacts by producing acid, and we have there- fore a case in which the organism produces changes in its environ- ment. And this is not by any means the only example of its kind : many bacteria tend to produce acids when cultivated in alkaline media and strongly basic amines when the media are acid so that, in either case, the pH of the medium is changed in the direction of physiological neutrality. PRODUCTION OF FUSEL OIL Alcoholic fermentation carried out by live yeast is attended by the production of a number of alcoholic substances other than ethyl alcohol and glycerol, and to these tho collective name of ‘fusel oil’ is applied. Theso substances usually account for less than I % of tho total alcohols, but aro of considerable industrial importance. They are interesting, too, because they are largely responsible for the characteristic flavours and bouquets of alco- hoho beverages. Heavy wines, such as port, contain considerable amounts of higher alcohols and their esters, especially iso-amyl alcohol, and these are responsible not only for the taste and 395 FtJSEL Oil. bouquet of the wine but also, in large measure, for the unpleasant effects of over-indulgence, since the higher alcohols aro powerful narcotics. Another interesting product of the same kind is the bitter principle of beer: this again is an alcohol, in tlu3 case tyrosol. These alcohols arise from amino-acids. The crude liquor con- tains amino-acids arising from grapes, hops and the like, and more are contributed by tho autolysis of dead yeast cells. They aro deaminated, apparently to furnish ammonia for the synthesis of the new yeast proteins which are required as tho cells grow and multiply, for if ammonium salts aro added to the fermenting liquor there is a marked fall in the yields of fusel oil. Yeast dcaminates amino-acids in a peculiar manner that is perhaps unique, the process consisting in an apparently simul- taneous decarboxylation and hydrolytic deamination: R.CHpiHJCOOH + 11,0 - R. 01,011 + Nil, + CO,. In this way the leucines, for example, give rise to tho corre- sponding amyl alcohols, while valine yields iso-butyl alcohol, e.g. leucine m-amyl ulcoW Tyrosol arises in the same way from tyrosine. 300 CHAPTER XVII ANAEROBIC METABOLISM OF CARBO- HYDRATES: MUSCLE AND LIVER INTRODUCTION Modern muscle biochemistry was founded at the beginning of the present century. Before that time, muscular contraction or any other kind of cellular activity was thought to depend upon the sudden decomposition of large, unstable molecules of a hypo- thetical stuff called * inogen \ In the case of muscle, this 1 inogen ’ was supposed to give rise to carbon dioxide and L-( + ) -lactic acid, furnishing the energy expended by the muscle. It was already well known that muscles produce carbon dioxide when they con- tract, and that lactic acid is produced in greater or smaller amounts at the same time. ‘The justification for considering muscle tissue especially, out of all the active tissues of the organism, lies in the fact that only in muscle can we come near to comparing the chemical changes going on with the simultaneous work done or the energy set free as heat. It is difficult to assess the work performed by a secreting gland, and the metabolism of such an organ can only be studied in elaborate perfusion experiments; great advances have been made in the Btudy of nerve tissue, but here the changes going on are so small as to make their detection only lately possible by modern methods. But certain muscles, and a variety of them, can bo removed from the body with absolutely no injury, and can be kept functional for days’ (D. M. Needham). Many different methods have been used to elucidate the prob- lems of muscular contraction. Histology, physiology, bio- chemistry, ^.-radiography and electron microscopy have nii played a part. From the point of view of the histologist we can distinguish between three main types of muscle. These are: { 1 ) the striped or striated voluntary muscle of the skeletal system, (2) the plain, unstriated or involuntary muscle of the visceral system, and (3) cardiac muscle. Most of the chemical work has 397 STRUCTU R£ OF MUSCLE been done on skeletal muscle, but tlicre seem to be few differences between the different types from the point of view of the chemistry of their contractile processes. The structural unit of striated muscle is the long, thread-like myofibril. Several of theso go to make up a muscle fibre, and many fibres to make up a wholo muscle. Under the microscope the musclo fibre shows alternating light and dark bands, or transverse striations. The differences between the appearances of the two kinds of bands are due to differences in their optical properties. The dark-looking striations or anisotropic bands show strong doublo refraction, and when the musclo contracts it can be Been that only theso anisotropio regions undergo shortening. The light-looking isotropic bands, which are not doubly refracting, do not. Chemically speaking, musclo consists chiefly of the muscle protein actomyosin, apparently in the form of a gel which accounts for 75-80% of the whole musclo substance. Now actomyosin is a protein of which the molecule is a thread-like or ‘fibrous’ particlo and is a complex formed by the association of two pro- teins, actin and myosin. A solution of actomyosin is singly refracting when at rest, when the molecules have a purely random distribution. But if the solution is made to flow along a glass tube, tho thread-like particles all become orientated In the same direction, pointing in tho direction of flow, and tho solution becomes doubly refracting. It is therefore probablo that tho anisotropic regions of tho musclo fibre are doubly refracting because all tho actomyosin molecules point in the same direction, while in the singly refracting, isotropic regions they lie perhaps entirely at random. There is now abundant evidence to show that tho molecule of actomyosin, like that of keratin, possesses con- tractile properties, and we know too that only tho anisotropio regions shorten when a muscle contracts. Itthorefore seems likely that the contraction of tho muscle as a wholo is in reality duo to summation of the individual molecular contractions of all tlio actomyosin particles which, lying parallel to one another, account for tho double refraction of the anisotropic, contractile bands. This is a rough picture of what is generally regarded as the contractile machinery. We now have two main problems to consider. First, what is the chemical source of the energy which FORMATION OF LACTIC ACID is expended when the muscle machine does its work and, secondly, how is this energy transformed into the mechanical energy of contraction? At the present time we have a considerable amount of information on the first of these problems, but only some intriguing hints and a considerable number of rival hypotheses about the Becond. The first really significant experiments on the chemistry of muscular contraction were carried out by Fletcher & Hopkins and published in 1007. Working on frog muscles, they showed that larger or smaller amounts of lactic acid are formed when muscle contracts. The general plan of the experiments was as follows. The sartorius or gastrocnemius muscles were removed from the hind legs of a frog and kept under identical conditions. One of the pair was made to do work by being stimulated, and this, tb© experimental muscle, and the unstimulated control were then dropped into ice-cold alcohol and finely ground with sand. These workers realized that a muscle is capable of doing a very large amount of work in a very short period of time, and that to injure a muscle amounts to stimulating it. By using small muscles, chilling and extracting them very quickly with ice-cold alcohol it was possible to inactivate the muscle enzymes very rapidly indeed, and so minimize the large-scale chemical changes which would otherwise result from injuries inflicted in the process of grinding. The chemical changes corresponding to the work done by the experimental muscle could then be found by analysing and comparing the extracts with those of control muscles. Other workers had done similar experiments already, but no precautions were then taken to cool the muscle before grinding, with the result that little difference could as a rule he detected between the experimental and control muscles, so grievous is the injury inflicted by grinding. Fletcher and Hopkins, with their new technique, confirmed and extended the older observations that lactio acid is formed when muscle contracts, and their results demonstrated with beautiful clarity the following points, (i) Muscle can contract in a perfectly normal manner in complete absence of oxygen, (ii) Lactic acid is produced during anaerobio contraction, and piles up with continued stimulation until in the end, the muscle becomes fatigued, (in) If the fatigued muscle is then put into FHOSrHAOEXB oxygen it recovers its ability to contract, and lactic odd simul- taneously disappears, (iv) Less lactic acid is formed in a muscle that is allowed access to oxygen than in one which works anaerobically. Shortly afterwards it was shown by Meyerhof that the lactic add is formed from glycogen and that, under anaerobic condi- tions, the amount of lactic acid formed is chemically equivalent to the quantity of glycogen broken down. A mass oflatcr work made it dear that thero is strict proportionality between tho amount of work done, tho heat produced, the tension developed in a muscle, and the quantity of lactic acid formed; and by 1027 it had. become evident that the energy expended in muscular contraction comes from the conversion of glycogen to lactic acid. A good deal of interest centred round tho phosphate com- pounds present in muscle, for it was already clear that phosphates play an important part in muscle glycolysis, just as they do in fermentation. Tho method in general use for extracting phos- phates from muscle tissue consisted in chilling tho material thoroughly and extracting it by grinding with ice-cold trichlor- acetic acid or some other protein precipitant. Ice-cold conditions were used here, as in the original work on lactio acid formation, in order to inactivate tho muscle enzymes as rapidly as possible. Once the extract had been prepared it was allowed to warm up, and estimations of the phosphate content were subsequently made. In 1027, however, it was discovered that the ice-cold filtrates from trichloracetic precipitation contain a hitherto undetected phosphate compound. This substance is exceedingly rapidly hydrolysed in acid solution and had not previously been noticed for that reason. In order to detect and estimate it, tho trichlor- acetic filtrate must bo kept ice-cold until it has been neutralized to a pH of about 8, at which the new compound is fairly stable. In duo courso the new substance was isolated and shown to ho c realine phosphate, to which tho name of phosphagen and the following formula have been assigned: ns— cc \\'.cn,cooir iff, crtalint fiotphaU 400 PHOSPHAOENS Later investigations showed that this compound is present in the striated, smooth and cardiac muscles of all classes of vertebrates, but absent from those of invertebrates (see p. 333 et seq.). In its place, most invertebrate muscles contain an analogous deriva- tive of arginine, arginine phosphate, with the following structure : HN— CNH (jay, CH.NH, iooH arginine phosphate In passing it should be noticed that both phosphagens contain high-energy phosphate, a feature which turns out to be of great physiological significance. In what follows we shall discuss mainly the creatine compound, but it may be assumed that what goes for this substance is also true of the arginine and other analogues. A wave of interest in the new compound soon developed, and within a few years it became known that it plays an important part in the chemistry of muscular contraction. Phosphagen, it was shown, breaks down during activity and is resynthesized during rest, aerobically and anaerobically alike, and, moreover, it breaks down far more rapidly than does glycogen. It was suspected by some that, since the breakdown of phosphagen precedes that of glycogen, it must be the immediate source of contraction energy, the more slowly acting process of glycolysis being used to resynthesize the phosphagen, rather as the lever of an air-gun is used to reset the spring after the trigger has been pulled. But this idea did not find much favour ; muscle chemists were still too much wedded to the older lactio acid hypothesis. In 1930, however, new evidence appeared. Lundsgaard, who was studying the pharmacological properties of iodoacetic acid, observed that in animals dying from iodoacetate poisoning the muscles went into rigor, and that, instead of becoming markedly acid as was to be expected, they actually became faintly alkaline. Closer examination showed that no lactio acid whatever had been produced. This discovery caused a good deal of surprise, 26 401 MUSCLE EXTBACTS since it demonstrated conclusively that muscle can contract icithoul producing any lactic acid at all. Further work on iodo- acetate-poisoned muscles showed that phosphagen breaks down when work is dono, the amount split being strictly proportional to the amount of energy expended. Further, phosphagen was not resynthesized if an iodoacetate-poisoned muscle was allowed to rest, and, with repeated stimulation, the muscle went into rigor aa soon aa its stock of phosphagen was exhausted. By this time it had begun to appear that muscle resembles yeast rather closely in its carbohydrate metabolism. Compounds such as the hexose phosphates could be detected as well in the one as in the other, while that all-important compound adenosine triphosphate was also detected in both. Host of the work so far described had been done on intact, isolated muscles, mostly of frogs, kept under anaerobic conditions, but in 1925 Meyerhof published a method for the preparation from muscle of extracts analogous to the yeast j uice that had been so valuable in the study of fermentation. Tho method employed is roughly as follows. The animal is anaesthetized and cooled to 0° C. The muscles are carefully cut away with the least possible injury, and care is taken to keep them cold. The tissue is put through an ice-cold mincer and allowed to stand for 30-00 min. with ice-water or isotonic KC1. After straining and centrifuging, a rather viscous liquid is obtained and stored in the refrigerator until required. Extracts prepared in this way contain all the enzymes and coenzymes required for the production of lactic acid from added glycogen, and will also break down creatine phosphate and ATP if these are added. Most of our knowledge of muscle chemistry has been acquired with tho aid of extracts of this kind. Dialysis removes tho coenzymes, just as it does in the case of yeast juice, and many experiments have been carried out with extracts previously dialysed or treated with fluoride or iodoacetate. One point that should be noticed is that, although they can convert glycogen into lactic acid, these extracts do not respire, so that it is possible to work on extracts in tho presence of air instead of having to take elaborate precautions to ensure anaerobiosis, as is necessary when isolated muscles are employed. Whereas the enzymes concerned in glycolysis are present in tho cytoplasm and come out in the extract, cytochrome oxidase and certain 402 GLYCOLYSIS other enzymes essential for respiration are not soluble bub remain attached to the insoluble cell debris, especially the mitochondria. Another important feature of these extracts is that, unlike intact muscle, they have no action upon glucose. FORMATION OF LACTIC ACID The reactions involved in glycolysis are very similar to those involved in alcoholio fermentation. The first step in muscle extract consists in the phosphorolyBis of glycogen, i.e. splitting of the glycogen molecule by the elements of phosphoric acid. The reaction, -which is reversible, is catalysed by muscle phos - phorylase and yields a-glucose-1 -phosphate: CH.01I CH.OH CH.OH + glycogen ®-OH CH.OH CH.OH CH.OH a-olucoie-l -phosphate The product is then converted into glucose-6-phosphate by phosphoglucomutase : Glucose-6-phosphate con also bo formed by the action of muscle hoxokinaso upon glucose. ATP is required in the usual -way but fresh muscle extracts, while they contain hexokinase, contain no ATP because powerful ATP’ases are also present. If the 403 26-2 GLYCOLYSIS latter are inhibited, e.g. with fluoride or by prolonged dialysis at O’ C, tho formation of gIucosc-6-phosphate from glucose can be demonstrated if ATP is added. Prom this point glycolysis and fermentation follow a common path until pyruvic acid is formed. Here the pathways diverge again, for muscle, unlike yeast, does not contain carboxylase. Co -carboxylase is present, but, like DPN and TPN, it collaborates with more enzymes than one, and its presence in muscle is in no way an indication that carboxylase itself is present. In yeast, it will be remembered, pyruvic acid is split into carbon dioxide and acetaldehyde, the latter then functioning as a hydrogen acceptor in the reoxidation of reduced DPN. In muscle extract, however, no carboxylase being present, pyruvic acid itself dis- charges this function and is reduced asymmetrically to L*(+)* lactic acid, under the influence of lactic dehydrogenase: err, I The overall effect of this reaction sequence is that, on the carbohydrate side, one 6-carbon unit of glycogen yields two molt' cults of lactic acid. DPN is alternately reduced and reoxidized just as it is in fermentation, but there are differences as far as the ATP/ AD P system is concerned. In yeast juice and in muscle extract alike tho sequence as a whole leads to the generation of four new high-energy phosphate groups for each 0-carbon unit metabolized. In fermentation, two of these new are used to compensate for those used in the preliminary phosphorylation of the glucose molecule, so that in thi3 case there is a net gain of two molecules of ATP for each glucose molecule fermented. JIuscle extract, however, starts from glycogen, not from glucose, and the first stage in glycolysis consists in the splitting of glycogen by phosphoric acid, not by ATP. Only one molecule of ATP has therefore to be used up in the production of each, molecule of frudo- 404 ENERGETI08 OF GLYCOLYSIS furanose diphosphate. Of the four molecules of ATP subsequently produced, only one is required to restore the status quo, so that in muscle glycolysis there is a net gain of three molecules of ATP for each G -carbon unit of glycogen metabolized , as compared with a gain of two molecules for each 6-carbon glucose unit metabolized in the case of fermentation. Calculations show that the conversion of glycogen into lactic acid, under biological conditions, is accompanied by a loss of free energy equivalent to approximately 57,000 cal. for each 6-carbon unit glycolysed. Three new high-energy phosphate radicals are formed at the same time, bo that the amount of energy ‘captured’ in the form of ATP is about 34,500 cal., which corresponds to approximately 60% of the total free-energy exchange. This, of course, is in muscle extract, and there are reasons for believing that an even higher proportion of the energy may be ‘captured’ in the intact muscle cell. This is really a re- markable performance when it is realized that the efficiency of even the most modern superheated steam turbines barely reaches 60 % or thereabouts. If glycolysis began with glucose instead of glycogen the free-energy change would be about 36,000 cal. per g.mol. and in this ease there would be a net gain of only two high-energy phosphates, as in alcoholic fermentation, correspond- ing to an energy-capture of 23,000 cal. per g.mol., or 64 %. Thus the convenience of having a localized store of readily available carbohydrate in the muscle costa very little in terms of efficiency. PARTS PLAYED BY ATP AND FHOSFUAGEN The reactions of glycolysis involve breakdown and resynthesis of ATP, just as do those of fermentation, and the resemblances between the two processes are very striking indeed.There is, how- ever, one very important difference between yeast and muscle, for whereas the latter contains phosphsgen, yeast does not. If an intact, isolated muscle is allowed to contract under anaerobic conditions there is a decrease in the amount of phos- pliagen present, together with corresponding increases in the amounts of free creatine and free inorganic phosphate. This sug- gests that muscle must contain an enzyme catalysing the hydro- lysis of creatine phosphate. Tim possibility was investigated by 405 ATP AND THOSPHAOEN Lohmann, who found that there is no hydrolysis if phosphagen is added to a dialysed extract, indicating that some dialysable factor is involved. This factor was identified with ADP, and it was then discovered that phosphagen is not hydrolysed, as had formerly been supposed, but that it reacts with ADP, a phos- phate radical being transferred, yielding free creatine together with ATP. ATP is split by a powerful adenosine triphosphatase that is present in tho extract, ADP is formed and phoaphorylatcd at the expense of the phosphagen and eo on, until no more phosphagen remains: ATP + n,0 ADP + HO©. ADP + C® ATP + C, OwraU: C® + 11,0 -f C + HO.®. Lohmann further discovered that adenosino triphosphatase can be inactivated by prolonged dialysis at 0° C. and, when the enzyme had thus been inactivated, he was able to show that the reaction between creatine phosphate and ADP is freely reversible : ADP + C® &=* ATP + C. Veiy little energy change is involved and no inorganic phosphate is set free in this, the Lohmann reaction. From the existence of this equilibrium system we can make certain deductions con- cerning the part played by phosphagen in the economy of the muscle. Anything that tends to decomposo ATP will force the reaction over towards the right and phosphagen will be broken down . If, on tho other hand, glycolysis is in progress so that new high-energy phosphate is being generated, fresh ATP will be formed, the reaction will swing towards the left and phosphagen will be resynthesized. Perhaps the most important outcome of Lohmann’s work was the realization that, until some ATP has been broken down to provide ADP, no decomposition of phosphagen can take place: in other words, the breakdown of ATP must take place even earlier than tliat of phosphagen. The breakdown of ATP, in fact, is the earliest reaction wo have so far been able to detect and henco is likely to be the mast immediate known source of contraction energy. This suggests that ATP must play some part in contraction, over and above the part it plays in glycolysis. 405 ATP AND CONTRACTION The probable nature of this additional function was first revealed by the work of Engelharfc & Lubimova, who found that the adenosine triphosphatase activity of muscle is in- separable from and apparently identical with ‘myosin*. This was before the discovery of actin and actomyosin, and there is not much doubt that their ‘myosin* consisted in reality of a mixture of actomyosin and myosin proper. Thus actomyosin, the actual contractile protein of muscle, appears to be identical with the enzyme that catalyses the decomposition of ATP and leads to the liberation of the free energy of its terminal high-energy phosphate. Experiments by D. M. Needham, J. Needham and othere have given results which are completely in harmony with the supposition that, when actomyosin and ATP come into contact, a sudden shortening of the molecules takes place, fol- lowed by decomposition of the ATP and a return of the protein molecules to their former length. These results were obtained by measurements of the viscosity and refractive indices of solutions of ‘myosin*. Threads prepared from actomyosin simi- larly contract if treated with ATP, and again, it is possible, by treating muscle with the collagenase of Clostridium welchit, to obtain isolated myofibrils which similarly contract on addition of ATP. The ability to contract in response to ATP is evidently a built- in property of the muscle proteins in situ for, if a fow fibres are stripped from the psoas muscle of a rabbit, they contract when treated with ATP, and the same results are obtained even when the excised tissue has been preserved in 50 % glycerol for many months in the cold. The contracted fibres can be made to relax again if a preparation of the ATP-creatine-transphosphoryl- ating enzyme (i.e. the enzyme catalysing the Lohmann reaction) is added. Exactly what happens in the process of shortening wo still do not know, but the problem is being studied intensively. The shortening of actomyosin and preparations containing it in an orientated form appears to be due to an ATp-induced disso- ciation or disaggregation of actomyosin into actin plu3 myosin. If we accept these results at their face value it follows that the first reaction known to take place when a muscle is stimulated consists in the decomposition of ATP, a process which is attended by a sudden shortening of the actomyosin molecules and, in 407 ATP AND CONTRACTION some manner which is still quite obscure at the present time, by the conversion of the free energy of its terminal ~(g> into the mechanical energy of contraction. This field is being very actively studied and there is no lack of hypotheses, but the nature of the contractile process itself is still unknown. There can be no doubt, however, that very intimate relationships exist between the source of contraction energy (ATP), the enzyme that catalyses the liberation of that energy (ATP’ase), the contractile material itself (actomyosin) and tho phosphagen. It is difficult, in view of the evidence, to escape the conclusion that ATP plays a most intimate part in muscle contraction, but as yet it has not been possible to demonstrate any direct decom- position of ATP in a stimulated muscle until tho muscle has reached a very advanced stage of fatigue and is on the very threshold of rigor. This we interpret as duo to tho extreme rapidity with which ADP, formed, as we believe it to be formed, in contraction is repliosphorylated by phosphagen through tho Lohmann reaction, but until such a breakdown of ATP can actuaQy be demonstrated in normal contraction there will still bo somo who will regard the ATP theory with suspicion if not with actual scorn. This vitally important demonstration might be forthcoming from studies of somo kind of slow-acting muscle, such as that of tho tortoise or toad, especially atlow temperatures as A. V, Hill has suggested, or by the discovery of somo selective inliibitor of the Lohmann reaction. If w© accept this hypothesis the function of phosphagen becomes clear. The muscle contains relatively small amounts of ATP, and these would soon be exhausted but for tho fact that tho ADP formed by its decomposition can be rapidly rophosphoiyl- ated at the expense of phosphagen, through tho Lohmann reaction. This means that tho muscle can go on acting several times longer than it could if no phosphagen was present, for tha amount of phosphagen present in a typical striated muscle is several times greater than that of ATP. These facts are illustrated in Table 33. Phosphagen, then , may be regarded as a reserve of high-energy phosphate. In creatino phosphate tho free-energy value of the phosphate has been estimated at about 13,000 cal. per g.mol., and that of arginino phosphate at about 12,000 cal. per g.mol. 408 ATP AND CONTRACTION Table 33. Phobphobtjs partition in striated muscle (mg. P per 100 g.) Ratio (Inorg. + PhOB phag en)-P Animal Inorganic-1’ Pbospbagen-P Pyro-P Available pyTO-P Prog 30 60 30 5-3 Rat 35 80 40 E-7 Rabbit 20 62 40 4-4 Notts. (1) TfiO ‘available’ pyrophosphate- Pis 50% of the total pjrophospha te-P of the ATP present, since only the terminal energy.rich bond is directly available through the activity of adenosine triphosphatase. (2) The inorganic phosphate found in trichloracetic extracts of muscle is mainly formed by the breakdown of some of the phosphagen during the extraction. The sum, {inorganic + pbosphagen)-P may therefore be taken aa an approximation to the true phosphagen-P of the resting muscle. ’ In all, three ways are known in which ATP can be synthesized anaerobically from ADP in muscle. The first of these, the Lohmann reaction, is probably used at the onset of contraction and enables the muscle to keep up a high level of immediately available energy in the form of ATP by drawing upon the stored of the phosphagen. The second source of supply consists in the new ~ @ generated in the course of glycolysis and this is the main ultimate source of energy when activity is prolonged. Glycolysis gets under way relatively slowly, however, and phos- phagen is used to tide the muscle over the interval between the onset of activity and the establishment of the glycolytic reaction sequence. But there is yet a third possible source of ATP, though this is probably used only when tho muscle is in extremis. In all the processes mentioned so far, only the terminal ~ (g) of ATP is involved. After this has been utilized there remains another such ~ ® in each ADP molecule, but this is not directly accessible. Muscle, howover, contains myokinase and this enzyme, acting upon two molecules of ADP, can catalyse the transfer of a phosphate radical from one molecule to a second, yielding a molecule of ATP, together with adenylic acid (see p. 116). As has been suggested, this way of producing ATP is probably only used as a last resort. The free adenylio acid produced is highly toxic and, once formed, rapidly undergoes deamination at the hands of the adenylic deaminase of muscle to yield inosinic acid, and the muscle goe3 into rigor. Inosinic acid cannot replace adenylic acid as a carrier of phosphate, nor does the action of the adenylia acid deaminase appear to bo reversible. ioo CHEMISTRY OF CONTRACTION Decent work has shown that myosin ATP’ase can split not only ATP but the triphosphates of inosino, guanosino, uridine and eytidino as well, but there is no evidence that any of these can act as alternative sources of contraction energy. CHEMICAL EVENTS IN NORMAL CONTRACTION Wo aro now in a position to make some sort of picture of the course of events in normal mtftcular contraction. It will bo convenient first to consider what takes place during contraction in a muscle previously poisoned with iodoacetatc. This drug, it will be recalled, abolishes the activity of triosephoaphatc dehydrogenase and therefore puts a stop to glycolysis and there-' fore to the oxidativo breakdown of its products, so that only two anaerobic sources of ATP are now available to the cells. On the arrival of a nerve impulse, ATP is broken down, giving rise, by way of unknown intermediary processes probably in- volving actomyosin or its components, to ADP and inorganic phosphate, and furnishing at the same time the contraction energy. The ADP is promptly converted again into ATP at the expenso of phosphagen and no change in tho ATP content of the muscle can be detected; some phosphagen disappears, how- ever, and is replaced by free creatine and free inorganic phos- phate. If repeated stimuli are applied to the muscle, these processes continue until, eventually, no phosphagen remains. At a last resort the myokinaso of the muscle is called into play and the last traces of ATP aro decomposed, giving, in tho end, adenylio acid, which is deaminated. The muscle goes into rigor, and the ammonia produced by the deaminase can be detected in the cells. In the case of a normal, unpoisoned muscle working anaero- bically, glycolysis also comes into the picture. Tho following phenomena can be observed during the period of anaerobic activity i AT P remains unchanged. Phosphagen disappears. Free creatine appears. Free inorganic phosphate appears. Glycogen disappears. Lectio acid fa formed. 410 CHEMISTRY OF CONTRACTION These changes continue as long as the muscle is active. There then follows a short period of anaerobic recovery and during tins interval, which amounts to about 30 sec., the following further changes take place: ATP remains unchanged. Phosphagon ia resynthesized. Free creatine disappears. Free inorganic phosphate disappears. Glycogen disappears. Lactic acid is formed. Thus glycolysis continues for a short time even after the cessation of muscular activity, and this * glycolysis of recovery ’ is attended by resynthesis of phosphagen and a return to the status gun of the resting muscle apart, of course, from the conversion of some glycogen into lactic acid. All these phenomena can be accounted for in terms of the reactions we have considered. While activity lasts, ATP is hroken down to provide the energy expended by the muscle. Phos- phagen is used up to maintain the level of ATP and corre- sponding amounts of creatine and inorganic phosphate are set free. The froe phosphate is taken up, for the phosphorolysis of glycogen in the first instance, and later for the conversion of glyceraldehyde-3-phosphate into the l:3-diphosphate. This is t followed by the generation of new high-energy phosphate in the usual way, the new ~ 12 6 9 16 7 10 17 14 . 11-6 13 1-2 . 22 23-2 The principal differences between different animal tissues from the point of view of their glycolytic mechanisms lie in the amounts of phosphagen they contain (Table 34). The larger and more powerful skeletal muscles usually seem to contain larger amounts of phosphagen, while the alow-acting, smooth muscle of the gastro-intcatinal canal, for example, contains only a 413 PIXOSPIIAGEN AND GLYCOLYSIS fraction, amounting to perhaps one-fifth, of the amount present in the average striated muscle. Cardiac muscle also contains rela- tively little. The only tissues known to contain phosphagen in concentrations comparable with those of striated muscle are the electric organs of certain fishes, e.g. Torpedo. Liko striated muscle, these organs are capablo of going into activity almost 4, CO im/ f tC.OH COOII Air *-COOII <0)1 cit, /\. tCOfi ADA I CH,Oll CR/Hl <» +®-OH CH.OH | \» ®oSr,.o v 0-'\ at ocn^a l/\ cn,( Nj |/Son nr.oe ^W^CK.0® co »■ iiroii f ADI ,Ofe coohV (IIJ'CH.OK coon Tr&naaldolaso M) Tranaketolaae (vii) Aldolaeo + phosphatase To what extent this somewhat circuitous route is followed it is difficult to be certain, but these reactions make it possible to account for the fact, winch has been realized for some time, that a part of tho total carbohydrate oxidation that takes placo in animat tissues goes on by soma route other than glycolysis followed by oxidation of pyruvate. Probably not more than 10% of the total oxidation proceeds by this, tho so-called ‘shunt’ routo. Having given some account of tho 'shunt* wo must now turn our attention to tho quantitatively more important oxidation of the pyruvate produced by glycolysis. Pyruvate is known to bo totally oxidized to CO. and water by most tissues, as long as conditions arc fully aerobic, and we have already dealt with tho glycolytic reactions which lead to its formation. Now tho muscles are the biggest consumers of carbohydrato and it is natural therefore that most of tho early work on tho aerobic metabolism of carbohydrates should have been done with muscle tissue. Muscle extracts cannot bo used for tills 442 OXIDATION OF CABBOHYDBATE purpose because, as will be remembered, they contain only the soluble enzymes of the tissues and do not respire. This fortunate circumstance made it possible to obtain a clear picture of anaerobic glycolysis before the more complex operations of aerobic glycolysis plus oxidation were investigated. The foundations of our present knowledge of aerobio meta- bolism were laid by Szent-Gyorgyi, using suspensions of minced pigeon-breast muscle as his material. This is a very active muscle and one which contains a good deal of myoglobin so that, in the minced form, it can keep itself weU supplied with oxygen. Indeed, Szent-Gyorgyi found, minced pigeon-breast muscle respires very actively and produces little or no lactic acid. He studied the rate of respiration of his preparations under a varie ty of experi- mental conditions and made the following fundamental observations: „ , , . r ~ (a) The rate of respiration is very high at first but falls off slowly with time. „ , , , ,, „ . (&) The fall in the rat© of respiration is paralleled by the rat at which succinate disappears from the mince, and (c) the respiratory rato of a failing preparation can be restored to its original high level by the addition of catalytic amounts of succinate or fumarate. (d) For each volume of oxygen consumed by the touoan equal volume of carbon dioxide is formed, indicating that the material undergoing oxidation is a carbohydrate of some kind. (CH,0), + "0, - "CO, + "3,0. From these observations Szent-Gyorgyi concluded that the oxidation of carbohydrate is in some way catalysed by succinate and fumarate and, since it was already well known that these two acids are interconvertible through the activity 0 hydrogenaso, he concluded that this enzyme must he mtnnate y concerned in the oxidation of carbohydrate mater, another important discovery. It had already been estabMwd that malonato is a powerful, competitive inhibitor of succinic • Tho ratio 0 7 and 0-8 respectively. 443 OXIDATION OF CARBOHYDRATE dehydrogenase, and Szent-Gyorgyi was able to show that malonate prevents the catalytic effect of succinate and fumarato in failing preparations and, moreover, baa a powerfully depres- sant action upon the respiration of the fresh minco. To account for these observations, Szent-Gyorgyi brought for- ward the suggestion that succinate and fumarato act together os a carrier system for hydrogen removed from carbohydrate materials of some kind. If wo represent these unknown sub- stances by AH, , his hypothesis can bo schematically represented as follows: '■'Y'ln CH.COOH X reduced cytochrome ( oxidued CH.COOH AH, dehydrogenate ■y" In this system, succinic dehydrogenase would have to work ‘backwards' instead of ‘forwards' but, ns was already known, dehydrogenases in general ore capable of acting reversibly, and in this, as well as in its general aspects, the scheme was consis- tent with the contemporary knowledge of biological oxidations. Moreover, in this system, everything depends upon succinic dehydrogenase, so that the dire effects of malonate on respiring muscle tissuo are readily explained. In the absence of any positive cluo to the identity of 'AH.', Szent-Gyorgyi suggested that tliis might bo triosophosphato, or perhaps, pyruvate. These results stimulated other investigators to study other materials and it soon became clear that Szcnt-Gyorgyi’s m alonato- sen8itive ej'stcm must bo as widely distributed in living tissues as are cytochrome and cytochrome oxidase, for colls and tissues of many kinds were found to hehavo in the same manner towards succinate, fumarato and the inhibitory substance malonate. Presently it was discovered by Szent-Gyorgyi himself that two Other C,-di carboxylic acids act in tho same way as succinate and fumarato, viz. malatc and oxaloacctate, and shortly after- wards Krebs announced tbo discovery that, in addition to succinate, fumarate, malate and oxaloacctate , a-ketoglutarate 444 OXIDATION OF CARBOHYDRATE and citrate also act catalytically on the respiration of minced muscle, and that the effects of all these substances are inhibited by malonate. The behaviour of a-kefcoglutarate could be explained readily enough, for it was already known that, being an a-keto-aeid, a-ketoglutario can undergo oxidative decarboxylation to yield succinic acid, and thus leads directly to Szent'Gyorgyi’s catalytio cycle: COOH Ah, in, Ao COOH +h,o Ah, 'Ah, Aoon co,. a-ketoglutaric acid The behaviour of citric acid too was explicable in terms of the widely distributed citric dehydrogenase, for this enzyme was believed to convert citric into a-ketoglutaric acid, from which sucoinio acid can then be formed. If, however, the formulao of citric and a-ketoglutaric acids are compared, it will bo noticed that whereas the ketonic oxygen of the latter is in the a-position, citrio acid contains a /?-oxygen atom, bo that the direct conver- sion of citrate into a-ketoglutarate seemed very improbable. This phenomenon was explained, however, by the discovery by Martius & Knoop of a new enzyme, aconitase, which catalyses the conversion of citrio into iso-citric acid through a common intermediary in the form of aconitic acid : COOH Ah. A{OH)COOH Ah, AoOH c Uric acid Ah =s C.COOK ! Ah, Aooh acanUtc acid COOH Ahoh Ah.cook Ah, Aooh lao-citric acid Reinvestigation of the old citrio dehydrogenase now showed that it is specific for iso-citric acid, and has no action upon citric acid 445 OXIJDATIOS or CABBOHYDEATE itself except in the presence of aconitase, with which the early preparations were invariably contaminated. j'so-Citric dehydrogenase, which collaborates with TPN, cata- lyses the dehydrogenation of teo-citric acid to yield oxalosuccinic (x'keto-/?-carboxyglutaric) acid. This compound then, nnder the influence of a specific oxalosuccinic decarboxylase, loses carbon dioxide and gives rise toa-fcetoglutaric acid. iso-Citric dehydro- genase and oxaloacetic decarboxylase are closely associated and, indeed, appear to be a single enzyme-protein, but the two reactions can be studied separately because the decarboxylase but not the dehydrogenase requires the presence of manganese ions for its activity: COOH Ahoh Au.cooh t COOH COOH COOH to to ±2H I . iCO, I CITCOO.n *=** CH, 1 (Mn-)1 CH, CH, toOH toOH OzaIot*ca*ic a-Ldo?? Blade odd cad The product can then be oxidatively decarboxylated to yield succinic acid, and thus leads to one of the primary catalysts of the Szent-Gyorgyi system. It became possible, therefore, to trace out metabolic con- nexions between all the substances known to act catalytically on the oxidation of carbohydrates by minced pigeon-breast muscle and by many other kinds of cells and tissues. Their action could be explained by their con vcrtibUity into succinate and fumarate, the two primary catalysts which, according to Szent-Gyorgyi, function as a hydrogen-carrying system- The effect of malonate was explained because malonate specifically inhibits the succinic dehydrogenase, upon which the carrier activity of the succinate- fumarate system depends. Thu3 all the phenomena observed in Szent-Gyorgyi’s original experiments, together with a number of later observations, could bo accounted for, apart only from the slow decline that takes place in the respiration of minced pigeon-breast muscle. Even this last phenomenon can be explained, however, by the slow, spontaneous /7-decarboxylation 446 CITRIC ACID CYCLE of oxaloacetate which takes place under physiological conditions, yielding pyruvate, which does not aot catalyticaUy. Oxaloacetate and the other catalytic substances therefore drain slowly away and, as their concentrations decline, the rate of respiration of the preparation falls off. In passing it should be noticed that the reaction chain leading from a-ketoglutaric, through succinic, fumaric, malic and oxalo- acetic acids to pynivio acid is an important link between carbo- hydrate metabolism and that of proteins, for a-ketoglutaric, oxaloacetic and pyruvic acids respectively are formed by the deamination of three of the commonest non-essential amino- acids, viz. glutamic acid, aspartic acid and alanine. THE CITRIC ACID CYCLE Before going on to consider more recent developments the reader will do well to study Fig. 35, in which the reactions just discussed are collected together in schematic form and the compounds shown in relation to some other metabolic products. The whole picture took on an entirely new aspect with the suggestion by Krebs that pyruvio and oxaloacetic acids might react together to form a 7-carbon compound, from which citrate (6C), a-ketoglutarate (5C), and the 4-C dicarboxylio acids were then re-formed. What had formerly been considered simply as a chain or series of reactions was now visualized as a cycle. In its simplest form this hypothetical Bcheme can be written as in Fig. 36. Pyruvic acid, with its three carbon atoms, enters this cycle by reacting with oxaloacetic acid. With each turn of the wheel one pyruvate molecule enters, three molecules of carbon dioxide are produced, and oxaloacetate is regenerated to take up a further molecule of pyruvate. Since a single molecule of oxaloacetate can be used over and over again in the oxidation of a theoretically unlimited number of molecules of pyruvate, it follows that oxaloacetate, or any substance lying on the cycle, can act catalyticaUy in the oxidation of pyruvate to carbon dioxide. The mcchanismspostulated involve succinate, fumarate, malato, oxaloacetate, a-ketoglutarate and citrate, all of which are known to act catalyticaUy on the oxidative breakdown of 447 CITRIC A.OID CYCLE carbohydrate. Succinic dehydrogenase is directly involved in the cycle in the oxidation of succinate to fumarate, so that the effect of malonate on the oxidation of carbohydrate is readily explained, while the slow failure of respiration in minced muscle COOK k C?OH]COOH 2 at, e CO COOH tJtloflutent COOH i„. . loOH 'In COOK COOH k = I CHOK toon ro«lic JchyJrw- CO // /> ,M X Fig, 35. Summary of react ions leading from citrate to pyruvate, rhowing iome metabolio interrelationships. preparations can again bo explained in terms of a slow, spon- taneous breakdown of oxaloacetate to pyruvate, which docs not act catalytically. Krebs’s hypothesis therefore goes much further than that of Szent-Gyorgyi. It accounts at once for the catalytia activity of all the di- and tricarboxylic acids, it explains the malonate effect and, abovo all, it accounts for the complete oxidation of 448 CITBIC ACID CYCLE pyruvate, a known and important intermediate in the oxidation of carbohydrate and many amino-acids, to carbon dioxide. Glu- cose and glycogen, we know, can be metabolically converted into pyruvate, and Krebs’s scheme, taken together with the known reactions of glycolysis, can account therefore for the complete oxidation of glucose and glycogen. Szent-Gyorgyi’s scheme, by contrast, could only account for the dehydrogenation of the un- identified carbohydrate intermediate which we have described here os ‘AH 2 \ Fig. 36. Outline of the citric cycle hypothesis. Krebs himself supplied the first evidence in favour of his ‘citric acid cycle’. Pyruvate and oxaloacetate were incubated together with minced muscle under Btrictly anaerobic conditions. By working anaerobically it was expected that, as the cycle is essentially an aerobic Bystem, some product might accumulate instead of being oxidized if, in fact, pyruvate and oxaloacetate do react together. After incubation, Krebs was able to demon- strate the formation of substantial amounts of citric acid. Many workers have since repeated and confirmed Krebs's observations on a variety of tissues, and there is no shadow of doubt that some reaction involving pyruvate and oxaloacetate does indeed take place. 29 449 CITRIC ACID OTCLE Other significant experiment-3 have been carried out on liver tissue. If pyruvate is incubated vritli liver tissue it is possible to demonstrate the formation of considerable yields of suc- cinic acid, together with smaller quantities of a-ketoglutaric acid. The discovery of the oxaloacetic decarboxylase of liver sug- gested a possible route for the synthesis of succinate from pyru- vate, since the latter, by /7-carboxyIation, yields oxaloacetic acid, from which succinic acid can bo formed by the reversed actions of malic and succinic dehydrogenases, together with that of fumaraso. In this case, however, the formation of a-keto- glutario acid is left unexplained. Krebs pointed out that if this latter route were followed it should, since it involves succinic dehydrogenase, bo inhibited by malonate. He therefore incu- bated pyruvate with liver tissue in the presence of malonate and found that even larger yields of succinate were obtained. Hence the synthesis must proceed by some route that does not involve succinic dehydrogenase. This, of course, is entirely in keeping with Krebs’s cyclical hypothesis, and tho evidence was further strengthened by the later demonstration that, in addition to succinate, substantial amounts of a-ketoglutarato and traces of citrate are formed at the same time. Much illuminating evidence regarding the Krebs cycle was later obtained by employing radioactive carbon as a ‘tracer’. If pyruvate is incubated with liver in tho preaenco of radioactive carbon dioxide, radioactive intermediates including citrate and a-ketoglutarate can subsequently bo isolated. Tliis and other similar evidence again argues in favour of tho cycle, and more precise information has been obtained by finding out precisely where, in tho a-ketoglutarate molecule, tho radioactive carbon was located. There is now good evidence in favour of every step in tho oyclo, with an exception in tho case of tho supposed 7-C in- termediate. Critical experiments designed to detect the for- mation of such a compound have failed and, indeed, there has never at any time been evidence that such a substance is formed. If a 7-carbon compound is produced it must loso one carbon atom, most probably by oxidative decarboxyl- ation, to yield citric acid: 450 OITBIO ACID CYCLE COOH in COOH L l(OH)COOH CH, toHjCOOH CH, CH, lo k. ioon iooln oxalocitracon ic acid L. t(OH)COOH iooH +co, citric acid Now arsenite ia known to be a powerful inhibitor of oxidative decarboxylation and it would therefore be anticipated that the formation of oxalocitraconic acid, the most probable 7-carbon intermediary, would readily be demonstrated by working in the presence of arsenite. Actually, however, experiments carried out in the presence of arsenite have led invariably to the isolation of the G-carbon compound, a-ketoglntarate, together with traces of citrate, but with no trace of any 7*carbon substance. More- over, oxalocitraconic acid itself is not metabolized. It seems very unlikely therefore that such a compound is formed. Probably, therefore, citric acid must arise directly, presumably by a reaction between oxaloacotate and Borne 2-carbon substance, rather than the 3-carbon compound pyruvic acid. It ia known that pyruvate readily undergoes oxidative decarboxylation to yield acetyl-Co A. It has also been found that the aerobic oxidation of acetate itself is inhibited by malonate, a feature which indicates that acetate is probably oxidized by way of the catalytic cycle. Moreover, isotopically labelled acetate can be incorporated into the cycle and yields correspondingly labelled products. Present opinion is, therefore, that pyruvate first undergoes oxidative decarboxylation to yield acetyl-Co a, the latter then reacting with oxaloacetate to yield citric acid directly. The pyruvate may, in fact, be said to undergo its oxidative decarboxylation before, rather than after, it combines with oxaloacetate. We have already considered some of the evidence for the occurrence of tho reactions leading from citrio acid to oxalo- acetate (summarized on p. 448) and the enzymes concerned have been dealt with in some detail in Part I of this book. Special 461 CiTRlC ACID CYOZ.S consideration must now be given to the fundamental synthetic reaction which leads to the formation of citrate. The formation of citrato from acetate and oxaloncetato has been demonstrated many times in tissue minces and homo- genates, and more recently such a synthesis has been demon- strated by means of a purified enzyme. Until this was achieved it was open to question whether citrato itself is the first product to be formed. Citrate could arise by an addition reaction between acetate and oxaloacetate; condensation, on the other hand, would yield aconitate, from which citrate could arise indirectly through tho action of aconitase : coon Jb coon in, :(OH)COOH Co A I + C(OH)COOH ATP I COOH coon ioon coon in bi f^OHjcoon Lcooir + 11,0 + ATP I CH, 6h, ioon ioon Ochoa was able to prepare from pigeon liver a preparation of the so-called ‘ condensing enzyme \ which was virtually free from aconitase and which, under suitable conditions, formed citrate in large yield from added acetate and oxaloacetate, thus demon- strating for the first time that citrate arises directly and not by way of aconitate. The precise mechanism of the reaction was still at tho time unknown, but tho achievement of tho synthesis of citrate from acetate and oxaloa.ee tate set the final seal of acceptance upon the citric acid cycle itself. To achieve this synthesis from free acetate, ATP and Co A must be present, just aa acetylation by acetate requirts tho presence of ATP and Co a (p. 432). That ATP is involved reawakened the long-standing suspicion that ‘active acetate’ is 452 OITBIO ACID CYCLE none other than acetyl phosphate. Acetyl phosphate itself can- not however replace free acetate plus ATP, but clearly enough Borne form of ‘active acetate’ was generated under the condi- tions of Ochoa’s experiments. The subsequent discovery of acetyl-Co A finally resolved the problem, for Ochoa found that this product can react with oxaloacetate in the presence of the ‘condensing enzyme’ to give almost quantitative yields of citrate, this time without the participation of ATP. Since Co a is now known to be an indispensable factor in most of the biological processes in which ‘active acetate’ is involved, it seems that the nature and identity of ‘active acetate’ have at last found their solution, and that ‘active acetate’, in the form of acetyl-Co A, arises from free acetate, with ATP as the eventual energy-source for its synthesis. The mechanism of the synthesis of acetyl-Co a has been investigated by a number of workers and the best available evidence points to adenylacetate as an intermediary: CH,COOH + ATP CH,CO.AMP + pyrophosphate, CH.CO.AMP + Co a ?==» CH.CO.Coa + AMP, Overall: CH.COOH + ATP + Co a j=~^ CH.CO.Co a + AMP + pyrophosphate. In addition, it will be recalled that acetyl-Co a arises as an intermediate in the oxidative decarboxylation of pyrnvio acid (p, 433) and this of course explains the synthesis of citrate from pyruvate and oxaloacetate in the early experiments. In the meantime the next reaction in the cycle had been studied extensively. Citrate ta a symmetrical substance and as such would be expected to undergo dehydration equally at either of two points, one in that portion of the molecule which arises from oxaloacetate and the second in that part which arises from acetate. Now, if radioactive carbon dioxide and pyruvate are incubated together with liver tissue, terminally labelled citrio acid ia formed and has been isolated. If it behaves symmetrically, this citrate would be expected to go on round the cycle to give an a-keto- glutarate carrying radioactive carbon in both its carboxyl radicals. This is shown by the equations shown on p. 454, in which 50 % of the citrate would be expected to follow route (A) and the remaining 60 % route (B). 453 OITKIC ACID CYCLE bs, is, (B) ^oii)cooa — ► itcoon - in, in ioon ioOH * iai.cooH- inoH ioon •COO! in. •coon *coon in, in, -^ in.coon — ►in, io io boo\ ioon ioon co, W. a-Ketoglutarate was accordingly isolated from liver tissue previously incubated with pyruvate and radioactive carbon dioxide in the presence of arsenite, and examined. By dccarb* oxylating the product with permanganate it was shown that all the radioactive carbon was present in the a-carboxyl group, for the carbon dioxide released was radioactive while the residual succinic acid was not. As an additional check, succinic acid also was isolated from the liver preparations and found not to contain radioactive carbon. These results show that citrate docs not react symmetrically and that only route (A) is followed. How- ever, cs Ogston was the first to point out, it does not follow that because citrate is a symmetrical substance it will necessarily behave in a symmetrical manner when combined with its enzyme, if only because the enzyme-substrate complex is exceedingly unlikely to be symmetrical. Indeed, the foregoing evidence proves conclusively that citrate does not, in fact, behavo a3 a symmetrical compound. This means that the dehydration of citrate by aconitaso takes place in that part of the molecule that 454 CITBIO ACID C5TOZ.2 arises from oxaloacetate; or, to be more precise, between the a and /? atoms of the original oxaloacetate. The same conclusion also follows from experiments in which citrate was formed from oxaloacetate and carboxyl-labelled acetate. In this case the succinate is radioactive and the carbon dioxide inert; again only route (A) is followed: COOH Ah. > A< Ah, . *ioon in Ahi CH, •Aoon COOH OH in.* COOH - Ah, 'Aook COOH COOH 1 Ao Ao COOH -Ah^cooh — ► Ah, — >- Ah,_ Ah, Ah, Ah, ♦Aoon *Aooh *Aooh We have travelled some way since the first hypothetical scheme was suggested by Szent-Gyorgyi, and there is little doubt that many details remain even yet to be established. For the moment, however, it will be convenient to recapitulate and summarize the reactions of the ‘tricarboxylic acid cycle’ as they are now believed to take place. A schematic summary is presented in Fig. 37 and the individual stages will now be briefly reviewed. (1) Pyruvio acid undergoes oxidative decarboxylation, yield- ing acetyl-Co a which (2) reacts with the enol- form of oxaloacetic acid. The product of this reaction is citrio acid which (3) lose3 water in the manner we have described to yield aconitic acid, which (4) takes up water and yields iso-citric acid under the influence of aconitaso. (6) iso-Citric acid is now dehydrogenated by its dehydrogenase, giving rise to oxalosuccinio acid which, in turn, (6) is decarboxylated by oxalosuccinio decarboxylase and yields a-ketogl utaric acid. Oxidative decarboxylation of the latter (7) gives rise to succinyl-Co a (omitted from cycle) and 453 OITEIC ACID CYCLE then co to Buccinio acid. This is dehydrogenated (8) to give fumaric acid, which, under the influence of fumarase, is hydrated (0) to yield malic acid. A further dehydrogenation (10) converts this into oxaloacetic acid which (II) cnolizes and re-enters the cycle. Fig. 37. Tho citric acid cycle, showing some important side-reactions and the effects of some inhibitors. For list of enzymes inrolred see Tablo 30. Nolt that acetate reacts as acctyl-coonzyme a. Taira of hydrogen atoms are transferred to tho appropriate hydrogen acceptors in reactions (1), (5), (7), (8) and (10), and pass on through the cytochrome system. In all, therefore, five pairs of H atoms are removed with each turn of tho wheel, and three molecules of carbon dioxide are removed at the earno time, one moleculo of pyruvic acid being used up. These quantities fit the theoretical equation for the complete oxidation of pyruvic acid: C,U 4 0, + 50 - 3CO, + 211,0. 450 CITRIC ACID CYCLE The five atoms of oxygen figured in this equation correspond to the five pairs of hydrogen atoms which, transferred through the cytochrome system, require five atoms of oxygen for their even- tual conversion to water, three molecules of which are consumed in the cycle. Table 36. Enzymes involved in the cubic ACID CYCLE ( see Fig. 37) Reaction no. Enzymes and coenzymes 1 Oxidative decarboxylation system* 2 'Condensing enzyme’ 3 Aconitaso 4 Aconitase 6 wo-Citric dehydrogenase; TEN 0 Oxaloauccinic decarboxylase; Hn* + 7 Oxidative decarboxylation system* 8 Succinic dehydrogenase 9 Fnmarase 10 Malic dehydrogenase; DPN 11 Spontaneous isomerization A Glycolytic sj^temsf B Fat-syntheeizmg enzyme systems J 0 Transaminase J D Transaminase! E L-GIutamic dehydrogenase + DPN F Oxaloacetic decarboxylase * See p. 437. f Seep. 414. J See p. 127 et eeq. j Seep. 485. Although it is certain that most of the reactions are reversible it is probable that the cycle is unidirectional in actual operation, since the reactions involving oxidative decarboxylation (3, 7) are, in all probability, irreversible. This scheme, every step of which is now well established, provides us with an explanation for the complete oxidation of pyruvate itself, of acetate, and of any substance that gives rise to pyruvate or to acetate. As we have already pointed out, acetate only reacts in the form of acetyl-Co a, but this can be generated from free acetate if ATP and Co a are present, as they may be presumed to be in the tissues. It can arise also by tho oxidative decarboxylation of pyruvate arising from carbohydrate sources and from many amino-acids, and, in addition, it is the primary product of tho oxidation of fatty acid3. The cycle accounts also for the com- plete oxidation of any substance lying on the cycle and for the complete oxidation of any substance that gives rise to a com- pound lying on the cycle. If, for example, a largo amount of 457 CITItIC ACID CYCLE succinate i3 added, only a catalytic amount remains in the cycle itself; the remainder passes round the usual reactions until it is converted into oxaloacetate, 'which breaks down to yield carbon dioxide and pyruvate by ^-decarboxylation. The pyruvate is then oxidatively decarboxylated and oxidized through the cycle. It follows that we have, in this catalytic system, a machine that can accomplish or complete the metabolic oxidation of a great diversity of important primary foodstuffs and the intermediary products of their metabolism. Citric acid itself tends to accumulate if the later reactions of the sequence are inhibited, e.g. by anaerobiosis, by arecnite or by malonate. It also accumulates in large quantities in the presence of fluoroacetate. The latter has no action upon tho ‘condensing enzyme ’ but is itself taken up by reacting with oxaloacetate to form afluorocitrale, which has a powerful inhibitory action upon nconitaso and so checks tho further metabolism of citrate, but not its synthesis. Practically quantitative yields of citrate hare also been obtained by incubating muscle tissue with pyruvic, or acetic, and oxaloacetic acids under strictly anaerobic condi- tions, suggesting that anaerobiosis retards or suppresses tho dehydrogenation of t so -citric acid (reaction (5)). This may be because iso-citric dehydrogenase requires TPN for its action and, once the available TPN has been reduced, no system can bo found to reoxidizo it with appreciable velocity, for relatively few of the known dehydrogenases can collaborate with this coenzyme. Tho next oxidative reaction in tho scqucnco (7) is an oxidative decarboxylation and, as such, is inhibited by arsenile, so that a-ketoglutario acid accumulates, but small amounts of citrio acid are found at the samo time. Succinyl-Co a is formed as an intermediate product in (7), Malonate inhibits the next reaction (8), in which succinic dehydrogenase is the responsible catalyst, and leads to tho accumulation of succinate, together with small amounts of a-ketoglutario acid and traces of citrate. It has now been shown that the complex system of enzymes and co-factora involved in the cycle are associated mainly with the mitochondria though some of tho components arc present in the cytoplasm. Suspensions of intact, washed mitochondria can catalyse all tho events which compose tho cycle, but tho particles can ho disintegrated to some extent, o.g. by treatment 453 CITRIC ACID CyCLE with fat solvents, liberating some of the enzymes. Several of the enzymic components have now been obtained in individual form, including the ‘condensing enzyme’, which has been crystallized. In addition to the reactions composing the cycle proper, atten- tion may now be paid to a number of important side reactions. Very important among these is the glycolytic reaction sequence (A) which leads from glucose and glycogen to pyruvate and hence to acetyl-Co a by oxidative decarboxylation. The cycle therefore allows us now to account for the complete oxidation of glucose, glycogen, and all the known intermediates involved in glycolysis. Furthermore, it has been shown in the last few years that the breakdown of fatty acids (B), also culminates in the production of acetyl-Co A, so that we can account for the complete oxidation of fat and its intermediary metabolites also. This remarkable mechanism also provides a Jink between the metabolism of carbohydrates and that of proteins. Among the amino-acids that enter into the composition of proteins there are BOme that are non-essential and can be synthesized in the animal body in seemingly unlimited amounts. Three such are alanine (C), aspartic acid (D) and glutamio acid (E). The corre- sponding a-koto-acids, pyruvic, oxaloacetio and a-ketoglutaric acids, arise, according to this scheme, as intermediary products in the course of carbohydrate metabolism, and would only require to be aminated or transaminated to yield the amino-acids in question. Particularly important among the side reactions is the /7-carb- oxylation of pyruvate to yield oxaloacetate (E). In musclo, which contains no oxaloacetio decarboxylase, oxaloacetate drifts slowly out of the system by spontaneous ^-decarboxylation, so that the rate of respiration of minced muscle slowly declines. Presumably the maintenance of a high rate of respiration in intact muscle also requires the provision of a constant supply of some one or other of the catalytic dicarhoxylic acids. In liver, by contrast with musclo, oxaloacetio decarboxylase is present, and here oxaloacetate will drift out of the system rapidly if the concentration of pyruvate is low. But if pyruvate is suddenly produced, e.g. by the administration of pyruvate to the intact animal or, again, by a sudden burst of glycolysis, oxaloacetate 459 CITEIC ACID CYCLE will be formed and the oxidation of pyruvate by way of acetyl* Co a can be initiated. Oxidation will continue so long as the concentration of pyruvate remains high, but when it falls again to a low level, oxaloacetatc will once moro drift out of tho system and, os tho catalytic carriers fall in concentration, respiration will again slow up. But oxidation will not be the only process tending to reduce the concentration of pyruvate: some will bo converted into glycogen, in all probability, some will bo oxida- tively decar boxylated and give rise to acctyl-Co A and hence to ketone bodies or fat instead of being immediately oxidized, while some may even bo transaminated and converted into alanine. Fixation of carbon dioxide leading to the formation of oxalo- acetate can also take place through an indirect route (not shown in Fig. 37) with malatc os an intermediate. Tho enzyme, which requires reduced TPN and lyin'*"*, catalyses a reductive carboxyl - ation of pyruvate to give malate: CO, COO-ET ch, "tei - I + TPN.H, I + TPN CO cnoH ioOH d:OOH This enzyme, malic decarboxylase, is particularly abundant in pigeon liver and is not identical with malic dehydrogenase, but the latter can oxidize malate produced in this way to yield oxaloacetatc. Here, therefore, we have a second mechanism, again involving pyruvate and COj-fixation, through which the catalytio reaotants of tho citric cycle can bo built up and main- tained, but again their formation depends upon tho production and presence of pyruvate. Another interesting reaction takes place between oxnloacctate and ATP: coon co, ini, cn. I + ATP |[ + ADP CO 0.0-© ioou ioou ATP can bo replaced by inosyl, guanosyl, uridyl and cytidyl triphosphates in this reaction, which may be important in tho 4 GO CITB1C ACID CYCLE formation of glucose or glycogen from cycle intermediates. But it also provides an accessory source of oxaloacetate if pyruvic acid phosphate concentration is high so that the reaction goes in reverse. The foregoing accessory reactions can he summarized as follows in relation to the rest of the cycle: While it is probable in the extreme that a great deal still remains to be discovered about this catalytio cycle and its various side reactions, there can bo little doubt of the great an un mental importance of the system as a whole, both as a clearing- house for the oxidation of the many products forme on e metabolic lines which converge upon it and as a meeting p ace for the main metabolio pathways of carbohydrate, fat an pro tcin. As has already been pointed out, there is evidence a cycle, in the form envisaged by Szent-Gyorgyi, is a ®J vri • distributed as are cytochrome and cytochrome oxidase, e of animals, plantB and micro-organisms appear, wit very ew exceptions, to contain succinic and malic dehydrogenases, gether with fumarase, while iso-citric dehydrogenase an aco tase, which together correspond to the citrio de y og originally described by Thunberg, seem likewise to have iav ry wide occurrence. Again, there is reason to believe that most cel have the ability to accomplish the oxidative decar oxy a 461 CITEIO ACID OrCLE a-keto-acids. It seems not by any means impossible that the carbohydrate metabolism of many living cells is organized on the same fundamental lines, glycolysis leading to the formation of pyruvate from glycogen, the pyruvate being metabolized in its tom through acetyl -Co a and the tricarboxylic add cycle under aerobio conditions, and linking up, as it does in mammalian liver, with the metabolism of protein and of fat. That we shall find variations on the general, fundamental theme we may be sure, and, indeed, wo havo already discovered in the phosphagen of muscular tissues a specialized chemical adaptation which admirably subserves the highly specific func- tions which muscle is called upon to discharge. Similarly, we may consider that the carboxylase of yeast is a specialization which permits that organism to live on carbohydrate even in the total absence of oxygen. It might, of courso, bo argued that fermentation is a simpler operation than respiration, and that the simpler must logically be the more primitive but, at the same time, there is evidence that evolutionary advancement and specialization may bo attended by tbo loss of old enzymes, as well as by tho acquisition of new, as is the case of tbo enzymes concerned with purine metabolism (p. 3G1). But beneath all the secondary and specific adaptations that wo are likely to meet In the future, there is every reason to think that we shall discover evidence of the existence of a common metabolic ground-plan to which living cells in general conform. Evidence already available indicates that tho citric acid cycle is present in living organisms of many different kinds. Primarily perhaps, its function was that of providing supplies of citrate, a-ketoglutarate, succinate and the other components of the cycle. From these, other substances such as glutamate, glut- amine and other sido products, many of which ore of enormous importance in intermediary metabolism, can then bo produced. Again, it is known that citrate can be enzymatically split into succinate and glyoxylate, which latter can then give rise to glycine, Berine and ethanolamino in that cider. From glycine and serine 1-carbon units of formate can be formed, while ethanol- amino can. give rise to choline. And there are many more possibilities. But, in animal tissues at any rate, the main func- tion of the cycle is apparently that of energy production, for ENERGETICS OF CARBOHYDRATE OXIDATION here, quite apart from producing many substances of metabolic importance and dealing with the oxidative disposal of many more, the cycle operates fast enough to be capable of supplying the greater part of the free -energy requirements of animal cells and tissues. We shall accordingly proceed next to consider the energetic aspects of the cycle. ENERGETICS OF CARBOHYDRATE OXIDATION The fall of free energy entailed by complete oxidation of glucose to carbon dioxide and water amounts to about 686,000 cal. per g.mol. The corresponding figure for the glycolysis of glucose, yielding lactic acid, is about 36,000 cal. per g.mol., and we know that a balance of 2 new high-energy phosphate radicals arises in the process (p. 405). In complete oxidation, therefore, we might expect a maximal yield of the order of i.e. some- where about 40 ~ © per g.mol. if we assume that free energy can be ‘captured’ with about equal efficiency under aerobic and anaerobic conditions alike. Under anaerobio conditions the glycolysis of 1 g.mol. of glucose gives a net yield of 2 ~. The subsequent energy-yielding stages may be summarized follows (see p. 210): Reaction Pyruvate -> acatyf-Co a -*• citrate wo-Citrate -*■ oxaloauccinate o-Ketoglntarote -*■ cuccinate Succinate -v fumarate Malato -* oxaloacetate Primary H-acceptor DPN TPN DFN Cytochrome DPN Estimated yield of »•© Since glucose yields 2 molecules of pyruvate the total yield of from this part of the process will be 30, making a grand total 463 ENERGETICS OF CARBOHYDRATE OXIDATION of 38 per g.mol. of glucose oxidized. These, of course, are imperfect calculations and the knowledge upon which they are based is still imperfect, but there is good agreement between this and the estimate arrived at in the first paragraph of this section. Many experiments have been carried out in order to determine directly the energy-yields of oxidative processes in various tissues. In one such series of experiments carried out on pigeon- breast muscle, the yields of ATP recorded ranged from 1 to 3 molecules of ATP for each atom of oxygen consumed. Since tho oxidation of one molecule of glucose requires 1 2 atoms of oxygen, these results indicate an energy -yield of 12-30 ~ per molecule of glucose oxidized. Bearing in mind the probability that somo ATP must have been broken down in tho course of incubation we may assume that at least 36 ~(£) are formed for each molecule of glucose undergoing oxidation and that, given ideal experi- mental conditions, the yields might conceivably be greater even than this. This again is in reasonable agreement with tho estimate of 38 reached by calculation and for which we can account by experimental data. We may therefore assume for the present that the true yield is probably not less than 3S for each g.mol. of glucose oxidized, and this allows us to calculate tho minimal efficiency of tho oxidative process as a whole. A bond-yield of 38 ~ © corresponds to an energy-capture of 38 x 1 1 ,600 cal., i.e. 437,000 cal. out of the 688,000 cal. to which the cell gains acccs3 by oxidizing one g.mol. of glucose. This corresponds to an energy -capture efficiency of about 64%. This figure may be compared with the 4G% efficiency calculated for alcoholic fermentation (p.389) and that of 64% for the glycolysis of glucose (p. 406), and suggests that aerobic metabolism is not much more efficient, from the point of view of energy-capture, than anaerobic. Even so, these considerations provide a further striking demonstration of tho remarkable efficiency of the organism in capturing and diverting to its own uses tho intrinsic free energy of its food materials. 464 CHAPTER XIX THE METABOLISM OF FATS TRANSPORT AND STORAGE OP PATS Aooordino to current opinion a large proportion of the food fat is absorbed in finely emulsified form from the small intestine by way of the lymphatic system and hence, through the thoracic duct, into the blood, where it appears in the form of minute droplets of neutral fat. The remainder undergoes hydrolysis to yield free fatty acids which also pass into the lymphatic The immediate fate of ingested fatty material has been studied with the aid of deuterated fats, i.e. fats containing heavy hydrogen. Deuterated fats may be prepared by catalytic hydro- genation of unsaturated fata, such as linseed oil, with hydrogen containing a high proportion of heavy hydrogen. Deutera c fat was fed to mice for several days and the animals were then killed. The fate from different parts of tho body were extracted from the carcasses, the water formed by their combustion being carefully collected and analysed for heavy water. 16 showed that tho hulk of the deuterium administered had found its way into the depot fats, and only small amoun J 11 other tissues. We must therefore suppose that t e ys ingested food fat after absorption is its deposition in e depots of the body. Of these the most important are ^ mesen- teries and the intramuscular and subcutaneous co Generally speaking, the kind of fat present in the of a given animal is fairly characteristic of the species, is always much the same in composition, while ^ always characteristically mutton fat. 'Vf , feeding lor instance, a dog is given large , .. ^ lay down a softer and much more unsaturated ep TRANSPORT AND STORAGE OF FAT characteristic of dogs as a whole. But it is none the less true that, in the ordinary way, each species lays down its own kind of fat, just as each species lays down its own kind of tissue pro- teins. The reason for this constancy 13 only partly covered by the tendency of animals to select a diet which is fairly constant in composition; not all the fat found in the depots is merely food fat that has been transported thither from tho alimentary canal, but fat which has been synthesized from non-fat sources. As every stock-breeder knows, animals can be fattened cheaply by feeding thorn an abundance of carbohydrates, and fat can also be synthesized from protein to some extent. It seems likely that the nature of the fat formed from these sources will depend upon the metabolic make-up of the particular species concerned, different animals starting from much the same raw materials but each manufacturing its own kind of fat. Tho synthesis of fats from non-fat sources is particularly im- portant in cattlo, sheep and other herbivorous animals, for hero cellulose bulks large as a foodstuff. In these animals, cellulose is digested by symbiotic micro-organisms which produce from it high yields of short-chain fatty acids, among which acetic and propionic acids predominate, together with some butyric and small amounts of other acids. Acetic and butyric acids are fat- formers, propionic acid yielding glycogen. In animals of this kind, therefore, it is probable that tho main reserves of fat aDd carbohydrate are built up almost entirely from short-chain fatty acids. The average animal is capable of laying down almost unlimited amounts of fat and, in point of fact, fat has certain definite advantages over proteins and carbohydrates as a form of reserve fuel. Fat is far richer in carbon and hydrogen than tho other primary foodstuffs, so that there is more combustible material In a gram of fat than in a gram of either protein or carbohydrate. From the point of view of energy, therefore, fat allows the greatest storage per gram of reserve material. If a gram of each of tho three main types of food is burned in a bomb calorimeter the heat produced is approximately as follows: 1 g. protein . . 0C00 cal. I g. carbohydrate 4200 col, 1 g. fat 9300 cal. 4 DO TRANSPORT AND STORAGE OF FAT Closely bound up with this is the fact that fat, when burned, gives rise to about twice as much water as the other foodstuffs on account of its high content of hydrogen: X g. protein 0-41 g. water 1 g. carbohydrate 0-65 g. water 1 g. fat 1*07 g. water This is an important feature of fat metabolism, especially among terrestrial animals, many of which live under conditions of acute water shortage. In such cases there is commonly a heavy em- phasis on the oxidation of fat; in this way the organism is better able to eke out its external supplies of water with metabolic water formed in its own tissues. As an example of this pheno- menon we may refer to the developing chick embryo. At laying, the hen’s egg is provided with a definito and limited amount of water, an amount which, by itself, would bo insufficient to see the embryo through development. But during the 3 weeks of incubation, rather more than 00 % of all the material oxidized by the embryo consists of fat. Again, the mealworm, an insect larva that can livo for long periods under the most arid con- ditions, metabolizes during starvation about 2| parts of carbo- hydrate for every part of protein, and no less than 8 parts of fat. The almost legendary ability of the camel to travel for days in the desert without a drink is similarly attributable to heavy fat metabolism with proportionately large-scale production of meta- bolic water. Bat which is on its way from the gut to the fat depots is carried mainly in the form of droplets of neutral fat, and a pro- nounced condition of lipaemia is regularly to bo observed after the consumption of a fatty meal. A small proportion of tho total fat apparently travels in tho form of phospholipids, for tho concentration of phospholipid materials in the blood shows a pronounced rise during absozption. When fat is being with- drawn from tho depots to be metabolized elsewhere there is no lipaemia, however, and it seems that most of the fat being trans- ported must travel in the form of phospholipids which, unliko the other lipids, are appreciably soluble in water. Phospho- lipids are normally present in the blood in small quantities, together with a certain amount of cholesterol and cholesterol 407 30 -s FAT TY LIVER esters, but neutral fat is normally only present as such while the condition of post-absorptive lipaemia persists. The mobilization of depotjfat can conveniently be studied in starving animals. After a short period of starvation the glycogen reserves of the liver are used up, and no more glycogen is forth- coming except through glyconeogenesis. Presumably because of the shortage of carbohydrate, largo amounts of fat appear in the liver. This condition, which is known as ‘fatty liver’, can also be observed in a variety of conditions other than starvation and it seems certain that the accumulation of fat in the liver repre- sents the first step towards its metabolic breakdown. The fat which appears in a fatty liver arises from the fat depots. As has been pointed out, the administration of deuterated fat to mice leads first to the deposition of most of the heavy hydrogen in the fat depots. But if the animals are allowed to starvo for several days before being killed and the body fats are then worked up for heavy hydrogen, it is found that the fat content of the liver is greater than at the beginning of starvation and, moreover, that the liver fat contains twice or three times as much deuterium as that in the depots. After its absorption, therefore, most of the ingested fat goes first to the fat depots, from tvliich it is withdrawn and transported to the liver as and when the need arises. The condition of fatty liver can be established by any treat- ment that tends seriously to diminish the power of the liver to Btoro, produce or metabolize carbohj-drato. In diabetes, for instance, the storage powers of the liver are impaired, and fatty liver is one of the features of this disease. Furthermore, in severe cases, the blood may contain three or four times as much fat os normal. In the pseudo-diabetic condition induced by phlorrhlzin the glycogen reserves of the liver are broken down and the glucoso thus set free is excreted by way of the urine. Here again fatty liver is to be observed. Small doses of liver poisons such, for instance, as carbon tetrachloride, chloroform, phosphorus and diphtheria toxin, also lead to fatty liver because they disturb the normal functions of tho liver with respect to carbohydrate meta- bolism. In all these cases tho establishment of a fatty liver is encouraged by feeding cholesterol and discouraged by the ad- ministration of choline or ethanolaminc. How the cholesterol 4CS FUNCTIONS OF FAT effect is produced we do not know, but when choline or ethanol- amine is given it seems not unlikely that their arrival in the liver encourages the formation of phospholipids which, being soluble in water, tend to he carried away. FUNCTIONS OF FAT: CONSTANT AND VARIABLE ELEMENTS It will be clear from, what has been said that the fat content of the depot tissues and of the liver — and the same is true of other organs — varies very widely with the nutritional condition of the organism. If an animal is allowed to starve for a long time, the amount of reserve fat in the body becomes very small, but, even at death from starvation, the tissues still contain large amounts of lipid material which, apparently, forms a part of the struc- tural material of the tissues and is not available for use as fuel. This part of the total body lipids is referred to as the * constant element*; constant because, being a part of the actual fabric of the organism, it is always present and always must be present. The remainder of the lipids comprise the ‘variable element*, so-called because they vary in amount with the nutritional state of the organism and the demands made upon them for energy production. The contrast between the constant and variable elements can be appreciated by comparing the effects of different doses of liver poisons upon the lipid content of the liver. Small amounts of carbon tetrachloride, for example, lead to the mobilization of fat from the depots and its deposition in the liver. Here we observe the movement of a part of the variable element from one place to another. If larger doses of the same poison are administered, the liver cells sufferserious damage and we observe the condition known 5.3 fatty degeneration. Again the cells ate rich in lipid materials, hut mainly because the other cell con- stituents have been broken down and dispersed. Indeed, the lipids present in fatty degeneration are all that remains of the structural materials of the cell, and represent the constant or structural element of the tissue lipids. Fats, then, have two main functions. They act as fuel reserves, and they form an important part of the structure of living tissues. METABOLISM OF FAT In addition, there are indications that certain fatty acids have special and specific functions, for it is known that rats kept on fat-free diets, or on diets from which particular fatty acid3 have been carefully removed, become ill and suffer from caudal necrosis, but we shall return presently to consider this condi- tion and its metabolio implications. METABOLISM OF FATS: GENERAL The evidence available regarding fat metabolism was until recently so contradictory and speculation so rife that almost any statement made might be contradicted by as many facta and arguments as could bo produced in its support. It baa long been believed that tho liver plays a predominant role in tho metabolism of fat, for it is to this organ above all that fat is transported when carbohydrate metabolism is subnormal and an alternative energy source is required. It has usually been assumed, though never proved, tliat fats are hydrolytically split into glycerol and free fatty acids before any oxidation takes place. This is not entirely an unreasonable supposition, for cells of most kinds 6eem to be furnished with lipolytic enzymes, the action of which is freely reversible. Glycerol, if administered to a diabetic or phlorrhizinized animal, is converted almost quantitatively to glucose. It also leads to the deposition of glycogen in the liver if administered to a starving animal. The pathway for its conversion into carbo- hydrate is through glycerol phosphate, formed by a specific pliosphokinoso at the expense of ATP, oxidation of glycerol phosphate to triosopbosphate by a glycerophosphate dehydro- genase, and hence, through tho normal glycolytic reaction sequence, back to glycogen or glucose. Fatty acids are normally completely oxidized to carbon dioxide and water. There is on abundance of evidence, wliicb wo shall presently review, that fatty chains are split into 2-carbon units consisting of some form of ‘active acetate*. Acetate itself can bo totally oxidized, e.g. by washed kidnoy-cortex homogenates in the presence of oxaloaceiato, and its oxidation is inhibited by malonato and other inhibitors of tho citric acid cycle, whence wo may conclude that it enters and is oxidized by way of this 470 METABOLISM OF FAT cycle. Confirmation of this is found in the fact that isotopically labelled acetate can be incorporated into the cycle and gives rise to correspondingly labelled intermediates in the presence of the usual inhibitors (p. 455). Long-chain fatty acids also can be completely oxidized by a variety of tissue preparations, including Blices, breis, homogenates and mitochondrial suspensions, and here again oxidation is dependent upon the presence of oxalo- acetate or some other intermediate of the citric cycle, and here too the oxidation is inhibited by malonate. These facts indicate that acetate and longer chains alike are probably metabolized through the same route and that acetate, and the 2-carbon units arising in the breakdown of longer chains, enter the cycle through a probably common intermediary. Now the entry of acetate itself into the cycle depends upon the presence of ATP and Co a and we know (p. 432) that in the presence of these substances acetate is converted into acetyl-Co a. The latter, we know, can react directly with oxaloacetate to yield citrate (p. 452), from which oxaloacetate is subsequently regenerated with formation of two molecules of carbon dioxide, equivalent to the two carbon atoms of the acetate entering the cycle (p.456). Since no form of ‘active acetate’ other than acotyl-Co a has so far been discovered wo may tentatively assume that the 2-carbon units formed from fatty acids arise in this form. In confirmation of this we have the further fact that fatty acids can he synthesized from acetate, presumably by way of the same common 2-carbon intermediate. These points may bo summarized: »- glycerol triosephosphate glycogen Fats — ►‘f glucose fatty acids acetyl-Co a acetate | + oxaloacetate citrate Now, in some tissues and under certain circumstances, fatty acids give rise to large amounts of other compounds, the ketone or acetone bodies. These are aceioacetic acid, together with fi-hydroxybutyric acid and small amounts of acetone. It is generally agreed that the parent substance of this group is 471 METABOLISM OF FAT acetoacetie acid. Under the influence of the 'widely distributed /Miydroxybutyric dehydrogenase andDPN, acctoaeetic acid and /?-hydroxybutyric acid3 are freely interconvertible. Acetone arises from acetoacctic acid by what appears to bo a spontaneous decarboxjdation, a process which takes place at an appreciable speed under physiological conditions of temperature and pH. Tho relationships between these three substances can briefly bo summarized as follows: CHjCOCHjCOOH actioaettic acid \ CIIjCIKOHJCH.COOH CIIjCOCUj+CO, fi-bydrozyhutyric acid acetone Acetoacetat-e, the parent member of the group, can bo formed from acetate, but tho synthesis is an endergonic process, indi- cating that the starting material must be a high-energy substance of somo kind. Moreover, the formation of acctoacctato from acetate implies acetylation, and there is evidence both that aeetyl-Co a is a high-energy compound and that it participates in tho biological acetylation of a number of different substances (p. 432). Hence wo may suppose that acetoacetate arises from acetate and from acids with longer chains by way of acetyl-Co A. Now acetoacetate, like acetate itself and like the long-chain fatty acids, can be completely oxidized by various tissuo prepara- tions in tho presence of oxaloacetate and other intermediates of the citric cycle, and once again oxidation is inhibited by malonate. Furthermore, isotopically labelled acetoacetate can gain admis- sion to the cycle, yielding correspondingly labelled intermediates . Once again, therefore, we must suppose that acetyl-Co A is involved as an intermediate, and again wo may summarize our conclusions : Arctoacclat* acetyl Co a acetate | + oxaloacetate citrate These considerations lead us to suspect that fatty acids and carbohydrates alike undergo final oxidation by way of a common 472 METABOLISM OF FAT 2-carbon intermediate, in the form of acetyl-Co A, and by a common mechanism, in the citric acid cycle. We may therefore summarize our summaries in a united form as shown in Fig. 38. Small quantities of ketone bodies are formed from ketogenio amino-acids when these are administered to diabetic or phlor- r h izi n ized animals, but that they arise mainly from fat cannot bo doubted. Fig. 38. Outlines of metabolism of fats and carbohydrates. Traces of ketone bodies can be detected in the blood of normal animals, but the amounts present are greatly increased when there is a heavy emphasis upon fat metabolism. The energy requirements of a typical animal are normally met by meta- bolizing fat and carbohydrate together, but if, for any reason, carbohydrate metabolism is subnormal, correspondingly more fat has to be metabolized. Thus ketone bodies tend to accumulate when food contains disproportionately large amounts of fat. They also accumulate in starvation, once the glycogen reserves of liver have been exhausted. These reserves can be experi- mentally drained in fed animals by injecting phlorrhizin, when ketone bodies again make their appearance. They are also formed if the liver's power to Btore and metabolize glycogen is seriously impaired, as it is in diabetes and in cases of poisoning by chloro- form, carbon tetrachloride, phosphorus and other liver poisons. There is a similar dependence upon carbohydrate metabolism 473 METABOLISM OH FAT in isolated, perfused livers. The blood leaving a perfused liver ordinarily contains small amounts of ketone bodies, but the amounts are greatly increased if fatty acids are added to the ingoing blood. The yield of ketone bodies js particularly high when butyric acid is used, but in all cases the yields are higher in livers that are poor in carbohydrate. Similar results have been reported from experiments with liver slices. Table 37. Enzymes involved in breakdown OF FATS AND CARBOHYDRATES {act Fig. 33) Reaction Enzyme or catalytio ■yitem 1 Lipase 2 Glycolytic enzymes 3 Oxidative decarboxylase 4 See p. 433 5 Enzymes of ^-oxidation G Oxaloacetic decarboxylase; mains diWMVftTjVw* v dehydrogenase 7 ‘Condensing enzyme' 8 Enzymes of citric cycle 9 Tnuisacetylaae 10 /J-Hydroxybntyric dehydrogenase 11 Mono (spontaneous) 12 Transacetylaso b ’ a r:ch,ch(oh)co~Co a The a -hydroxy-acids must pass back through the unsaturated to the ^-hydroxy-compounds to be further metabolized. (3) Oxidation of (i-hydroxyacyl-Co a is catalysed by fi-hydroxy- faity-acyl-Co a dehydrogenase, a DPN-specifio enzyme (p. 187). This is present in and can be extracted from liver and acts upon substrates with 4-12 and probably more carbon atoms: R.CH(OH)CH,CO~Coa + DPN R.CO.CH 1 CO~Co a + DPN.H,. (4) Breakdown of a-keto-derivatives. We must now enquire how the ft-'ketO'Compounds are fragmented. We know that the products are a 2-carbon unit, probably acetyl-Co a, and a fatty acid derivative with 20 less than the starting material. Here the classical view was in favour of simple hydrolysis to liberate acetic acid: CD, CO; . CH,COOH + H;.OH — ► CH.COOH + CH,COOH, The objections to this hypothesis were, first, that /hke to -acids are not prone to this kind of hydrolysis at physiological pH, and second, that the biological formation of acetic acid from aceto- acetic acid or any other fi-ketonic acid in significant quantities bad never been demonstrated. Knoop disposed of the latter difficulty by the supposition that acetate is removed as fast as it is formed, and, as we now know, acetate can indeed be rapidly incorporated into the citric cycle. It has in any case long been certain that acetic acid or some form of ‘active acetate’ does arise in the body because many substances containing — NH 2 groups are excreted in acetylated form after administration to experimental animals. Moreover, small amounts of acetic acid have been isolated from liver tissue in the form of the 2:4-dinitrophenylhydrazide . If now the fatty acid-Co a derivative undergoes oxidation in the /^-position, the produot could undergo fission, perhaps by hydrolysis, to yield acetyl-Co a: HOj.H CO : ;.CH,CO~Co A — — COOH + CH,CO~ Co a 483 31-5 0XIDAT10K OF FATTY ACIDS Tills product, a3 we know, is capable of direct reaction with oxaloacetate to yield citrate. Now, if the /7-oxidative split is hydrolytic in character, this reaction would lead to the dissipation of the energy associated with the a:/? bond. The suggestion may be made that Co a is again involved, this time in the fission of ^-koto-acids, perhaps as follows'. — cir,co;.cn,co~Co* + Coa — ► — cn.co-co* + cii,co~coa. Thi3 so-called t biolysis of /?-keto-acyl-Co a compounds can actually take place in the presence of a liver enzyme known ns fi-ketothiolase. This is an interesting operation since it means that the 'prim- ing energy ’ originally provided from ATP through the mediation of cocnzyme a is recovered in the high-energy ~ Co a of the acetyl-Co a unit split off, wliile the residual fatty chain is left already ‘primed’ for the next step in its degradation. Suoh a mechanism leads, moreover, to the eventually complete degradation of naturally occurring, even-numbered chains into 2-carbon units consisting of acetyl-Co a. If now we combine the ideas developed in the last few pages we can arrive at an overall picture of the process of ^-oxidation as a whole. Such a picture is presented in Fig. 30. It is not possible in this case to refer to a ‘cycle’ because the final product of each Bequcnco of operations is not identical with the starting material but contains 2 carbons less. But the process can bo likened to a clock spring. The free fatty acid is converted into its Co a derivative and enters the outer coil of the spring and then undergoes desaturation, hydration and dehydrogenation under the influence of the enzymes wo have already described. Acetyl-Co A is split off from the /?-keto-compound and the residual aeyl-Co a continues on along tho next coil, undergoing tho same sequenco of reactions, loses acetyl -Co a again, and so on round Ike rest of the coils. Thus palmitic acid, with 1&C, would require a spring of 7 coils and would yield 8 molecules of acetyl-Co a if completely broken down. One point of the greatest possible importance must bo emphasized here. AU the reactions invoiced in the (t •oxidative process are reversible and it is. in fact, possible to start with 484 FORMATION OF AOETOAOETATE acetyl-Co a and synthesize fatty acids by travelling backwards along the spring. The acetyl-Co a unite formed can, aa we know, enter freely into and undergo rapid oxidation by the citric acid cycle and can enter freely into reactions of biological acetylation also. But under conditions in which oxaloacetate is in Bhort supply, i.e. in conditions in which carbohydrate metabolism is suppressed or Fig. 39. yJ-Oxidativo breakdown of fatty acids. subnormal, there will be a tendency for acetyl-Co a to accumu- late for lack of oxaloacetate with which to combine and, more- over, there will be an increased production of this substance at the same time. Under such conditions as these ketone bodies make their appearance. FORMATION OF ACETOACETIO ACID It has already been emphasized that no theory of fat metabolism stands much chance of survival unless it can account adequately for the formation of ketone bodies, and a great deal of atten- tion has been paid to the problem of ketone-body formation. Jowett & Quastel, for example, used the liver-slice method, and with its aid confirmed in a general manner the results of earlier experiments carried out by the older techniques. In particular they carried out quantitative determinations of the yields of ketone bodies (expressed in terms of acetoacetato) from a scries 485 FORMATION OF AOETOACETATE of fatty acids, and discovered that some fatty acids give rise to more than one molecule of acetoacetic acid for each molecule of fatty acid metabolized. Other-workers, using similar techniques, obtained rather different figures but nevertheless confirmed tho observation that, in certain cases, one molecule of fatty add can product more than one molecule of accloaceiate. Feeding experi- ments carried out on starving rats have added further confirma- tion and, in the case of octanoic acid, which wo may take as typical, two molecules of acetoacetato wero obtained from each molecule of the C 8 acid. This corresponds to a complete conver- sion of fatty acid-carbon to ketone-body-carbon. Kow according to the theory of /1-oxidation, as formulated by Knoop, a C 8 acid would lose two pairs of carbon atoms in the form of presumptive acetic acid, leaving butyric acid which, undergoing /1-oxidation in its turn, could be converted into one molecule and no more of acetoacctate. Ono way of accounting for the observed high yields of ketone bodies is to invoke the alternative theory of multiple alternate oxidation. According to this scheme the fatty chain is supposed to undergo oxidation to form keto-groups at the /?-carbon atom and at every alternate carbon atom along the chain, thus: fi a CH.cn, on, CH,CH,cn,cn t coo« l fi a tCH,CO.CH,CO-;.CH,CO.CH,COOH The chain is supposed then to split into 4-carbon fragments, each fragment coming away in tho form of a molecule of aceto- acetic acid. From every molecule of octanoic acid therefore, wo should expect to obtain two molecules of acetoacetic acid and this, of course, is what is actually observed. But to accept this hypothesis means totally ignoring and setting aside all the considerable mass of evidence in favour of ^-oxidation: indeed, the only sound evidence in favour of multiple altemato oxida- tion seems to consist of precisely those facts which it seta out to explain. Furthermore, there is an alternative possibility which is entirely consistent with the theory of /?-oxidation, i.e. that pairs of the 2-carbon products of /?-ox:dation react together to form acetoacctate. 480 FOEMATION OF ACETOACETATE - Clearly it is important to consider the possibility that accto- acefcate may arise primarily from the 2-carbon units removed from the fatty chain by ^-oxidation. These, as we have seen, consist of acetyl-Co a. Many experiments have been carried out in order to elucidate the fate of acetate itself when added to various tissue preparations. Feeding, perfusion, tissue-slice and brei experiments have all been used and, while different quanti- tative data have come out of different experiments, it has been established beyond reasonable doubt that acetic acid is at any rate partially converted into aeetoacetate by liver and other tissues. That part which is not so converted disappears and is presumably oxidized. Moreover, the yields of aeetoacetate are greatest in livers that are poor in glycogen and it therefore appears that two possible fates await acetic acid in the liver. Either (a) pairs of acetate molecules react together in some way and give rise to aeetoacetate, from which the other ketone bodies subsequently arise, or else (6) acetate is completely oxidized. The factor determining the extent to which condensation and oxidation respectively shall take place appears to be the avail- ability of carbohydrate. With low-glycogen livers there is much condensation and little oxidation, while in high-glycogen livers there is much oxidation and little condensation. Here, therefore, as in fat metabolism generally, we find that the oxidation of fatty metabolites depends intimately upon the availability of carbohydrate or carbohydrate metabolites. Convincing evidence for the formation of aeetoacetate from acetate has been obtained by the use of isotopio carbon, for if isotopically labelled acetate is inoubated with liver tissue and the resulting aeetoacetate is isolated, it is found that the isotopic carbon has been transferred to the aeetoacetate. The behaviour of octanoio acid in particular has been studied by the isotope method. Octanoio acid was prepared with 13 C in the carboxyl radical, the product was incubated with rat liver tissue in tho form of slices, and the aeetoacetate formed was isolated and analysed for “C. Now, according to the classical theory of/?-oxi- dation, aeetoacetate would be formed only from the butyrate left after two 2-carbon units had been removed, and the product should therefore contain no 13 C. According to the theory of multiple alternate oxidation, the 8-carbon chain would give rise 487 rORMATIOJ* OK ACETOAOETATK to two molecules of acetoacetato, of ivliich only one would con- tain U C, located in tlie carboxyl group. If, however, pairs of carbon atoms were removed in the form of acetic acid or some highly reactive derivative thereof, followed by random condensa- tion of pairs of these 2-carbon units, “C should bo present both in the carboxyl and the ketonio groups of tho product, but not in the methyl or — CH a — radicals: It was found that, in fact, isotopio carbon was present in the carboxylic and ketonic but not in the other two radicals, indi- cating that the acetoacetato formed must have been produced from tho 2-carbon units split off from tho molecules of octanoic acid by /7-oxidation. If, however, acetoacetato arises by purely random condensa- tion of 2-C fragments, equal amounts of isotopic carbon would bo expected to bo present in the ketonio and carboxyl radicals. Actually, however, the carboxyl group is always richer than tho ketonic. This lias been explained on tho supposition that the free 2-C fragments can become (probably rovcreibly) attached to /7-kcto-acids, which subsequently lose Jour instead of only two carbon atoms, yielding acetoacetato directly, o.g. : — c n.CH.cii.CH.coo 11 — cn.cn, co .cn.coon -cii.cn.oooH + cn.coon i it. -co.cn,coou + cir,coou — ► — co;cn,co.cn,coou —coon + cn.co.cn, cooit If this process, for which there is good evidence, takes place together with purely random condensation of pairs of 2-C units, 488 OXIDATION OP ACETOACETATE the relatively low isotope content of the ketonic radical can he accounted for. The formation of ketone bodies from free acetate has, of course, been repeatedly demonstrated in tissue preparations of variouskindsbutis.nevertheless, an endergonioprocess.Weknow that the 2-carbon product of ^-oxidation consists of acefcyl-Co a, a high-energy compound and at the same time a natural acetylating agent, admirably qualified to serve as a source of acetoacetate : CH,CO~Co a + CH,CO~Co a — ► CH,CO . CH,CO ~ Co A + Co a. How the terminal ~ Co a group is removed from the product is uncertain, but some molecules at least can transfer it to succinic acid, forming succinyl ~Co a: acetoacetyl~ Co A + succinate ?=* acetoacetate + succinyl/- Co a. In the main however, the overall equation is probably: acetoacetyl'vCo a + ADP + HO.® — *• acetoacetate + Co A + ATP. OXIDATION OP A OETO ACETIC AND /l-HYDROXYBCTYRIO AOIDS Much attention has been given to the problem of the oxidative metabolism of acetoacctio and /l-kydroxybutyric acids, and it is now well established that they are interconvertible through the action of /?-hydroxybutyric dehydrogenase together with DPN, both of which are widely distributed in animal tissues. Many tissues are able to oxidize added acetoacetate and /1-hydroxy butyrate. Other compounds examined include the following : CH.CH.CH.COOH Cn,CH=CH,COOH CH,Cn(OH)CH.COOH CH.(OH)CH,CH,COOH Cn,(OH)CH,CH(OH)COOH CHr=CH.CH,COOH All these substances were rapidly oxidized to carbon dioxide and in every case the oxidation was inhibited by malonate, which suggests that the oxidation of these substances, like that of the fatty acids themselves, takes place by way of the citric acid cycle. Butyric acid Crotoaic acid /f-Hydroxybutyric acid y-Hydroxybutyric acid st-y-Dibydroxy butyric acid Vmylacetie acid 4b0 OXIDATION OF ACETOACETATE Now if isotopically carboxyl-labelled acetoooetato is incubated with oxaloacotato in tissue homogenates, correspondingly labelled citrate can bo formed, and similarly labelled derivatives can bo obtained by the use of the usual inhibitors of the cycle. In this respect acetoacctate behaves like other /Nketo-acids, the two terminal carbon atoms being split off and incorporated into citrio acid. Probably free acetoacetato first requires conversion into acctoacetyl-Co a , which, being a ^-koto-compound could undergo t biolysis in the usual way: CH.CO.CH, CO~Co a + Co a * CII.CO-Coa + CH.CO-Coa. This initial formation of acctoacetyl-Co a might tako through reactions with Co a and ATP similar to those involved in the formation of an acyl-Co A from a free fatty acid (p. 480). To some extent, however, acetoacetato and some higher /1-keto- acids can react with succinyl-Co a: acetoacctAt* + *uccinyl~Co a aceto&cetyl~Co A + •occlnatc. This linkage with an intermediate of the citrio acid cycle is most intriguing. Since the oxidation of acetoacctate can bo accounted for it follows that the oxidation of substances which, liko butyrate, crotonato and /7-hydroxybutyrate, are known to give rise to acetoacetato, can also bo accounted for. It remains to bo explained why acetoacctate, /7-hydro xy- butyrate and related compounds are freely oxidizablo in certain tissues other than liver, even when carbohydrate is in short supply. Unlike the liver, kidney and muscle contain no oxalo- acetic decarboxylase, so that oxaloacetato is only spontaneously (a3 opposed to catalytically) degraded to pyruvate and carbon dioxide, and its concentration in these tissues is not immediately dependent upon the availability of carbohydrate. How these tissues maintain their stocks of oxaloacetato is not known ; some oxaloacotato could arise by transdeamination of aspartic acid, and a-kctoglutarate, another reactant in the cycle, could bo formed by the deamination of glutamate. In this connexion it is perhaps significant that E-glutomic aspartic tnmsaminaso is present in the tissues generally. 490 SYNTHESIS OP FATTY ACIDS If we accept the scheme just proposed to account for the formation of acetoacetate, together with that proposed for the intermediate stages of /?-oxidafion (Fig. 39, p. 486), we have a hypothesis that can account for the complete oxidative de- gradation of fatty acids by way of the citric acid cycle, for the formation of ketone bodies by way of acetoacetate, and for the oxidative breakdown of any substance that can give rise either to fatty acids or to acetoacetate or /?-hydroxybutyrate. These are the salient features that have to be taken into account in any theory of fat metabolism. Evidence is accumulating meanwhile that the oxidative degradation of the fatty acids is coupled up to the generation of new high-energy phosphate and the synthesis thereby of ATP, perhaps at each successive stage of /?-oxidation, and certainly in the subsequent oxidative metabolism of the 2-carbon, acetyl- Co a units to which each such stage gives rise (see pp. 4G3-4). SYNTHESIS OF FATTY ACIDS It has long been known that fat can be synthesized from fatty metabolites such as acetoacetate, and also from carbohydrate sources. The naturally occurring fatty acids contain an even number of carbon atoms, practically without exception, and it follows that the starting materials for their synthesis must pro- bably also contain even numbers of carbon atoms. It has from time to time been suggested that the raw material for the synthesis might be acetoacetate itself, with 4 carbon atoms, but In this case it might be anticipated that fatty acids with 4, 8, 12, 16 and 20 carbon atoms would predominate in nature. In fact, however, the 14-, 16- and 18-carbon acids are the most common, and all occur in similar proportions. It therefore seems probable that fatty acids must be synthesized from 2-carbon units. The usnal explanation in the past has been that fatty acids arise from acetaldehyde by multiple aldol condensation, a re- action which readily takes place in vitro, followed by ^-reduction , according to some scheme such as i3 shown on p. 492. This hypothesis, at best, has always been purely speculative and with no factual basis, apart from the fact that aldehydes readily undergo aldol condensation under in vitro conditions. There is 491 SYNTHESIS OF FATTY ACIDS no reason to believe that acetaldehyde arises in quantity in the tissues of animals. CH.CHO + CHjCHO — CH.CHIOHJcn.CHO /?.rcduction~ CH.CH, CH.CHO + en.CHO — CH,CH,CH,CU(OH)CII,CiIO fi reduction CII.CIItCnjCH.CH.CHO + en.CHO -* CH.cn, CH.CH.CH.CniOUlCII.CHO etc,'*' Recent developments in our ideas about fat metabolism show that the starting material is not acetaldehyde but acetyl-Co A. The latter is known to be derivable from pyruvic acid by oxida- tive decarboxylation and, in addition, is the predominant pro- duct of tho /?-oxidativo fission of fatty acid chains. It can also arise from free acetate by reacting with Co a and ATP (p. 4S0). Work carried out with the aid of isotopes bears out tho truth of tho supposition that fatty acids can bo synthesized from 2-carbon units in the form of acetate, though without giving any indication of the intermediate processes. Acetic acid, containing deuterium in the methyl group and 1S C in the carboxyl radical, was fed to rats for 8 dayB. The liver fats wero then collected and analysed, with the result that deuterium and 13 C were found in alternate — CH, — radicals throughout tho chain, while 13 C also appeared in the terminal carboxyl group. Fat oxidation and therefore presumably fat synthesis is mainly localized in tho mitochondria, but fatty acid synthesis has nevertheless been demonstrated in cell-free extracts of that remarkable fat-producing organ, tho mammary gland. It lias now been shown that all tho enzymes concerned with /7-oxidation can act reversibly and it seems certain therefore that fatty acids can bo synthesized from. ooctyl-Co A by tho reverse process of /l -reduction. Synthesis would ho favoured by a high concentration of reduced DPN such as could arise by the active oxidation of many other metabolites. This same active oxidation could also produce tho free energy needed for tins cndcrgonic synthesis. Finally, synthesis would also be favoured by removal of the 402 SYNTHESIS OT LIPIDS fatty acida produced and this can be achieved by their conver- sion into neutral fats or into phospholipids. Neutral fat can be formed by reactions between free fatty acids and a-glycero- phosphate, which is itself formed by a glycerophosphokinase: cn.oii I choh 1 CH.OH CH.O© CHOH I cn.oH Enzymes are known which will catalyse the formation of of phoaphatidic acida: Njh phosphatidie acid (I) etcaryl~Co a + a-glycerophojphato -*• monoatearylphosphatidic acid + Co a, (ii) Btearyl~Co l + rnonostearylphoapli&tidic acid -► disteaiyJphcwphatidic acid + Co a. The enzymes seem to be fairly specific for chains of 10, 17 and 18 carbon atoms. The next stage involves removal of the phos- phoric acid radical and its replacement by a third fatty acid chain, but the mechanism of this reaction is at present uncertain. Alternatively the diacylphosphatidic acid, having lost its phosphate radical, apparently through the action of a phospha- tase, can react with cytidine diphosphate choline, which transfers choline phosphate to tho diglyceride to yield a phospholipid : CH,O.B, 01,0. R, iuO.R. + CDP-eholino ->■ AhO.R, n CH,OH CH,0 — P — 0 — choline ''•Oil 493 CARBOHYDRATE FROM FAT A similar reaction can take place in which CDP-tlhanolamint replaces ihe choline derivative. Both CDP compounds arise by similar reactions: CTP 4 choline phosphite CDP-cholino 4 pyrophosphate, CTTP 4 ethanolamine phosphate «=» CDl’-ethanolamino 4 pyrophosphate. These latter reactions bear a close formal rcsemblanco to one in which uridine derivatives are involved: UTP 4 a-glucwc-l-phosphale p=* UDP-glnco«o 4 pyrophosphate. CONVERSION OF FAT TO CARBOHYDRATE While there can bo no doubt whatever that fats can be syn- thesized from carbohydrate sources, tho reverse process takes place to a very limited extent and, in fact, it has been denied that the conversion of fat into carbohydrate takes place at all. Much of tho earlier work on this problem gave results which were somewhat vague, and some of it has actually been dis- credited. Inasmuch ns they yield glycerol on hydrolysis, fats certainly are potential sources of glycogen, for glycerol is quantitatively converted into glucoso when administered to diabetic or phlorrhizinized animals. Apart from glycerol, fats aro not known to give rise to anything but acetyl-Co A and the free acetate and ketone bodies which can be derived from it. Experiments havo been performed in which the incubation of sliced rat liver with butyrio acid gavo small but apparently significant increases in tho glycogen content of tho tissue. But, as wo now know, glycogen and fat alike are oxidized through a common metabolite in the form of acetyl-Co A and it may well be that tho addition of butyrate merely spares the oxidation of glycogen by giving rise to this common metabolite. Since acetyl-Co a can bo formed by tho oxidative decarboxyl- ation of pyruvate and can itself give riso to the synthesis of fat, there is not much doubt that this is an important route for tho production of fat from carbohydrate sources. It may bo asked whether this process is rcvcrsiblo. There is, however, no evidence that tho stage of oxidativo decarboxylation can bo reversed, at any rate in animal tissues, so that, while pyruvate freely give* riso to acetyl-Co a, tho latter is not convertible back into 494 CARBOHYDRATE FROM FAT pyruvate. It has been shown that, if lactate or pyruvate con- taining isotopic carbon is administered to animals, the carbon isotope can be partly recovered in the liver glycogen. If acetate similarly ‘labelled’ is used instead, however, very little trans- ference of the isotope to the liver glycogen can be detected. It may therefore reasonably be assumed that the conversion of acetate to pyruvate takes place to a limited extent at most if, indeed, it takes place at all. In short, there is no convincing evidence for any large-scale production of carbohydrate from fatty sources and, in so far as such a conversion takes place at all, it must probably be attri- buted in the main if not entirely to the formation of glycerol as an intermediary metabolite of fat katabolism. 49G BIBLIOGRAPHY The list of references that follows makes no claim to bo exhaustive. It is designed only aa an introduction to tho literature, but a wealth of further references will bo found in tho books (marked with an asterisk) and articles listed hero. Preference has in general been given to recent publications, but for tho roost recent work tho Annual Revictc of Bio- chemistry should bo consulted: hero will bo found many valuable and critical appraisals of the newest work, together with exhaustive lists of references to recent papers. Short but useful reviews of specific subjects will bo found annually in Federation Proceedings, the Annual Reports of the Chemical Society and else who ro. REFERENCES Chapters Alberty, R. A. (1056). Enzyme kinetics. Adv. Enzymol. 17, i. i-m Ajujon, D. I. Hoaoland, D. R. (1914). Plant nutrition. Biol. Rev. 19, 65. IX Axelrod, B. (1050). Enzymatic phosphate transfer. Adv. Enzymol. 17, 1 59. V Bacon, J. S. D. (1053). Transfructcwjlation. Ann. Rep. Chem. Soc. 50, 231. V Baldwin, E. (1030). Arginoso. Biol. Rev. II, 247. XII •Baldwin. E. (1040). An Introduction to Comparative Biochemistry (3rd ed.). Cambridge. XII, XIII Bell, D. J. (1053). Biological synthesis and in tercon ver- sion of some monosaccharides. Ann. Rep. Chem. Soc. 50, 324. V, XVII BerqmaNn, M. (1042). A classification of proteolytic enzymes. Adr. Enzymol. 2. 40. IV Beromann, M. & Fruton, J. S. (1941). Tho specificity of proteinoses. Adv. Enzymol. 1, 03. II’ Blasctiko, H. (1045). Tho amino-acid decarboxylases of animal tissues. Adv. Enzymol. 5, C7. IV, XVIII •Bloob, W. R. ( 1 0 13). Biochemistry of the Fatty Acids atul their Com pounds. .the Lipids. Now York.. XIX •Brand, T. von (1046). Anaerobiosis in Invertebrates. Normandy, Miss. XVI, XVII B exit AN AN, J. M» 4: Hastings, A. B. (1046). The use of ieotopically marked carbon m tho study of inter* General, mediary motnlioliKm. Physiol. Rev. 26, 120. VITI •CntHNALL. A. C. (1930). Protein Metabolism in the Plant. New Ilavcn. X. XI BIBLIOGRAPHY Davidson, J. N. (1950). The Biochemistry of the Nucleic Acid*. London. ♦Dixon, SI. (1949). Multi-enzyme Systems. Cambridge. Doyle, IV. L. (1943). Nutrition of the Protozoa. Biol. Rev. 18, 119. Edelman, J. (1950). The formation of oligosaccharides by enzymic tronsglycosylation. Adv. Enzymol. 17, 189. Engelhaudt, V. A. (1946). Adenosinetriphosphatase properties of myosin. Adv. Enzymol. 6, 147. Gaebler, O. H, (editor) (1956). Enzyme a, Units of Biological Structure and Function. New York. ♦Gale, E. F. (1951). The Chemical Activities of Bacteria (3rd ed.). London. Gale, E. F. (1946). The bacterial amino-acid decarboxyl- ases. Adv. Enzymol. 6, 1. Green, D. E. (1941). Enzymes and trace substances. Adv. Enzymol. 1, 177. Green, D. E. (1054). Fatty acid oxidation in soluble Bystems of animal tissue. Biol. Rev. 29, 330. Green, D. E. & Beinebt, H. (1955). Biological oxida- tions. Ann. Rev. Biochem. 24, 1. Gunbalus, I. C., Horeoker, B. L. & Wood, W. A. (1955). Pathways of carbohydrate metabolism in micro- organisms. Bact. Rev. 19, 79. XVI, XVII ♦Haldane, J. B. S. (1030). Enzymes. London. I, II Haldane, J. B. 8. (1956). Biochemistry of Genetics (2nd ed.}. London. General Hassid, \V. Z. & Dotjdoroff, M. (1950). Synthesis of disaccharidos with bacterial enzymes. Adv. Enzymol. 10, 123. V Hildebrandt, F. M, (1947). Recent progress in industrial fermentation. Adv. Enzymol. 7, 557. XVI Horecxer, B. L. & Mauler, A. H. (1955). The path of carbon in photosynthesis. Ann. Rev. Biochem. 24, 244. XV Jeeneb, R. (1056). Ribonucleic acids end virus multi- plication. Adv. Enzymol. 17, 477. XIV Johnson, M- J. & Berger, J. (1942). The enzymatic properties of peptidases. Adv. Enzymol. 2, 09. IV Kennedy, E. P. (1956). Biological synthesis of phospho- lipids. Canad. J, Biochem. dr Physiol. 34, 334. XIX Knight, B. C. J. G. (1945). Growth factors in micro- biology. Vitam. ct* Harm, 3, 108. IX Korxes, 8. (1956). The shunt mechanism. Ann. Rev. Biochem. 25, 703. XVH2 Korkes, S. (1956). Photosynthesis. Ann. Rev. Biochem. 25, 728. XV Chapters XIV General IX V XVII General General IV, XI General XIX vi, vn 3* 497 BIBLIOGRAPHY Chapters Laotfeu, M. A.. Price, TV. C. &■ Pmr, A. TV. (1049). Tho nature of viruses. Adv. Enzymol. 9, 171, XIV •Lehkincer, A. L. (1051). The organized respiratory VIII, activity of isolated rat-liver mitochondria. In XVHJ, Eruymcs and Enzyme Systems. Cambridge, Mass. XIX LectOwitz, J. &. Hestrxk, S. (1045). Alcoholio fermenta- tion of the oligosaccharides. Adv. Enzymol. 5, 87. XVI •LiXDnEP.a, O. & Ernsteh, h. (1054). Protoplasmato- login, Uandbuch der pTOtoplasmaforsehu-ng, 3, part A 4, pp. 1-130. Vienna. General Lifmann, F. (1041). Metabolic generation and utiliza- III, XVI- tlon of phosphate bond energy. Adv. Enzymol. 1, DO. XIX I.rpHANtt, F. (1040). Acetyl phosphate. Ado. Enzymol. Ill, XVIII, 6, 231. XIX McAnally, R. A. (10-47). Insect nutrition. Bbl. Rev. 22, 148. IX Vox E. 1L J. (1937). The specificity and collaboration of digestive enzymes in Metazoa. Did. Rev. 12, 245. IX WetkbocsE, S. (1954). Formation of acetyl groups from pyruvic odd. Ann. Rev. Diochem, 23, 132. XVIII •Williams, It. T. (1947). Detoxication Mechanisms, London. XI 500 INDEX OF SUBJECTS Abomasum, 83 Absorption, of amino-acids, 240 • — of carbohydrates, 241 — of fats, 245 — of fatty acids, 248 — of glucose, 242 — of mono saccharides from small intestine, 243 — of oleic acid, 248 — of olive oQ, 248 — of pentoses, 243 — of products of digestion, 230-50 — of proteins, 239 Absorption spectrum, of catalase, 26 — - of catolase-azide, 26 — of catalaso-azide-peroxide, 26 — of CO — compound of Atmungs • ferment, 162 — of chlorocruorin, 163 — of cytochrome oxidase, 170 — of cytochromes, 164 — of DPN and TPN, 178 — of peroxidase, 20 — of peroxidoso-peroxides, 20 Acetaldehyde, from pyruvate, 41, 105, 386, 440, 402 — in fermentation, 385 Acetic add, acetoacetate formation from, 472, 492 — entry into the citric acid cycle, 452 — formation by fermentation, 395 — formation of acetyl-Co a from, 480 — formation of citrato from, 481, 490 — from acetoacetate, 472 — from cellulose, 00 — from fatty acids, 466 — in detoxication, 131, 271 Acetoacetlc add, acetone from, 218 — after phlorrhizin, 218, 473 — formation of, 485 — from acetyl-Co a, 471, 475, 488 — * from amino-acids, 218, 250, 473 ~ from butyrio acid, 218, 472 — from fatty acids, 218, 472 — • from -hydroxy bntyric acid, 184, 207, 472 — from octanoic acid, 487 — from products of /7-oxidation, 486 — from tyrosine, 291 — in diabetes, 473 — m liver poisoning, 473 — in starvation, 473 — oxidation of, 460 — see aUo Ketone bodies Acetoacetyl-Co a, formation of, 480 Acetone, from acetoacotate, 214, 472; see also Ketono bodies Acetone bodies, gee Ketone bodies Acetone powders, 22G, 259 Acetyl phosphate, energy-rich bond in, 04, 430 — formation of pyruvate, 431 Acetylation, acetylcoenzyme A in, 131. 433, 492 — activation by cysteine, 432 — ATP in, 131, 433 — coenzymes of, 432 — detoxication by, 131, 271, 282 — in synthesis of mercapturio acids 432 — inhibition by iodoacetato, 432 — inhibition by oxidation, 432 — of acetate, 472, 492 — of aniline, 131, 271, 431 — of choline, 131, 326, 433 — of sulphamlamide, 131, 271, 431, 433 — see also Acetylcoenzyme a A cetylcholine, 432 — formation, 326 — from choline, 131 — hydrolysis of, 97 Acetylcoenzyme a, acetoacetate from, 472, 489 — entry into citric cycle, 45? 480, 485 — from acetate, 453, 480 — from fatty acids. 471 — from pyruvate, 434 - — from pyruvic acid, 432 ACE— AER [Acetylcocnzyme a,] Identity with ‘Active Beet Ate 433 — In Acetylation, 131, 433, 492 — In adenylncet&te synthesis, 453 — in fat metabolism. 471 — in oxidative decarboxylation, 432 — in synthesis of citrate, 132, 434 — tec alto Activo acetate A’-Acetylsulphanllamide, 432 Aconitase, flurodtratc aa inhibitor of, 453 — properties of. 102, 130, 445 Acrylic add, 203 ACTH, tee Adrenocorticotrophie hor- mone Actln, in muscle, 393 Actinia equina, 320 Activation, of acetylation, 432 — of aminopeptidaaca, 76 — ol amyloao, 40 — of carboxypopt idase, 70 — of chymotiypainogcn, 75 — of dipeptidasca, 76 — of enclose, 112, 379, 334 — of enterokinase, 76 — of enzymes by accessory sub- stances, 36-44 — of enzymes by cysteine, 39 — of enzymes by de-inhibition, 38 — of enzymes by reduced gluta- thione, 39 — or enzymes by reducing agents, 39 — of enzymes by trace elements, 39 — of enzymes by unmasking, 38, 75 — of glyoxalase, 103 — of haem by globin, 35 — of knthepsina, 84 — of papain, 19, 85 — of pepsinogen, 76 — of peptidases, 84 — of substrates, 35, 75 — of trypsinogen, 38, 75 Activators, 30, tee alto Activation, Coenzymes Active acetate, identity with acetyl- cocnzymo a, 432 Actomyosln. 393 Aeyl-Co A, desaturation of, 4S2 — unsaturaled, hydration of, 432 Adding enzymes, 71, 102 Adfnase, 101 — distribution of, 359 Adenine, occurrence of, 357 — mononucleotide, 193 Adenosine diphosphate, as co- factor in pyruvate oxidation, 431 — energy -rich bond in, 63 — in transphosphorylation, 353, 4 10 Adenoslne-3'>monophosphate, we Yeast adenylic odd Adenoslne-5'-monopho*phate t *re Adenylic acid Adenosine triphosphatase, asso- ciation with myosin, 67 — in electric tissue, 67 — in muscle, 93 — in snake venom, 67 Adenosine triphosphate, function of, 353 — in fermentation, 370 — in formation of DPN, 355 — in formation of flavin adenine ilinucleotido, 335 — in formation of flavin mono- nucleotide, 355 — m formation of ribulo*e-l:5-di- phosphate, 363 — in formation of e no/pymvie acid phosphate, 428 — in methionine activation, 354 — m muscle, 407 — production of, 67, 102, 107, 205, 3S6, 414 — properties and functions of, 62 — structure of, 353 Adcnyl acetate. In acetyl-Co a synthesis, 453 — synthesis of, 433 Adenylic add, in formation of DPN, TPN and flavin-adenine dinucleotide, 354 — la transphoepborylation, 69 — structure, 352 — synthesis from adenosine, 332 — synthesis of, 346 Adenylic deaminase, of muscle, 101 — properties of, 101 ADP, tee Adenosine diphotpliate Adrenaline, 155 — action oa blood glucose, 423 — formation, 252. 292 — structure of, 253 Adrenaline oxidase, 155 Adrenocorticotrophie hormone, 422 Aerobes, strict, 376 Aerobic dehydrogenases, 163 502 Aerobic metabolism, of carbo- hydrates, 424 Aerobic oxidases, tee Oxidases aerobic AFDN, tee Adeninoflavin dinucleo- tide Agmatlne, 12, 289 - — detoxication of, 156 — distribution of, 336 Alanine, special metabolism of, 270 /I -Alanine, in anserine, 294 ■ — in camoeine, 293 — structure of, 277 /J-AlanylhisUdlne, tee Camoeine Albinism, 160, 292 Alcaptonnrla, 216, 292 Alcohol, tee Ethyl alcohol Alcohol dehydrogenase, 184 — in fermentation, 385 Alcoholic fermentation, tee Fer- mentation Aldehyde oxidase, a a flavoprotein, 195 — in liver, 13, 165 — in milk, 13 Aldolase, in fermentation, 381 — in glycolysis, 415 — properties of, 107 Alkmtoin, from uric acid, 162 Alloxan, 217 Almonds, bitter, amygdalin in, 92 — emulsin in, 02 Amine oxidase, 155 Amino groups, asparagine in storage of, 286 — storage of, 264 Amino-add, absorption of, 239 • — bacterial attack and detoxication, 270 — deamination by yeast, 390 — decarboxylation of, 427 — detoxication of, 271 — • formation of, in plants, 371 — in diabetic or phlorr h iz in ized dog, 255 — non-essential, 269 — nutritional status of, 252 — optical isomerism and nomen- clature of, 272 — oxidative deamination by kidney extract, 259 — - rates of de amina tion by rat kid- ney, 258 AER— ANA — transdeamination in deamination of, 261 Amlno-add decarboxylases, pro- perties of, 104 d- A mino- add, occurrence of, 0 D- Amino-add oxidase, 163, 169 L-Amlno-add oxidase, 164 o-Amlno-acryllc add, 103 y-Amlnobntyric add, formation, 287 2 - Amino - 6 -hyd roxypurine , tee Guanine, 353 5 - Amin o - 4 - lmlnazole carb ox - amide, in purine synthesis, 318 a-Amlno-nltrogen, fate of, 266 2-Amino-6-oxypurlne, synthesis of, 323 Amlnopeptldases, 75, 240 — activation, 76 — in digestion, 76 — specificity of, 81 Amlnopborase, tee Transaminase 6-Amlnopurlne, tee Adenine Ammonia in chick. 307 — origin in kidney, 204 — toxicity of, 250, 299 Ammoniotellsm, definition of, 300 Amoeba, digestion in, 7 AMP, see Adenylio acid Amphibia, nitrogen excretion in, 304 AraygdaUn, in bitter almonds, 92 Amylase, digestive, 89 — in barley, 235 — pancreatic, 89 — salivary, 89, 242 — specificity of, 85 a- Amylase, mode of action, 85 ^-Amylase, mode of action, 85 Amylo-l ;6-gluco9ldase, from muscle, 89 Amylolytlc enzymes, linkages at- tacked by, 86 Amylomaltase, in Etcheriehia coli, 114, 388 Amylopectln.coloration with iodine, 87 — composition, 86 — structure, 87 Amylophosphorylases, 120 Amylosc, composition, 80 Amytosucrase, in Neisseria per- fava, 113, 388 Anabolism, definition of, 64. 238 603 ANA— BET Anaerobes, facultative, 375 Anaerobic metabolism, contrasted with aerobic, 374 — of carbohydrates, see Fermenta- tion, Glycolysis Aneurfne (Vitamin B,), set Thi- amino Anenrine diphosphate, see Co- ear boxy Uoe Anisotropic bands, in muscle, 303 Anserinase, 85 Anserine, 85 — /7-alanine in, 277, 204 — distribution of, 339 Anterubln, distribution of, 333 Afiysia, cellulose in, 90 Apples, browning of, 140 Arubans, 241 Ana floor, 329 — tetramethy lone digu&nidinei n, 337 Areal n, distribution of. 337 Arglnaae, 309 — action of, 2S8 — distribution of, in liver and kidney of vertebrates, 310 — in liver of ineotebc animals, 310 — In telcos tean fishes, 313 — inhibition of, 100 — properties of, 100 — specificity of, 0, 12, 100 — stereochemical specificity of, 0 — substrate specificity of, 12 Arginine, distribution of, 333 — formation from citrulline, 315 — in Invertebrate phosphagens, 289 — In tranaamidmation, 2S9 — special metabolism of, 283 Arginine phosphate, function of, CS — in muscle, 401 Arsenical smokes, 181 Arsenite, as inhibitor of oxidative decarboxylation, 451 — inhibition of dtrio acid cycle, 458 Ascorbic oxidase, properties of, 151 Asparaginase, properties of, 101 Asparagine, in storage of amino groups, 230 — - storage of ammonia, 205 Aspartase, properties of, 107 Aspartic add, in ureogenmit, 286 — formation of aspargino from, 205 — special metabolism of, 280 — synthesis by legume symbionts, 566 Aspartic transaminase, 129 Asperyilhie, saccharoses in. 112 - — maltase in, 02 ATP, *« Adenosine triphosphate Autolysis, 55 Autolytlc enzymes, function of, 66 Autotrophes, definition of, 230 Aves, nitrogen excretion, 3 16 — uricogeneeis, 317 Azide, inhibition of catalase by, 15S Bacillus dtlbrucBii, lactic dehydro- genase of, 10 — oxidative decarboxylation in, 430, 434 BacHlue pyoeyaneue, 173 Bacillus subtil is, 105 Bacteria, activity in tbo intestine, 160, 270, 290 — activity in urine, 213, 210 — amino-acid decarboxylases of, 104 — chemosynthetic, 230 — cytochrome in. 164 — detoxication of amino-acids, 270 — digestion of cellulose by’, 00, 241 — in legumes, 267 — nutritional requirements, 233 — photosynthetic, 230 — production of methane by, 00 — production of phenols by, 271, 293 — production of polysaccharides by, US — production of trimethylamine by, 323 — symbiotio in herbivores, 00, 01, 241, 410, 406 Bacteriostatic activity, of tub phanilamido, 34 UAL, or, British anti-lewisite, set 2.3-Dimereaptopropanol Balanoglosms, phosphagens in, 333 Barley, a- and /T-amylasra in, 235 Bee, cytochrome in wing muscle*, )M Benzedrine, 156 Benzidine, test for peroxidase, J37 Benzoylated peptides, 73 Benryloxy carbonyl chloride, in synthesis of peptides. 74 Bergmann's method, for peptMs synthesis, 74 Betaine aldehyde, formation from choline, 182 501 BET— CAR Betaines, distribution of, 328 Bile salts, glycine in, 278 — • in fat absorption, 246 — bydrotropic action of, 248 — taurine in, 281 Biological energy cycle, 60 Biological methylatlon, see Trans- methylation Biological synthesis, see Enzymic synthesis Biological value, of proteins, 25-1 Biolumlnescent organisms, 67 Blood, cholesterol eaters in, 467 — lactic acid in, 425 — menstrual, 327 — phospholipids in, 250, 407 — pyruvic acid in, 430 — scrum composition of, 221 — transport of glucose in, 123 — transport of bpids in, 250, 467 Blood cells, red, see Erythrocytes Blood charcoal, definition of, 160 — ‘models’, 161 — systems, 161 Blood sugar level, enzymic control of, 421 — in diabetes, 422 — influence of adrenaline, 423 — normal value, 421 — of insulin, 422 Bomb calorimeter, 373 Bond energy, 208 Bone, ossification of, 08 Brain, metabolism in thiamine de- ficiency, 420 — metabolism of pyruvate in, 420, 430 — oxidative decarboxylation in, 429, 430 Branching factors, 122, 417 Braseica, ascorbic oxidase in, 151 Brels, use in metabolic studies, 223, 224 Brewer’s yeast, flavokinase from, 193 Brittle* stars, phospbagens in, 334 Bromelin, S3 Bromogorglc acid, 293 Butyric acid, from cellulose, 00 — ketone bodies from, 218, 474 — • synthesis of fatty acids from, 466 Butyryl-Co a dehydrogenase, FADN aa prosthetic group of, 183 — properties of, 183, 196 y-Butyrobetaine, distribution of, 329 Cadaverine, bacterial formation, 104 — detoxication, 156, 291 Caffeln, 345 Calcium Ions, in fermentation, 379 Calcium phosphate, in bone forma- tion, 98 Calorific value, of foodstuffs, 57, 466 Calorimeter, bomb, 67 Calorimetry, animal, 56 Camel, 467 Carbamyl-aspartic acid, in purine synthesis, 286, 315, 319, 320 — in pyrimidine synthesis, 347 Carbamylglutamlc acid, in uero- genesis, 288 Carbamyl phosphate, in citrulline formation, 314 — in purine synthesis, 286 Carbohydrases, 85 Carbohydrates, alcoholic fermenta- tion of, 375 — amino acids from, 256, 416 — calorific value of. 467 — digestion and absorption of, 241 — from non-carbohydrate material, see Glyconeogcnesis — glycolysis, in liver, 416 — glycolysis, in muscle, 403 — linkage with fats, 473 — oxidation, 441 — synthesis of, from fata, 406 Carbohydrates metabolism, aero- bio metabolism of, 424, 441 — anaerobic, see Fermentation, gly- colysis — energetics of, 389, 463 — linkage with fat metabolism, 473 — linkage with protein metabolism, 218. 448 — relationship to fatty liver, 4C8 Carbon, atomic structure of, 227 Carbon dioxide, fixation, reaction network in photosynthesis, 367 — fixation, reduced TPN in, 364 — production by yeast juice, 377 — respiratory origin of, 426 Carbon, iso topic, see Iso topic carbon 505 CAR— Cl! O Carbon monoxide, compound of cytochrome oxidase, absorption ipoctmm of, 170 — inhibition of evtochroma oxidase by, 166 — Inhibition of respiration, 101 Carbonic add, 105 Carbonic onhydrasc, properties of, 103 Carboxydlsmutase, it* Ribulose diphosphate carboxylaao Carboxylase.coenrymo requirement of. 41, 370, 427 — distribution of, 105 — in fermentation, 335 — in yeast, 4 1 — production of acetaldehyde by, 4 1, 395 ^-Carboxylase, tte Oxaloacetic do- carboxylase /1-Carboxylatlon, tee Oxaloacetic decarboxylase Carboxylation enzyme, it* 1U- buloro diphosphate carboxylase Carboxypcptldase, activation, 70 — in digestion, 75, 210 — properties, 81 — specificity of, 81 Carnitine, distribution of, 329 Camoslnaso, 85 Carnoslne, ^ -alanine in, 277, 293 — distribution of, 338 — inethylation of, 291 Cartilage, ossification of, 08 Casein, 83 Caseinogen, 83 Catalase, absorption spectrum of, 20 — affinity for substrata, 158 — inhibition of. 20 — peroxidase activity or, 138 — properties, 158 Catalyst, amount required, 3, 4 — definition, 1 — heavy metals as, 2, 4 — inhibition, 3, 4 — initiation of now reactions by, 4 — reversibility of action, 3 — speciflcity, 6 — thermostable, tee Activator*, Co. enzymes, Myokin&ao — • water as, 2 — tee alto Knxymca Catalytic functions of the B group of vitamins, 232 Catechol, action of polyphenol oxi- dase on, U9 — os hydrogen -carrier. 149 — in ‘browning* of plant tissues, 119 Caudal necrosis, 470, 476 Cell nuclei, isolation of, 202 Cell-free extracts, preparation of, Celloblases, 93 Cellobiose, 66, 03 Cellulose, distribution of, 90 - — extracellular, 91 — of Cdlulomonat, 01 — of Clottndium ifiermoctUun, 91 — of Spyrojyru, 0 1 — of VampyrtUa, 01 Cdlulomonat, 91 Cepballns, ethanolamine in, 219, 320 Ccphalochorda, pbosphagens in, 335 Charcoal, os adsorbent, 25 Charcoal ‘models *, tee Blood char- coal ‘models', 101 Chclonla, nitrogen metabolism. 305 Chcmosynthetlc bacteria, 230 Chick, ammonia in. 307 Chick embryo, fat metabolism, 467 Chlmpunzce, pbenyUoetylglut- ammo in, 283 Chi tin, 01 Chllinasc, 01 ChlortUa, respiration of, 174 Chloride ions, in araylaso activity, 89 Chlorocruorin, 163 Chlorophyll, bacterial counterparts or, 230 — in photosynthesis, 230, 363 CholamJnc, ««• Ethanolamine Cholesterol, In fat absorption, 216 — production of fatty liver by, 463 Cholesterol esters in blood, 467, 163 Cholic add. In bflo salts, 216 Choline, acetylation of, 326 — as a biological methylating agent, 326 — es vitamin. 323 — distribution of, 325 — special metabolism of, 323 Choline dehydrogenase, properties of, 182 500 CHO— CYS Choline esterases, 97 Chromoprote!n3, visual, 44 Chylomicrons, 250 Chyme, 242 Chymo trypsin, in digoation, 75 — precursor, 76 — specificity of, 78, 80, 84 — synthetic action, 84 Chymotrypslnogen, activation of, 75, 76, 239 Citric acid, acetyl-Co a in synthesis of, 434 ■— anaerobic formation of, 449 — ea primary product in citric acid cycle, 451 — asymmetric dehydrogenation of, 453 — breakdown, summary, 418 — conversion to iso-citric acid, see Aconitaao — formation of, from acetate and oxaloacetato, 452 wo-cltrlc acid, conversion to citrate, see Aconitaao Citric acid cycle, enzymes involved in, summary of, 457 — aide-reactions of, 456, 459 — summary of inhibitors, 460 — Bummary of reactions, 455 wo-Citrlc dehydrogenase, diatri- bution and properties of, 188, 446 Citrulllne, in ornithine cycle, 314 — special metabolism of, 289 Citrullus yu Irjarit, citruliine in, 311 Clcidoic eggs, 307 Clostridium Ihtrmoeellum, 01 Clostridium welchii, toxin of, 85, 97 Cobalt, as activator, 82 Cobra, 97 Co-carboxylase, as coenzyme of carboxylase, 105 — in cozymasc, 379 — in oxidative decarboxylation, 430 — in transketolation, 132 — in yeast, 41 Co-decarboxylase, see Co-carb- oxylase, Pyridoxal Go-dehydrogenase, see Di- and Tripbosphopyridine nucleotides Cod muscle, trimethylomino oxid- mq in, 156, 327 Coenzyme requirements of de- hydrogenases, 180 Coenzyme i, see Diphosphopyridino nucleotide Coenzyme n,see Tripbosphopyridine nucleotide Coenzyme a, acetylation of, 131, 432 — in acetylation, 131, 432 — in formation of citrate, 433 — in oxidative decarboxylation, 432 — structure of, 356 Coenzyme r, 320 Collagenase, 85 Compositae, inulin in, 370 Condensing enzyme, 452 Copper, as prosthetic group of poly- phenol oxidases, 148 — as prosthetic group, 183 Co-transaminase, pyridoxal phos- phate as, 262 Cozymase, composition of, 379 — »es also Diphosphopyridino nu- cleotide Creatine, distribution of, 330 in Lohmann reaction, 115 — synthesis of, 227, 331 Creatine phosphate, distribution, 334 — free energy of hydrolysis of, 64 — function of, 68 — in muscle, 400 Creatinine, 331 Creatone, formation from creatine, o-Cresol, detoxication of, 293 Crotonbetalne, distribution of, 329 Crotoxln, 97 . Cyanide, inhibition of catalase by, _ inhibition of cytochrome oxidase, 166 Cyanide-stable respiration, cyto- chrome 6 in, 167 Cyclical dextrans in micro- organisms, 114 Cysteic add, taurine from, -81 Cysteine, decarboxylation of, zw — from serine, 278 _ in enzyme activation. 39, 84, 432 _ in formation of mercaptunc acids, _ special metabolism of, 279 Cysteine desulphydrase, 279 Cysteine sulphlnic add, -7 , - 507 CAR— CHO Carbon monoxide, compound of cytochrome oxi d aao, absorption spectrum of, 170 — inhibition of cytochrome oxidase by, 160 — Inhibition of respiration, 181 Carbonic add, 105 Carbonic anhydrnse, properties of, 105 Carboxydismutnse, tee Ribolose diphosphate carboxylase Carboxylase, coenzymo requirement of, 41, 370, 427 — distribution of, 105 in fermentation, 3S5 — in yeast, 41 — production of acetaldehyde by, 41, 385 ^-Carboxylase, see Oxaloacetic de- carboxylase /7-CarboxyIation, tee Oxaloacetic decarboxylase Carboxylation enxyme, tee Ri- buloso diphosphate carboxylase Carboxypeptidase, activation, 76 — in digestion, 75, 210 — properties, 81 — specificity of, 81 Carnitine, distribution of, 329 Camoslnase, 85 Carnoslne, ^-alanine in, 277, 293 - — distribution of, 338 — mcthylation of, 291 Cartilage, ossification of, 98 Casein, 83 Caselnogen, 83 Catalase, absorption spectrum of, 20 — affinity for substrate, 158 — inhibition of, 26 — peroxidase activity of, 158 — properties, 158 Catalyst, amount required, 3, 4 — definition, 1 — heavy metals as, 2, 4 — inhibition, 3, 4 — initiation of now reactions by, 4 — tevncHihOity of action, 3 — - specificity, 5 — thermostable, tee Activators, Co- enzymes, ilyokinase — -water as, 2 — see alto Enzymes Catalytic functions of tbs B group of vitamins, 232 Catechol, action of polyphenol oxi- dase on, 149 — as hydrogen -carrier, J49 — in 'browning' of plant tissues, 149 Caudal necrosis, 470, 470 Cell nuclei, isolation of, 202 Cell-free extracts, preparation of, 225 Celloblases, 93 Celloblose, 88, 93 CeLluIase, distribution of, 90 — extracellular, 91 — of CtUvlomcmas, 91 — of Clostridium Ihermoeellum, 91 — of Spyroyyra, 91 — of Vampyrdla, 91 Celiulomonas, 91 Cephalins, ethanolamine in, 219, 328 Cephalocborda, phosphagena in, 335 Charcoal, as adsorbent, 25 Charcoal ‘models’, tee Blood char- coal ‘models', 161 Chelonla, nitrogen metabolism, 305 Chemosynthetic bacteria, 230 Chick, ammonia in, 307 Chick embryo, fat metabolism, 407 Chimpanzee, phenylocetylglut- araino in, 28S Chi tin, 91 Chitlnase, 91 ChlortUa, respiration of, 174 Chloride Ions, in amylase activity, 89 Chlorocruorln, 163 Chlorophyll, bacterial counterparts of, 230 — in photosynthesis, 230, 363 Cholamlne, see Ethanolamine Cholesterol, in fat absorption, 216 — production of fatty liver by, 4C8 Cholesterol esters In blood, 467, 488 Cholic acid, in bile salts, 246 ChaUae, acetylation- of, 326 — as a biological methylating agent, 320 — os vitamin, 326 — distribution of, 325 — special metabolism of, 325 Choline dehydrogenase, properties of, 182 50G CHO— CYS Choline esterases, 07 Chromoprotelns, visual, 44 Chylomicrons, 250 Chyme, 242 Chymo trypsin, in digestion, 75 — precursor, 75 • — specificity of, 78, 80, 84 — synthetic action, 84 Chymotrypsinogen, activation of, 75, 76, 239 Citric acid, acetyl-Co A in synthesis Of, 434 — anaerobic formation of, 449 — as primary product in citric acid cycle, 451 — afiymmetrio dehydrogenation of, 453 — breakdown, summary, 448 — conversion to wo-citric acid, see Aeonitaso — formation of, from acetate and oraloQcetato, 452 iso-cltric acid, conversion to citrate, see Aconitose Citric acid cycle, enzymes involved in, summary of, 467 — Side-reactions of, 466, 469 — summary of inhibitors, 466 — summary of reactions, 455 wo-Cltrlc dehydrogenase, distri- bution and properties of, 188, 446 Cltrulllne, in ornithine cycle, 314 — special metabolism of, 289 Citrullua vulgaris, citrulline in, 311 Cleldolc eggs, 307 Clostridium. thermoeeUum, 91 Clostridium \cdchii, toxin of, 85, 97 Cobalt, as activator, 82 Cobra, 07 Co 'Carboxylase, as coenzyme of carboxylase, 105 — In cozymase, 379 — in Oxidative decarboxylation, 430 — in transketolation, 132 — in yeast, 41 Co -decarboxylase, see Co -carb- oxylase, Pyridoxal Co-dehydrogenasc, see Di- and Triphosphopyridine nucleotides Cod muscle, trimetbylamino oxid- ase in. 156, 327 Coenzyme requirements of de- hydrogenases, 180 Coenzyzne i, tee Diphospkopyridme nucleotide Coenzyme n, see Triphosphopyridine nucleotide Coenzyme a, acetylation of, 131, 432 — in acetylation, 131, 432 — in formation of citrate, 433 — in oxidative decarboxylation, 432 — structure of, 356 Coenzyme r, 320 Collagenase, 85 Composltae, inulin in, 370 Condensing enzyme, 452 Copper, as prosthetic group of poly- phenol oxidases, 148 — as prosthetic group, 183 Co-transamlnase, pyridoxal phos- phate as, 262 Cozymase, composition of, 379 — see also Diphoephopyridine nu- cleotide Creatine, distribution of, 330 — in Lohmann reaction, 115 — synthesis of, 227, 331 Creatine phosphate, distribution, 334 — free energy of hydrolysis of, 61 — function of, 68 — in muscle, 400 Creatinine, 331 Creatone, formation from creatine, 332 p-Cresol, detoxication of, 293 Crotonbetalne, distribution of, 329 Crotoxln, 97 Cyanide, inhibition of catalase by, 168 — inhibition of cytochrome oxidase, 160 Cyanide -stable respiration, cyto- chrome 6 in, 167 Cyclical dextrans in micro- organisms, 124 Cystelc acfd, taurine from, 231 Cysteine, decarboxylation of, 282 — from serine, 278 — in enzyme activation, 39, 84, 432 — in formation of mercapturic acids, 282 — ■ special metabolism of, 279 Cysteine desulphydrase, 279 Cysteine sulphlnlc add, 279, 281 507 CYS— DIG Cyxteinyl-tyroslne, aa substrate, 81 Cystine, special metabolism of, 279 Cystlnurla, 2S3 Cytldine diphosphate choline, syn- t hes is of, 493 Cytochrome, absorption spectra, 154 — flavoproteina and the reduction of. 192 — system (general), 160 Cytochrome a, properties of, ICS Cytochrome . autoiidizability, ICS — reactions with carbon monoxide, 163 — relation to cytochrome oxidase, 108 Cytochrome b, properties of, 167 Cytochrome b t , relation to lactic dehydrogenase, ICS Cytochrome c, properties of, 1C7 Cytochrome c-peroxldase, 157 Cytochrome oxidase, carbon mon- oxide compound, absorption spectrum of, 170 — i n hi bition by carbon monoxide, ICC — inhibition of, by carbon monoxide, 39. 166 — inhibition by cyanide, 160 — inhibition by hydrogen sulphide. 166 — iron in, 153 — properties of, 166, 167 Cytochrome reductase, 195 Cytldlne nucleotide, synthesis of, 347 Cytidyilc add, 317 Cytosine, 345 Dalmatian coach-hound, 339 Deamination, hydrolytic, 101, 257 — of amino- acids in an i m a l tissues, 270 — of wnono, 103 — tmdatrte, 157 Deamlnidases, 101 Decamethylene dlguanindine, m tr eatm ent of diabetes racllitaS, 337 Decarboxylation, oxidative, cn- carbcxyhuw in. 41, 431 — - mechanisms of, 430 — of B. delbruckii, 430 — of a-ke toglutnric add, 445j see also In dividual a-keto-acids — of malic add, 428 — of oxalosucdnic add, 423 — summary of reactions, 439 Decarboxylation, spontaneous, of acetoacetate, 472 — of oxaloacet&te, 2S6, 428 Decarboxylation, straight, cocarb- oxylase in, 41, 335 — pyridoxal in, 101, 232 Dehydrogenases , coenzyme require- ments of, ISO — dtscovery, 151 — requiring DPN, 183 — requiring TPX, 1 86 — systems, reversibility and coupling of. 197 Deoxyribonuclease, properties of, 99 2-DesoxyrJbose, synthesis of, 319 Detoxication, by acetylation. 271 — of amino-acids, 271 — of aromatic adds by glycine, 276 — of aromatic acids, glutamine in, 288 — of cadaveriDC, 291 — of p-cresol, 293 — of histamine, 294 — of indoxyb 296 — of phenol. 271, 2S2, 293 — of phenylethylomino. 293 ~ of potresdne, 290 — of tyramine, 293 Deuterium, tee Isotopic hydrogen Dextrao sucrose, in Leueonottoe dexlranievm, 113 Dextrlnogenic amylase, tee Endo- amjlaso Diabetes, diabetogenic hormone of the anterior pituitary in, 217,422 — glycogen in, 217, 422 — - hexokinase in, 422 — insulin in, 422 — use in metabolic studies, 217 TWasaime oxidise , 15&, 2S& Diaphoroses i and n, properties of, 194 Dibenzoyl ornithine, in onus of birds, 290 Dibromotyroslne, of Gorymia, 293 Digestion, extracellular, 236 — by l-gulactoaidaae, 242 SOS DIG— ENE [Digestion,] by glncosaccharaee, 212 — by a-glucosidase, 242 — definition of, 235, 238 • — in amoeba, 7 — in lamollibranch molluscs, 236 — in platyholminth worms, 236 _ — in protozoa, 230 — in epongea, 236 — of carbohydrates, 24i — of fata, 216 — of galactans, 241 — • of glycogen, 61 — of nucleoproteins, 346 — of proteins, 236 Digestive enzymes, see Digestion Dlhydroxyacetone phosphate, from fructofuranose-l: -diphos- phate, 107, 186 3:4-DlhydroxyphenyIacetlo acid, 160 Dlhydroxyphenylalanlne, from ty- rosine, 161 3 :4 - Dlliyd roxyphcnyl-lactlc add, 160 6:8-Dihydroxypurlne, 164 Di-iodotyroslne, in thyroid tisano, 293 2^-DImercaptopropanol, 281 6:6-Dlmethyl-wo-aUoxazine, in flavoproteina, 192 Dimethylsucdnlc acid, 181 Dlnitropbenol, action on oxidative phosphorylation, 207 Dipeptidases, activation, 76 — in digestion, 76, 82, 240 — occurrence, 75 Diphosphopyrldlne nucleotide, ab- sorption spectrum of, 178, 201 — as cofactor in pyruvate oxidation, 431 — as coenzyme of phosphogalacto- isomer aae, 418 — as hydrogen acceptor, 42 — formation, 176, 355 — functional behaviour of, 177 — structure of, 176, 364 Dlphospbothiamlne, see Co-carb- o xylose Dipnoi, 304, 309 Disaccharldes, digestion of, 242 — excretion after injection, 242 — fermentation of, 388 Dlthlonitc, 193 DMA, tee Desoxyribonucleic acid Dopa qulnone, 161 DPN, see Diphosphopyridino nucleo- tide Duodenum, digestion in, 239, 240, 247 — pH of contents, 239, 247 Dynamo, energy, 69 Dytiscus, 165 Echidna, nitrogen metabolism of, 306 Echlnochrome, 173 Echlnodermata, phosphogens in, 334 Eck’s fistula, 218, 256 Efficiency, biological, 373 Eggs, cleidoic, 307 — nitrogen metabolism of, 307 — of lobster, 43 — of sea-urchin, 173 Egg-white (ooflavm), 192 Elasmobranchs blood, urea in, 303, 327 — nitrogen excretion of, 301, 304 — phosphatase of, 99 Electric organs, of fishes, 67, 414 Electrical energy, 67 •Electron-transporting’ flavo- proteln, 196 Electron transporting particles, composition of, 203 — fractionation of, 204 — from beef heart muscle, 203 Embryonic development, chemical recapitulation in, 307 — fat metabolism in, 467 — of chick, 308 — of frog, 305 Emulsln, in bitter almonds, 02 Endergonlc reactions, definition of, 63, 238 Endo- and exo-amylases, see a- and /?- Amylases Endopeptldases, definition of, 77 Energetics, of carbohydrate meta- bolism, 463 — of fermentation, 389 — of glycolysis, 463 — of synthetic reactions (general), 60 Energy, associated with oxidation of lactic acid, 374 - — free, concept of, 50 — of hydrolysis of phosphoric acid derivatives. Tab" ~ 509 ENE— EXE Energy,] production by chemosyn- thetic bacteria, 230 Energy cycle, biological, 88 Energy dynamo, 60 Energy-poor phosphate radicals, definition of, 63 Energy-rich phosphate radicals, definition of, 83 Enolase, properties of, 102 Enteroklnase, activation of tryp- smogen by, 38, 75, 239 Enteropneusta.phosphagensin, 335 Entropy, definition, of, 51 Enzymes, activation of, *e« Activa- tion — active groupe of, 22 — activity, measurement of, 14 — adding, tee Adding enzymes, 102 — adding or removing carbon di- oxide, 104 — as ampholytes, 20 — as zvnttenoo, 24 — autolytio, function of, 56 ■ — chemical nature of, 14-22 — concerned in photosynthetio car- bon-fixation, 368 — definition of, 7 — extracellular, 7 influence of pH on, 19 — influence of protein procipitants on, 21 — influence of temperature on, 16 — inhibition of, tee Inhibition • — intracellular, 7 — involved in breakdown of fata, 474 — ionization of, 20 — isoelec trio pH of, 20 — localization of, 7 — Miohaebs constants of, 28, 47-9, 158 — nomenclature and classification, 7, 8 • — precursors, 38 — properties of, 7 — prosthetic groups of, tee Proathetio groups — purification of, 228 — - respiratory, intracellular organiza- tion of, 201 — specificity of, 8-14 ■ — 1 stereochemical specificity of, 9 — - Table of pH optima of, 46 — transferring, 71 — turn-over numbers of, 197 — union of, with substrate, 24 Enzymic synthesis, of adenylic add, 352 — of creatinine, 331 — of cytidine diphosphate cholme, 493 — of cytidine nucleotide, 317 - — of fatty acids, 491 — of gentiobioso, 94 — of hyporanthine 321 — of nucleosides and nucleotides, 348 — of phosphatidic acids, 493 — of polysaccharides by transglyco- sylation, 113 — of purines, 346 — of pyrimidines, 347 -— of trimetbylamine oxide, 322 — of urea, 303 — ■ of urio acid, 316, 321 — - of xanthine, 321 Enzymic synthetic reactions , ener- getics of, 60 Ephedrine, 160 Erepsin, 72, 75 Erythrocytes, carbonio anhydrwo in. 105 J — glncose-6-phosphate dehydrogen- ase in, 175, 441 — respiration of, 175 Etchallot, cytochrome In, 165 Esclterichia coli, nmylomoJtaso in, 114, 388 — decarboxylation of pyruvate by, 434, 436 Eskimos, fat tolerance of, 474 Essential nmlno-adds, distribu- tion, 621 — function, 262 — glycogenic, 255 — botogenlc, 255 Esterase activity of peptidases, 83 EthanolamlflCi effect on fatty liver, 488 — from choline, 326 — in cephalins, 249, 326 Ethereal sulphates, 282 Ethyl alcohol, production of In fer- mentation, 385 ETP, tee Electron transporting par- ticals Exergonlc reactions, definition of, 63, 238 510 Exo-amylase, specificity of, 87 Exopeptidases, definition of, 77 Extracellular cellulases, 01 Extracellular digestion, 236 FADN, see Flavin adenine dinu- cleotide Fat depot, composition of, 465 Fats, acetyl -Co a from, 475 — conversion to carbohydrate, 404 — deficiency disease, 470, 476 — desaturation of, 477 — digestion and absorption of, 245 — enzymes involved in the break- down of, 474 — functions of, 469 • — linkage with carbohydrates, 473 — metabolic water from, 467 — metabolism in chick embryo, 467 — metabolism of, 465, 470 — specific nature of, 466 — synthesis of, from carbohydrates, 4C6 — tolerance of, 474 — transport and Btorage of, 465, 467 Fatty adds, acetylcoenzyme a from, 471 — absorption of, 248 — from carbohydrates, 472 — ketone bodies from, 471 — metabolism, mechanisms of, 470 — multiple alternate oxidation of, 487 — oxidation by citrio cycle, 480 — /^-oxidation of, 478 — scheme of oxidative breakdown of, 485 — Synthesis of, 491 Fatty acyl -Co a dehydrogenases, FADN as prosthetic group, 182 — properties of, 182, 100 Fatty liver, 468 Feeding experiments, in metabolic studies, 213 Fermentation, alcoholic, adenosine triphosphate in, 370, 387 — by yeast colls, 3D1 — calcium ions In, 379 — coenzymes of, summary, 337 — energotics of, 339 — • emtyraos of, summary, 337 — formation of acetio acid, 395 — formation of glycerol In, 392 EX O— FLU — fructofurano8e-l:6-dlphosphate in, 377 — fructofuranose-6-pho8phatem,377 — glucopyranose-6-phosphate in, 377 — inhibitors of, 37 B — inhibitors, summary, 387 — inorganic phosphate in, 377, 379 — magnesium ions in, 379 — micro-organism in, 375 — Neuberg’s three forms of, 393 — of glucose, 375 — potassium ions on, 379 — production of fusel oil, 395 — reactions, summary, 38Q — yeast juice in, 376 Fidn, 83 Fig, 83 Fire- fly, 67 Fishes, electric organs of, 414 — nitrogen excretion of, 301, 360 Flavin adenine dinucleotide, as prosthetic group of aldehyde oxidase, 155 — aa prosthetic group of butyryl- Co a dehydrogenase, 183 — ns prosthetio group of O -amino acid oxidase, 159 — as prosthetio group of fatty acyl- Co a dehydrogenases, 182 — formation of, 193, 355 — structure of, 355 Flavin mononocleotlde, as pros- thetio group of lactic dehydro- genase, 182 — formation, 103 — from riboflavin, 3 55 — structure of, 355 Flavokinase, from brewer's yeast, 193 Flavoprotelns, classification of, 193 — the reduction of cytochrome, 192 Flavoproteln dehydrogenases, properties of, 104 Flavoproteln oxidases, properties of, 104 Fluoride, Inhibition of catalase by, 158 — inhibition of enolaso by, 102, 384 — inhibition of fermentation by, 384 — inhibition of glycolysis by, 415 Fluoroacetate, 4G8 FI u o rod t rate, aa inhibitor of aco- nitaso, 458 611 FMN — GLU FMN, ate Flavin mononucleotide Food chains, definition of, 233 Formaldehyde, os sourco of methyl groups, 284 — in punne synthesis, 319 Formic add, from cellulose, 90 • — ■ from glyoxylio acid, 215 — in punne synthesis, 321 Free energies, of respiratory car- riers, 203 Fructofuranosans, 241 Fructofuranose-l:6-dlphosphate, action of aldolaso on, 107 Fmctofuranose-l -phosphate, syn- thesis by aldolase, 107 Fructofuranose-6-phosphate, in fermentation, 377 Fructosaccharases, specificity of, 93 Fructose, phosphorylation of, 417 Fumarase, m citric acid cyde, 450 — properties of, 103 Fumarlc add, conversion to malic odd, 103 — role in citric acid cyde, 458 Functions and late ol proteins and amlno-acfds, 251 Fusel oil, 395 Galactans, 241 Galactogen, 9 Galactoklnase, action of, 36S D- Galactose, absorption of, 243 — formation of m plants, 370 — synthesis, UPDO in, 371 x.-G3lactose, 0 n-Galactosidases, specificity of, 94 /?-GnlactosIdasea, specificity of, 12, 94 — of intestinal juice, 242 a-Galactosides, 92 /?-Galactosidcs, 93 Galactowaldenase, see Phospho- galactoisoraentso Galleria, cytochrome in wing muscles of, 175 Gas-gangrene organisms, see C7o- ttridia Gastric Juice, digestive functions of, 239 — hydrochloric acid in, 75 — lipase in, 98, 245 *— pepsin in, 6, 75, 239 — pepsinogen in, 75, 239 — rennin in, 83 Gelatin, biological value of, 255 Gentloblose, occurrence, 88, 93 — synthesis of, 94 Geodia ffiyaa, 330 Geometrical isomerism, 10 Globiu, influence of, upon haem, 35 Glucogenesls, in diabetes, 217, 258, 209, 422, 470 Glucogenic amino-adds, defini- tion of, 258 Gluconeogenesls, ate Diabetes, Gly- coneogenesis D -Gluconic add, from dglucono- lactone, 155 Gluconic add-6-phospbate, formation from glucose-O-phos- phate, 163 Glucopyranose-6-phosphate, in fermentation, 377 Glucosaccbarases, 03, 2 12 Glucose, absorption of, 243 — alcoholic fermentation of, 375 — excretion in diabetes, 217, 250, 289, 422. 470 — free energy changes in metabolism, tee Free energy — from amino-acids, 26S, 417 — from glycerol, 218, 268. 422, 470 — from glycogon, «e» Glyeogonolysis — from lactato. 218, 268, 417 — from propionate, 263, 417, 486 — from pyruvate, 268, 417, 440 — oxidative metabolism of, 441 — phosphorylation of, 01, 417 — use in physiological salines, 217 Glucose dehydrogenase, properties or, 185 Glucose-l:6-glucosldc, 88 Glucosc-4-/?-glucoslde, 93 Glucose-6-^-glucoside, 03 Glucose-oxidase, 155 — of Pentcillium, 185 Glucose-6-phosphate, action of glucose-6-phosphato dehydro- genase on, 188 Glucosc-6-phosphate dehydro- genase, distribution and pro- perties of, 188 ■ — - in erythrocytes, 441 a-Glucosidase, 12. 01. 92. 242 /7-GlucosIdases, 12, 02 012 GLU— GUA Glutamic add, from proline, 287 — in ureo genesis, 288 — special metabolism of, 287 Glutamic-a-decarboxylase, 287 {.•Glutamic dehydrogenase, co- factor requirements, 260 — properties of, 186 Glutamic transaminase, 127, 262 Glutamlnase, 101, 2S8 Glutamine, in detoxication of aro- matic acids, 288 — in purine synthesis, 320 — storage of ammonia, 206 — synthesis of, 124 Glutathione, as activator, 103 — m prosthetic group of triose- phosphato dehydrogenase, 180, 383 — structure, 276 — synthesis, 126 Glyceraldehyde, optical activity of, 272 D-Glyceraldehydc-3-phosphate, formation, 107, 186 — utilization in fermentation, 331 G!yceraldehyde-l:3-dIphosphate, formation, 186 Glyceric acid, from carbon dioxide and ribulo8o-l:5-diphosphate, 365 Glyceric add-2-phosphate, con- version to end-pyruvic acid phos- phate, 102 — dehydration of, 68 Glycerol, formation of in fermenta- tion, 392 a-Glycerophospbate dehydrogen- ase, distribution and properties of, 180, 183 — soluble, in yeast juice, 382 Glyceryl monostearate, in fat ab- sorption, 246 Glycine, detoxication of aromatic acids by, 270 — in bile salts, 276 — in creatine synthesis, 332 — in porphyrin synthesis, 275 — in purine synthesis, 276 — oxidase, 276 — serine from, 278 — specific metabolism of, 275 Glycine betaine, distribution of, 328 Glycocyamlne, in ereatino syn- thesis, 331 Glycocyamlne phosphate, 336 — synthesis of, 228 Glycogen, action of amylases on, 87, 241 — composition, 87 — digestion of, 61 — ■ from amino-acids, 263 — from propionic acid, 419 — in diabetes, 422 — in muscle, 420 — metabolism of aerobic, 424 — metabolism of anaerobic, 403 — occurrence, 420 — tee alto Glycogenolysis, Glycolysis Glycogen phosphorylase, see Amyl oph osph ory loses Glycogenosis, definition of, 268 Glycolysis, as a reversible operation, 416 — - by muscle extracts, 377 — energetics of, 463 — in tissues other than muscle, 412 — summary of coenzymes, 415 — summary of enzymes, 415 — summary of inhibitors, 415 — summary of reactions, 414 Glyconeogenesls, definition, 416 — from amino acids, 208, 417 — from fatty acids, 466 — from glycerol, 218, 208, 422, 470 — from lactate, 218, 268, 417 — from propionate, 208. 317, 460 — from reactants of glycolytic cycle, 414 — in diabetes, 217, 260, 269, 422, 470 — in herbivores, 460 — in liver, 418 — in muscle, 418 — see alto Glucogenesis Glycosidases, properties of, 112 — specificity ©f, 85, 91 — transglyrosylation by, 112 Glycylglydne, preparation of, 74 Glyoxatase, properties of, 103 Oorgonio, tyrosine of, 293 Group transference, CO GTP, tee Gnanosine triphosphate Gualacum, test for peroxidase, 167 Guanase, distribution of, 359 — properties of, 101 Guanidine, origin of, 333 • Guanidine bases, distribution of 330 GUA— IIYD Guanine, occurrence of, 358 — jn spider*, 353 — synthesis of, 323 Guanosine triphosphate, 68, 117 Guanyllc add, synthesis of, 346 Guinea-pig, cytochrome In heart muscle, 165 Gymncttu, electric organ of, 67 Haem, ‘activation’ of, by globin, 35 — of catalase, 35, 158 — of chlorocruorin, 163 — of cytochromes, 167 — of haemoglobin, 35, 163 — oxidation to liae matin, 35 Hncmatln, from haem, 35 • — as prosthetic group of poroxidasee, 35 — peroxidase activity of. 159 Haemocyanln, prostbetio group, 43, 147 Haemoglobin, globin of, 35 Hair keratin, 281 11 alia parthenopcca, hallochrome in 173 Hallochrome, 173 Heart muscle, cytochrome in, 176 — cytochrome oxidoso In, 109 — diaphornso in, 195 — /?-bydroxybutyno dehydrogenaso in, 184 Heat engines, 61 Heavy hydrogen, eee Isotopio hy- drogen Heavy nitrogen, sec Isotopio ni- trogen Heavy oxygen, tee Isotopio oxygen miix, cellulose in, 00 Iiemicelluloses, 241 Heterotrophes, definition of, 230 Hexoklnase, In diabetes, 422 — in fermentation, 380 — properties, 65 — reaction, energetics, 65, 66 Hexose diphosphate, sc s Fructo- furanose-1 : 6 -diphosphate Hexose monophosphate, see Fruc- tof uranoae- 6-monophosphato, Glucose- 1 -monophosphate. Glu- cose- C- monophosphate Hexose monophosphate dehydro- genase, tee Glucose- 6 -phosphate dehydrogenase High-energy phosphate radical , m product of oxidation, 205 Hippuric add, 124, 276 Histamine, oxidation of, 156 — bacterial formation, 101 — detoxication of, 29 1 — in histidine, 294 HJstidase, 294 Histidine, essential, 252 — histamine in, 294 — in synthesis of purines, 294 — special metabolism of, 293 Histidine decarboxylase, 294 Hlstohacmatln, see Cytochrome Homaririe, distribution of, S30 Homocysteine, 280 Homogenate, preparation of, 201, 224 — use of, 223 Homogentlslc add, excretion in alkaptonuria, 216, 292 — in ketogenesis, 292 Honey bee, tronsglucosylation in, 112 Hopkln’s aphorism, 60 Hormones, tee Aeetyl choline, AC7TH. Adrenaline, Diabeto- genic hormone, Insulin, Thy- roxine Hydrogen , bacterial production from cellulose, 00 — isotopic, tee Isotopio hydrogen Hydrogen acceptors, 37, 42, 141 Hydrogen donators, 37, 42, 141 Hydrogen peroxide, fato of, 167 Hydrogen sulphide, inhibition of catalase by, 168 — inhibition of cytochrome oxidase by. 160 Hydrolases, 71 Hydrolytic deamination, of ode- nine. 357 — or gttanine, 358 Hydrolytic deaminldascs, classifi- cation, 101 Hydrolytic glycosldases, proper- ties of, 1 12 3-Hydroxyanthranllic add, from kymirenino, 205 /Mlydroxybutyrlc add, as sub- strate for oxidative phosphoryl at ion studies, 207 — oxidation of, 483 — tee alto Kctono bodies 514 HYD— ISO ^-Hydroxy butyric dehydrogen- ase, distribution and properties of, 184 ^-Hydroxy fatty-acyl-Co a de- hydrogenase, properties of, 187 y- Hyd roxygt utamlc acid from hydroxyproline, 297 p-Hydroxyphenyl-lactlc acid, 216 p-Hydroxyphenylpyruvlc add, 216 Hydroxyproline, non-essential, 252 — special metabolism of, 297 Hypoxanthlne, from adenine, 357 — synthesis of, 321 Imbedllltas ‘oligophrenia’ phe- nylpyruvlca, 292 Iminazole bases, distribution of, 338 ct-Imlnoproplonlc add, 103 Inborn errors of metabolism, 202; see alto Albinism, Alcnptonuria, Cyslinuria, Imbecillitas, Tyro- sinosis Indigo, natural, 290 Indole, 295 Indophenol oxidase, 1G9 Indoxyl, detoxication of, 296 Inhibition, of cytochrome oxidase by carbon monoxide, 39 — of enzymes by heavy metals, 21, 84 — of enzymes by products of re- action, 15, 33 — of enzymes, competitive, 34 — of enzymes, selective (‘specific’), ice Individual inhibitors — of fatty acid absorption by iodo- acetate, 249 — of fatty acid absorption by phlor* rbizin, 249 — of fermentation by fluoride dialy- sis, 384 — of liver phosphoiylaso by glucose, 421 — selective (‘ specific ’) , tee Individual inhibitors Inoslne, 350 Inoslne triphosphate, in trans- phorylation, 117 Inoslnlc add, 320 Insnlln, composition of, 262 — in diabetes, 422 Intermediary carriers, of hydro- gen, see Flavin adenine dinu- cleotide, DPU, TFN, Cyto- chromes, Flavoproteins Intracellular organization, oi re- spirstory enzymes, 201 Intracellular peptidases, 83 Invertases, see Saccharoses Inulin, digestion of, 91 — in Compositae, 370 Iodoacetate, influence on absorp- tion, 244 — inhibition of acetylation, 432 — poisoning of muscle, 401 lodogorglc add, 293 Irldocytes, 358 Iron, as oxidation catalyst, 161 — in Atmungiftrmtnt, 161 — in catalase, 158 — - in cytochrome oxidase, 163 — in cytochromes, 107 — m haemoglobin, 35 — in peroxidaso, 167 — in prostheiio group of oxidases, 147 — in urieo-oxidase, 152 — in yeast, 196 cu-tram Isomerism, 10 Isomerizlng enzymes, 71, 135 Isotopes, 060 in metabolic studies, 227 ; tee alto Isotopic carbon, etc. Isotopic carbon (radioactive), in acetate metabolism, 470, 492 — in acetoacetate formation, 487 — • in adrenaline formation, 292 — in carbon dioxide fixation, 460 — in citrate formation, 460, 454, 490 — - in fat absorption, 250 — in fatty acid formation, 492. 495 — in glycine metabolism, 275 — in glycogenesia, 495 — in ketogeneeia, 291, 487 — in ec-ketoglutarate formation, 450, 454 — in methionine metabolism, 292, 328 — • in phenylalanine metabolism, 291 — in purine synthesis, 317 — in pyruvate metabolism, 450 — in sarcoaine formation, 328 — in snccinate formation, 454, 455 — - in transmethylation, 292, 328 — in tyrosine metabolism, 291 33< d 615 ISO— LEO Isotopic hydrogen (heavy), m creatine formation, — in absorption or fat, 240, 465 in ^-oxidation of fatty acids, 4*9 — in storage of fat, 465, 463 — - in synthesis of fatty acids, 492 Isotopic nitrogen (heavy), in ammo-group metabolism, 2C3 — in creatine formation, 331 — m creatinine format ion, 331 — in glycocy amino synthesis, 228 — in, transamination studies, 263 — in area excretion, 265 Isotopic oxygen (heavy), la photo- lysis of water, 363 Isotopic sulphur (radioactive), In cysteine formation, 280 — In cyBtine formation, 280 — m methionine metabolism, 2S1 • — in taurine formation, 232 Isotropic bands, in muscle, 393 Jack-bean, urease in, 100, 235 Janus green, as mitochondrial stain, 202 Jerusalem artichoke, 241 Katabotlsm, definition of, 54, 238 Kathepstn, m kidney and spleen, 83 a-KctO-acids, decarboxylation of, see Decarboxylation, oxidative and ‘straight’ /?-Keto-adds p in oxidation of fatty acids, 477 — see alto Acetoacetio acid, /3-Oxida- tion, Oxaloacetic acid a-Keto-^-carboxyglutarlc add, tee Oxalosuccinio acid Ketogenesls, definition of, 208 Ketogenlc amlno-adds, classifica- tion of, 25B a-Ketoglutarlc acid, as respiratory catalyst, 445 — conversion to oxalosuccinic acid, 106 — formation from pyruvate, 450 — in transamination, 261 — oxidative decarboxylation of, 445 Ketone bodies, excretion in diabetes. 217 — from fatty acids, 471 — * In liver poisoning, 463 — in starvation, 408, 487 — in subnormal carbohydrate meta- bolism, 468, 487 - — see also Acetoacetio acid /?-KetothloIase, action of, 484 Kidney, D-amino-acid oxidase in, 153, 259 — arginaso in, 310 — deamination in, 258 — glutaminaae in, 265 — histidine decarboxylase In, 294 — /?-hydroxybutyric dehydrogenase, 164. 489 — kathepsm In, 83 — mammalian, L-amino-acid oxidase from, 260 ■ — xanthine oxidase in, 317 Kreb's physiological saline, 221 Kynurenlc add, 294 Kynurcnine, 29 1 — 3-hydroryanthronibe acid from, 295 Laccase, 148 Lachrymatora, 281 Lactase, see /J-Galactosidaac Lacteals, transport of fat In, 250 o-Lactlc add, formation by micro- organisms, 4. 10, 182 L-Lactic add, change of free energy with oxidation, 374 — from methyl glyoxol, 103 — in blood. 425 — in muscle fatigue, 399 Lactic dehyd rogenase, flavin mono- nucleotide os proetbetio group or. 182 — from muscle, 179 — in Bacillus delbr&ckii, 10 — in musclo, 404 — of muscle, stereochemical speci- ficity of, 0 — of yeast, 106 — properties of, 182, 183 Lactoflavln, 102 Lactose, 04, 242 Lamellibranch molluscs, digestion in, 236 Laminaria, 86 Lancelots, phosphagens in, 335 Latex, 83 Ledthinase a, of cobra venom, 07 — of rattlesnake venom, 07 Leucine, special metabolism of, 285 CIO LEU— MET iio-LeucIne, special metabolism of, 285 Lruconoatoc dezlronicvm, dextran su- craso in, 113 Levan, 91 Levan sucrase, in spore-forming aerobes, 113 Light energy, 67 Limit dextrin, colour with iodino, 87 Lipase, in ffidniu seeds, 336 — occurrence and specificity, 95 a-Lipoic acid, in pyruvic acid oxida- tion, 435 Lipothiamide diphosphate, struc- ture of, 435 Liver, anaerobio metabolism of car- bohydrates, 397 — fat metabolism in, 468 — fatty, see Patty liver — glycogen storage in, 416 *— glycogenosis in, 410 — glycogenolyaia in, 416 — glyconeogeneais in, 416 — perfusion in metabolic studies, 219, 261, 308, 478 Lohmann reaction, 64, 406 Lombrlcine phosphate, in obgo- cbaetes, 336 Lupinua ItUeue, nitrogen metabolism in seedlings of, 205, 260 Lysine, special metabolism of, 290 Lysoleclthln, 07 Magnesium, as activator for amino- peptidases and dipeptidasea, 76 — as activator of enolaae, 102 — as activator of phosphatase, 99 — as cofactor in pyruvate oxidation, 431 — effects of deficiency, 430 — in fermentation, 379 Magnesium fluoro phosphate, 102 ilolapterunu, electric organ of, 67 Maleic add, 10 Malic acid, catalysis of respiration by, 444 — conversion to fumaric acid, 103 — from pyruvate, 105 — in citric acid cycle, 449 Malic decarboxylase, 105, 428 Malic dehydrogenase, properties of. 181, 189 — in citric acid cycle, 449 Malonic add, as Inhibitor of suc- cinio dehydrogenase, 13, 33, 443 — inhibition of citrio acid cycle, 458 Malt, maltose in, 9