Chemical Pathways of Metabolism

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Chemical Pathways of Metabolism

Table of contents :
Front Cover......Page 1
Chemical PathwaysofMetabolism......Page 4
Copyright Page......Page 5
Table of Contents......Page 8
CONTRIBUTORS TO VOLUME II......Page 6
LIST OF ABBREVIATIONS AND SYMBOLS......Page 10
Contents of Volume I......Page 11
II. DEAMINATION......Page 12
III. DEAMIDATION......Page 37
IV. TRANSAMINATION......Page 40
VI. UREA SYNTHESIS......Page 47
VII. SUMMARY REMARKS......Page 54
ADDENDUM......Page 56
CHAPTER 10. Carbon Catabolism of Amino Acids......Page 58
I. SCOPE OF THE CHAPTER......Page 59
II. AMINO ACIDS LINKED WITH THE CITRIC ACID CYCLE......Page 60
III. GLYCINE......Page 63
IV. THE β-HYDROXYAMINO ACIDS......Page 65
V. THE ALIPHATIC BRANCHED-CHAIN AMINO ACIDS......Page 70
VI. CERTAIN AMINO ACIDS OF UNCERTAIN BIOLOGICAL SIGNIFICANCE......Page 81
VII. THE SULFUR AMINO ACIDS......Page 83
VIII. LYSINE......Page 87
IX. ARGININE AND ORNITHINE......Page 90
XI. THE AROMATIC AMINO ACIDS......Page 91
XII. TRYPTOPHAN......Page 101
XIII. HISTIDINE......Page 112
ADDENDA......Page 122
I. INTRODUCTION......Page 124
II. THE GLYCINE-SERINE INTERCONVERSION......Page 125
III. FORMATION OF PHOSPHATIDE BASES......Page 127
IV. INTERCONVERSIONS OF GLUTAMIC ACID, ORNITHINE, AND PROLINE......Page 134
V. BIOSYNTHESIS OF BRANCHED-CHAIN AMINO ACIDS......Page 138
VI. LYSINE BIOSYNTHESIS......Page 141
VII. BIOSYNTHESIS OF AROMATIC AMINO ACIDS......Page 142
VIII. REACTIONS INVOLVING TYROSINE......Page 145
IX. SYNTHETIC REACTIONS INVOLVING TRYPTOPHAN......Page 149
X. BIOSYNTHESIS OF HISTIDINE......Page 156
ADDENDA......Page 158
I. BIOLOGICALLY IMPORTANT,SULFUR COMPOUNDS......Page 160
II. RELATIONSHIPS OF METHIONINE AND CYSTEINE......Page 162
III. OXIDATION OF SULFUR-CONTAINING AMINO ACIDS......Page 166
IV. THE DESULFHYDRASE REACTION......Page 173
VI. REACTIONS OF SULFUR-CONTAINING COENZYMES......Page 174
CHAPTER 13. Enzymatic Syntheses of Peptide Bonds......Page 184
I. THERMODYNAMIC CONSIDERATIONS......Page 185
II. CLASSIFICATION OF ENZYMATIC PEPTIDE SYNTHESES ACCORDING TO THE SIGN AND MAGNITUDE OF THE FREE ENERGY CHANGE (–ΔF)......Page 193
III. PEPTIDE SYNTHESES WHERE –ΔF IS NEGATIVE AND LARGE: COUPLED WITH HIGH-ENERGY PHOSPHATE......Page 209
IV. MECHANISM OF AMINO ACID INCORPORATION INTO PROTEINS......Page 214
V. ADDENDUM......Page 230
CHAPTER 14. Purines and Pyrimidines......Page 234
I. THE PURINES......Page 235
II. THE PYRIMIDINES......Page 259
CHAPTER 15. Nucleotides and Nucleosides......Page 274
I. NUCLEOSIDES......Page 275
II. NUCLEOTIDES......Page 281
III. DEAMINATION OF NUCLEOSIDES AND NUCLEOTIDES......Page 288
IV. THE ENZYMATIC SPLITTING OF CERTAIN COENZYME NUCLEOTIDES......Page 290
V. THE ENZYMATIC SYNTHESIS OF CERTAIN COENZYME NUCLEOTIDES......Page 291
CHAPTER 16. Metabolism of Heme and Chlorophyll......Page 298
I. INTRODUCTION......Page 299
II. COMPOUNDS OF THE BIOSYNTHETIC CHAIN LEADING TO THE FORMATION OF PROTOPORPHYRIN......Page 306
III. BIOCHEMICAL LESIONS IN THE PORPHYRIN METABOLISM OF HUMAN BEINGS......Page 319
IV. TRACER STUDIES OF PROTOPORPHYRIN BIOSYNTHESIS......Page 322
V. THE IRON BRANCH OF THE BIOSYNTHETIC CHAIN AND SOME GENERAL PROPERTIES OF HEME COMPOUNDS......Page 330
VI. THE MAGNESIUM BRANCH AND THE BIOSYNTHESIS OF CHLOROPHYLL......Page 340
VII. DECOMPOSITION OF IRON PROTOPORPHYRIN TO BILE PIGMENTS......Page 342
Author Index......Page 354
Subject Index......Page 370
ERRATA: VOLUME I......Page 395

Citation preview

CHEMICAL PATHWAYS OF METABOLISM VOLUME II

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Chemical Pathways of

Metabolism EDITED BY

DAVID M. GREENBERG Department of Physiological Chemistry School of Medicine University of California Berkeley, California

VOLUME II

1954 ACADEMIC PRESS INC., PUBLISHERS NEW YORK, N. Y.

Copyright 1954, by ACADEMIC PRESS INC. 125 EAST 23RD STREET NEW YORK 10, N.Y.

All Rights Reserved No part of this book may be reproduced in any form, by photostat, microfilm, or by any other means, without written permission from the publishers. Library of Congress Catalog Card Number: 54-7613

PRINTED IN THE U N I T E D STATES OF AMERICA

CONTRIBUTORS TO VOLUME II

H.

Kerckhoff Laboratories of Biology, California Institute of Technology, Pasadena, California

BORSOOK,

P. P. COHEN, Department of Physiological Chemistry, Wisconsin, Madison, Wisconsin S.

GRANICK,

University of

Rockefeller Institute for Medical Research, New York, New

York D. M. GREENBERG, Department of Physiological Chemistry, School of Medicine, University of California, Berkeley, California A. H E P P E L , National Institutes Service, Bethesda, Maryland

LEON

of Health, U. S. Public Health

P. SCHULMAN, Department of Biochemistry, State University of New York Medical School, Syracuse, New York

MARTIN

V

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CONTENTS CONTRIBUTORS TO VOLUME I I

v

L I S T OF ABBREVIATIONS AND SYMBOLS

ix

C O N T E N T S OF V O L U M E I

x

9. Nitrogen Metabolism of Amino Acids BY P. P COHEN I. II. III. IV. V. VI. VII.

Scope Deamination Deamidation Transamination Amino Acid Racemases Urea Synthesis Summary Remarks Addendum

1 1 26 29 36 36 43 45

10. Carbon Catabolism of Amino Acids BY DAVID M. GREENBERG I. II. III. IV. V. VI. VII. VIII. IX. X. XL XII. XIII.

Scope of the Chapter Amino Acids Linked with the Citric Acid Cycle Glycine The /3-Hydroxyamino Acids The Aliphatic Branched-Chain Amino Acids Certain Amino Acids of Uncertain Biological Significance The Sulfur Amino Acids Lysine Arginine and Ornithine Proline and Hydroxyproline The Aromatic Amino Acids Tryptophan Histidine Addenda

11. Synthetic Processes Involving Amino Acids BY DAVID M. GREENBERG . . . I. II. III. IV. V. VI. VII. VIII. IX. X.

1

Introduction T h e Glycine-Serine Interconversion Formation of Phosphatide Bases Interconversions of Glutamic Acid, Ornithine, and Proline Biosynthesis of Branched-Chain Amino Acids Lysine Biosynthesis Biosynthesis of Aromatic Amino Acids Reactions Involving Tyrosine Synthetic Reactions Involving Tryptophan Biosynthesis of Histidine Addenda vii

47 48 49 52 54 59 70 72 76 79 80 80 90 101 Ill 113 113 114 116 123 127 130 131 134 138 145 147

CONTENTS

Vlll

12. Metabolism of Sulfur-Containing Compounds BY DAVID M. GREENBERG . . 149 I. II. III. IV. V. VI.

Biologically I m p o r t a n t Sulfur Compounds Relationships of Methionine and Cysteine Oxidation of Sulfur-Containing Amino Acids T h e Desulfhydrase Reaction Thiosulfate and Thiocyanate Reactions of Sulfur-Containing Coenzymes

13. Enzymatic Syntheses of Peptide Bonds BY H. BORSOOK

149 151 155 162 163 163 173

I. Thermodynamic Considerations 174 II. Classification of Enzymatic Peptide Syntheses According to the Sign and Magnitude of t h e Free Energy Change (— AF) 182 III. Peptide Syntheses Where — AF Is Negative and Large: Coupled with High-Energy Phosphate 198 IV. Mechanism of Amino Acid Incorporation into Proteins 203 V. Addendum (David M. Greenberg) 219 14. Purines and Pyrimidines BY M A R T I N P . SCHULMAN

I. The Purines II. The Pyrimidines 15.

Nucleotides and Nucleosides BY L E O N A. H E P P E L

I. II. III. IV. V.

Nucleosides Nucleotides Deamination of Nucleosides and Nucleotides The Enzymatic Splitting of Certain Coenzyme Nucleotides The Enzymatic Synthesis of Certain Coenzyme Nucleotides

16. Metabolism of Heme and Chlorophyll BY S. GRANICK

223

224 248 263

264 270 277 279 280 287

I. Introduction 288 II. Compounds of t h e Biosynthetic Chain Leading to t h e Formation of Protoporphyrin 295 III. Biochemical Lesions in t h e Porphyrin Metabolism of H u m a n Beings. . 308 IV. Tracer Studies of Protoporphyrin Biosynthesis 311 V. The Iron Branch of the Biosynthetic Chain a n d Some General Proper­ ties of Heme Compounds 319 VI. T h e Magnesium Branch and the Biosynthesis of Chlorophyll 329 VII. Decomposition of Iron Protoporphyrin to Bile Pigments 331 AUTHOR I N D E X

343

SUBJECT I N D E X

359

E R R A T A : VOLUME I

384

LIST OF ABBREVIATIONS AND SYMBOLS adenosine-5-phosphate adenosine diphosphate adenosine triphosphate diphosphopyridine nucleotide mase, coenzyme I) reduced form of above DPN red , D P N H TPN OI , TPN+ TPN red , T P N H triphosphopyridine nucleotide zyme II) flavin adenine dinucleotide FAD coenzyme A CoA, CoASH sulfhydryl compounds RSH SH S

AMP ADP ATP DPN OI , DPN+, D P N

L \

/

SH Kcal.

,L

/I

\ S

Kj. 'ΐθ2? Vacetatej ClC.

AF AF° RNA PNA Pi PPi PPPi

(cozy(coen­

lipoic acid, thioctic acid kilocalories kilojoules metabolic quotients expressed in μΐ. metabolite/mg. dry weight/hr. increment of free energy standard free energy change ribose nucleic acid pentose nucleic acid, desoxyribose nu­ cleic acid inorganic phosphate inorganic pyrophosphate inorganic triphosphate

Contents of Volume I 1. Free Energy and Metabolism b y ARTHUR B . PARDEE 2. Enzymes in Metabolic Sequences b y DAVID E . G R E E N 3. Glycolysis by P . K. STUMPF

4. The Tricarboxylic Acid Cycle by H. A. K R E B S 5. Other Pathways of Carbohydrate Metabolism by SEYMOUR S. COHEN 6. Biosynthesis of Complex Saccharides by W. Z. HASSID 7. F a t Metabolism and Acetoacetate Formation by I. L. CHAIKOFF a n d G. W. BROWN, J R .

8. Sterol a n d Steroid Metabolism by D . K. FUKUSHIMA a n d R O B E R T S. R O S E N F E L D

Author Index Subject Index

CHAPTER 9

Nitrogen Metabolism of Amino Acids P. P. C O H E N Department

of Physiological

Chemistry,

University of Wisconsin,

I. Scope I I . Deamination 1. Oxidative a. L-Amino Acid Oxidases (Dehydrogenases) b. D-Amino Acid Oxidases (Dehydrogenases) c. Specific Amino Acid Oxidases (Dehydrogenases) d. Amine Oxidases (Dehydrogenases) 2. Nonoxidative Deamination a. Amino Acid Deaminases b . Cysteine and Homocysteine Desulfhydrases c. Other Nonoxidative Deaminases I I I . Deamidation a. Amino Acid Amidases IV. Transamination V. Amino Acid Racemases VI. Urea Synthesis VII. Summary Remarks Addendum

Madison,

Wisconsin Page 1 1 1 4 11 14 16 22 22 24 25 26 26 29 36 36 43 45

I. SCOPE This chapter is concerned primarily with the metabolic reactions in­ volved in transforming or transferring the amino, amine, and amide nitrogen moiety of amino acids, amino acid amides, and amines. Although reference may be made to ill-defined enzyme systems, emphasis will be placed on those systems which have been at least partially purified and reasonably well studied. Recent reviews of certain aspects of this subject have appeared.1>2'3 II. DEAMINATION 1. OXIDATIVE

A large number of enzyme systems have been recorded in the literature in support of the concept that amino acids as a group are oxidatively 1 2

3

Krebs, H . A., Enzymes 2 (pt. 1), 499-535 (1951). Tarver, H., In D . M. Greenberg, Amino Acids and Proteins, pp. 769-908, Charles C Thomas, Springfield, Illinois, 1951. Tarver, H., Ann. Rev. Biochem. 2 1 , 301 (1952). 1

2

P. P. COHEN

deaminated in accordance with the following general over-all reaction: RCHCOOH + O - -> RCCOOH + NH:i I II NH, O

(1)

In a general way these enzyme systems may be classified as follows: 1. L-Amino acid oxidases (or dehydrogenases). 2. D-Amino acid oxidases (or dehydrogenases). 3. Specific amino acid oxidases (or dehydrogenases). This classification indicates that the L- and D-amino acid oxidases have broad substrate specificities, whereas the other enzymes have a narrower specificity. For the purpose of simplicity the above classification will be used. The amino acid oxidase systems may be subclassified according to the nature of the hydrogen acceptor under two categories, aerobic and anaerobic. The former have been more commonly referred to as amino acid oxidases, whereas the latter are frequently referred to as dehydrogenases. The term oxidase is usually considered to represent a system in which oxygen is an obligatory hydrogen acceptor. The term dehydrogenase is used for that enzymatic component of an oxidative reaction system which is concerned with the activation of the substrate and its dehydrogenation. Thus, the distinction between the succinoxidase system and succinic dehydrogenase is clearly recognized under this terminology. In the case of the amino acid oxidative enzymes, the distinction is somewhat more difficult to establish, owing chiefly to the fact that most of the measure­ ments of activity have been based on oxygen consumption and thus it is not certain in most instances that the rate-limiting reaction is the dehy­ drogenase step. It is clear from the formulation of the mechanism of the oxidation of amino acids and amines that the primary reaction is that of a dehydrogenation and that the ultimate fate of the hydrogen (or electrons) is determined either by the autoxidizability of the primary acceptor, i.e., coenzyme, or by its participation in the chain of hydrogen or electron transport systems of the cell. The term oxidase will be retained in referring to most of the amino acid oxidizing enzyme systems be­ cause of precedent and for the reasons given above. The term dehydro­ genase will be included parenthetically in those instances in which in the author's opinion the term is preferable to the more commonly used term, oxidase. It is beyond the scope of this discussion to reclassify and rename the many enzymes mentioned. It should be clear to the reader, however, that there is a great need for a more systematic basis for nomenclature.

NITROGEN

METABOLISM

OF ΑΜΙΝΟ

3

ACIDS

Reaction 1 should be considered as a two-step reaction as follows: RCH—COOH ;=± RC—COOH + 2H' NHRi

(2)

NRi

RC—COOH ; = L ± RCOCOOH + N H ^ ||

NR!

(3)

-H2O

Thus, the primary oxidative step is a dehydrogenation (and thus the term dehydrogenase is preferable) to form an imino acid which in turn is nonenzymatically hydrolyzed to form the keto acid and ammonia or a deriva­ tive thereof. In the formulation of reactions 2 and 3, Ri is intended to represent either a hydrogen atom or a substituent, such as an alkyl group. If Ri is a hydrogen atom, ammonia is formed; if Ri is an alkyl group, an alkyl amine is formed. The aerobic oxidases, as far as they have been investigated, are all flavoproteins, and the hydrogen acceptor for this group is molecular oxygen. The nature of the oxidative steps following reaction 2 may be represented as follows: R

R

1

N

sea R

H

v

^ 1

II 0

H

H

R

C=0 /NH

.

H

X^ II 0

C=0 1

(4)

+ H202

(5)

C II 0

c

II 0

T h e net effect of reactions 4 a n d 5 is: 2H + O 2 - + H 2 0 2

(6)

Thus, the aerobic oxidases form hydrogen peroxide. In the absence of catalase, the peroxide formed may react with the keto acid as follows: RCOCOOH + H202 -> RCOOH + C02 + H20

(7)

The over-all formulation of amino acid oxidation by an aerobic oxidase in the absence of catalase is as follows:

4

P. P.

COHEN

RCH—COOH + 0 2 -+ RCOOH + NH2Ri + C02 NHR!

(8)

In the presence of catalase which decomposes the hydrogen peroxide, the over-all reaction is: RCH—COOH + y202 -> RCOCOOH + N H ^ I

(9)

NHRi

Keilin and Hartree 4 demonstrated the coupling of D-amino acid oxidase with ethyl alcohol in the presence of catalase and were able to show that in the presence of catalase ethyl alcohol was oxidized in preference to the α-keto acid to form acetaldehyde as follows: catalase

CH3CH2OH + H202

> CH3CHO + 2H20

(10)

The total coupled system is thus formulated as follows: RCH—COOH + 0 2 + CH3CH2OH -> RCOCOOH + NH2R1 + CH3CHO + H20 NHR!

(11)

and exhibits the theoretical oxygen uptake without destruction of the α-keto acid. The anaerobic oxidases (dehydrogenases) represent enzymes which have as coenzymes nonautoxidizable hydrogen acceptors and thus are linked with the cytochrome system. These systems do not produce hydro­ gen peroxide. a. h-Amino Acid Oxidases (Dehydrogenases) Three L-amino acid oxidases have been partially purified and studied, all from such widely different sources as snake venom, rat kidney, and molds. (1) Snake Venom or Ophio L-Amino Acid Oxidase (Dehydrogenase). Zeller and Maritz (see5) described an enzyme originally discovered in snake venoms and later shown to be present in various tissues of both venomous and nonvenomous snakes, which they termed ophio L-amino acid oxidase. 4

Keilin, D., and Hartree, E. F., Proc. Roy. Soc. (London) 119B, 114 (1936).

NITROGEN METABOLISM OF AMINO ACIDS

5

The enzyme is widely distributed in a large number of snake venoms. 5 The L-amino acid oxidase from moccasin venom has been highly purified and shown to be a homogeneous protein with a molecular weight of 62,000.6 It has a turnover number of 3100, a Q0i of 68,400, and a pH optimum of 7-7.5, with a sharp decline in activity on either side.6 The prosthetic group has been shown to be FAD, which is present in a concentration of 1 mole per mole of protein. 7 (a) Specificity. Early specificity studies have been reviewed by Zeller5 and more recently carried out by Bender and Krebs. 8 In Table I are listed the reactivities of different amino acids with four different amino acid oxidases, as reported by Bender and Krebs. 8 In general, ophio-L-amino acid oxidases, although showing variation in substrate specificity from one species to another, are highly specific for L-amino acids. Zeller5 has summarized the specificity requirements as follows: "The substrate must possess a free carboxyl group, an unsubstituted α-amino group and an organic radical. A second amino or carboxyl group inhibits a substance otherwise suitable as a substrate for the enzyme." A study of twenty-two amino acid analogs using cottonmouth snake venom 9 revealed that all were oxidized with the exception of two which had a tertiary α-carbon. A variety of ß-arylalanines was found to be oxidized at rates comparable to that of the corresponding naturally occurring amino acids, supporting the generalization of Zeller5 that the ß-group exerted relatively little influence on the substrate activity of different amino acids. (b) Mechanism of Action. With the clear demonstration by Singer and Kearney 7 that FAD is the coenzyme of ophio-L-amino acid oxidase, the oxidative steps are those represented by reactions 2, 4, and 5. In the absence of catalase, the over-all reaction is that shown by reaction 8; in the presence of catalase, it is that shown by reaction 9. The reaction pro­ ceeds more rapidly in pure oxygen than in air.6 On the basis of pH activity data and reversible inactivation by phos­ phate and other ions, Kearney and Singer 10-12 have suggested that one of the active groups of the enzyme is an ionizable imidazole. 5

Zeller, E. A., Advances in Enzymol. 8, 459 (1948). Singer, T. P., and Kearney, E. B., Arch. Biochem. 29, 190 (1950). 7 Singer, T. P., and Kearney, E. B., Arch. Biochem. 27, 348 (1950). 8 Bender, A. E., and Krebs, H. A., Biochem. J. (London) 46, 210 (1950). 9 Frieden, K, Hsu, L. T., and Dittmer, K., J. Biol. Chem. 192, 425 (1951). 10 Kearney, E. B., and Singer, T. P., Arch. Biochem. 33, 377 (1951). 11 Kearney, E. B., and Singer, T. P., Arch. Biochem. 33, 397 (1951). 12 Kearney, E. B., and Singer, T. P., Arch. Biochem. 33, 414 (1951). 6

TABLE I

COMPARISON OF REACTIVITY OF AMINO ACIDS WITH D I F F E R E N T AMINO ACID OXIDASES 8

L-Amino acid oxidase of cobra venom 6-C > 8-C > 5-C

13-C rapidly oxidized 4-C slowly oxidized 5-C, 6-C, and 7-C not at­ tacked Ornithine > lysine

/3-Quinolyl (2) alanine > ß-quinolyl (4) alanine

Unclassified amino acids

Total number of amino acids oxidized out of 38 tested

Proline > methionine > serine > threonine > cystine

31

D-Amino acid oxidase of N. crassa 4-C > 5-C > 6-C > 3-C > 18-C 8-C, 11-C, 12-C not at­ tacked Leucine > isoleucine, valine

Only 13-C oxidized

7-C > 6-C > 13-C > 5-C > 4-C All oxidized

5-C > 7-C > 4-C > 6-C 13-C not attacked

Not attacked

Ornithine > lysine

Not attacked

3-C, 4-C, 11-C, 12-C, 18-C not attacked Only leucine oxidized

DimethylaminophenylTyrosine > aminophenalanine > phenylala­ ylalanine and phenyl­ nine > aminophenylalanine > dimethylalanine > tyrosine aminophenylalanine Tyrosine, aminophenylTyrosine, aminophenylalanine, dimethylaminoalanine, dimethylaminophenylalanine, e-iV-sulphenylalanine, t r y p t o fanilyllysine consumed phan, and J3-furyl(2)> 1 atom 0 alanine consumed > 1 atom 0 Histidine and e-iV-sulfanilyllysine not attacked ß-Quinolyl (4) alanine and ß-Quinolyl (4) alanine > ß-quinolyl (2) alanine /3-quinolyl (2) alanine attacked at similar rates Only methionine oxidized Serine > cystine > methionine > threonine

19

Proline and piperidine-a;carboxylic acid not at­ tacked Cystine consumed > 1 atom 0 33

p-Aminophenylalanine > jS-pyridyl (4) alanine > phenylalanine > tyrosine p-(ß-Aminoethyl)phenylalanine, p-aminophenylalanine, p-dimethylaminophenylalanine, pyridyl(4)alanine, tyro­ sine consumed > 1 atom of 0

Only methionine at­ tacked

23

COHEN

Tyrosine > aminophenylalanine > dimethylaminophenylalanine > phenylalanine Dimethylaminophenylalanine consumed > 1 atom 0 €-iV-Sulfanilyllysine not attacked

L-Amino acid oxidase of N. crassa 4-C > 5-C > 6-C > 3-C > 8-C 11-C, 12-C, 18-C not at­ tacked Leucine > isoleucine > valine

P. P.

Diaminomonocarboxylic acids Amino acids with cyclic substituent

D-Amino acid oxidase of sheep kidney 3-C > 6-C > 4-C > 5-C > 8-C 11-C, 12-C, 18-C not at­ tacked Valine > isoleucine > leucine

6

Type of amino acid Straight-chain ali­ phatic monoaminomonocarboxylic acids Branched-chain monoaminomonocarboxylic acids Aliphatic monoaminodicarboxylic acids

NITROGEN METABOLISM OF AMINO ACIDS

7

(c) Inhibitors. Zeller and co-workers 5 ' 13-17 have studied a variety of carboxylic and sulfonic acids as possible inhibitors of ophio-L-amino acid oxidase. Benzoic, salicylic, mandelic, iodoacetic, some aliphatic and aromatic sulfonic acids, and sulfonamides inhibit competitively. Carbonyl reagents such as hydroxylamine and semicarbazide are reported also to be competitive inhibitors. This latter effect has suggested the presence of a carbonyl group on the enzyme surface essential for substrate activation. Singer and Kearney 7 found that riboflavin analogs were potent inhibitors of ophio-L-amino acid oxidases. Riboflavin at 10~3 M inhibited 100%. Atabrine, proflavin, and alloxazine were found to be weak, whereas lumazine and dimethylumazine did not inhibit at all. The inhibition by these analogs is instantaneous and is not prevented or reversed by adding substrate or FAD. An interesting noncompetitive inhibition by di- and trivalent ions was reported by the same workers. 10 According to these investigators, ophio-L-amino acid oxidase exists as an equilibrium mixture of active and reversibly inactive enzyme. Although the rate of inactivation is not pH-dependent, the rate of reactivation is. Inactivation was enhanced by multivalent ions in high concentration and prevented by univalent ions. 11 Of importance was the finding that consistently higher Qo2 values were obtained with ophio-L-amino acid oxidases from different species if tris(hydroxymethyl)aminomethane, imidazole, or veronal buffers (or no buffer at all) were used than with 0.04 M phosphate buffer at pH 7.2. This observation necessitates a reassessment of the values for enzyme activity previously reported, most of which were obtained using a phosphate buffer. The reversible spontaneous inactivation has been interpreted as being due to the enzyme being in equilibrium with an inactive form, the latter differing from the active form by the presence of a new acidic group, probably imidazole in nature. On the basis of the nature of the pH activity curve and the considerations noted above, these investigators have schematically represented their interpretation of the probable changes involved. 12 (2) Rat Kidney iu-Amino Acid Oxidase (Dehydrogenase). Studies by Krebs 18 clearly indicated that different systems existed in animal tissues for the oxidation of D- and L-amino acids. However, while considerable success has been achieved in the purification of mammalian D-amino acid 13

Zeller, E. A., and Maritz, A., Helv. Chim. Ada 27, 1888 (1944). Zeller, A. E., and Maritz, A., Helv. Physiol. et Pharmacol. Ada 3, C48 (1945). 15 Zeller, E. A., and Maritz, A., Helv. Chim. Ada 28, 365 (1945). 16 Zeller, E. A., Maritz, A., and Iselin, B., Helv. Chim. Ada 28, 1615 (1945). 17 Zeller, E. A., Iselin, B., and Maritz, A., Helv. Physiol. et Pharmacol. Ada 4, 233 (1946). 18 Krebs, H. A., Biochem. J. {London) 29, 1620 (1935). 14

8

P. P.

COHEN

oxidase, this has not been so in the case of the L-amino acid oxidase. The only well-defined mammalian L-amino acid oxidase purified to date is that reported by Blanchard et αΖ.19·20 and Green et αϋ.,21·22 which was isolated from rat kidney. The purified enzyme was electrophoretically homogeneous, but two components were revealed in the ultracentrifuge. The lighter component has an approximate molecular weight of 120,000 and contains 2 moles of flavin mononucleotide (FMN) per mole of protein. The heavier component has a molecular weight in excess of 430,000 and is con­ sidered to represent a tetramer of the lighter component with 8 moles of F M N per mole. Both components have equal catalytic activity. The enzyme has a pH optimum of 10.0, a Qo2 of 52, and a turnover number of 6. The enzyme is low or absent in cat, dog, guinea pig, rabbit, pig, ox, or sheep tissues, the best source being rat liver or kidney. The limited distribution of this L-amino acid oxidase and its extremely low turnover number make it highly doubtful that this enzyme plays any major part in mammalian amino acid metabolism. (a) Specificity. All naturally occurring monoaminomonocarboxylic acids of the L-series except threonine and serine are oxidized. Neither the dicarboxylic-monoamino acids, glutamic and aspartic acids, nor the diamino acids, lysine and ornithine, are attacked. Proline and iV-monomethylamino acids are oxidized. An enigmatic feature of the purified enzyme is its ability to oxidize L-a-hydroxy acids at a significant rate. (b) Mechanism of Action. With the demonstration of F M N as the prosthetic group, it would appear that the mechanism of oxidation of L-amino acids is essentially the same as that of ophio-L-amino acid oxidase. In view of the relatively low activity of the purified enzyme, the question has been raised 23 as to whether this enzyme system represents but one component of a more complex system. Thus it is suggested that molecular oxygen is not the physiological hydrogen acceptor for this oxidase but possibly rather a cytochrome-coupled system. This hypothesis is also supported by the difference in behavior of inhibitors on the crude and purified enzymes. (c) Inhibitors. Although the oxidation by crude enzyme preparations of L-amino acids is strongly inhibited by KCN and octyl alcohol, as 19

20

21 22

23

Blanchard, M., Green, D . E., Nocito, V., and Ratner, S., J. Biol. Chem. 155, (1944). Blanchard, M., Green, D . E., Nocito, V., and Ratner, S., J. Biol. Chem. 161, (1945). Green, D . E., Nocito, V., and Ratner, S., J. Biol. Chem. 148, 461 (1943). Green, D . E., Moore, D . H., Nocito, V., and Ratner, S., / . Biol. Chem. 156, (1944). Cohen, P . P., and McGilvery, R. W., In H . A. Lardy, Respiratory Enzymes, 226-254. Burgess Publishing Co., Minneapolis, (1949).

421 583

384 pp.

NITROGEN METABOLISM

OF ΑΜΙΝΟ

ACIDS

9

originally reported by Krebs, 18 the purified rat kidney enzyme was found by Blanchard et al.20 to be unaffected by 0.002 M KCN or saturated octyl alcohol. Ammonium ions were inhibitory in high concentration (96% inhibition with 0.066 M NH4 + ). Of interest was the finding that the oxidation of lactic acid by the purified rat kidney L-amino acid oxidase was not affected by N H 4 + ions. (3) Mold li-Amino Acid Oxidase (Dehydrogenase). The presence of an L-amino acid oxidase in both the mycelium and culture fluid of Neurospora crassa was reported by Bender, Krebs, and Horowitz 24 and by Bender and Krebs. 8 I t was found that the quantity of enzyme varied from strain to strain, and further was dependent on the composition of the medium. Under identical conditions some wild types produce only D-amino acid oxidase, some produce only L-amino acid oxidase, while others form both enzymes. 24 However, Thayer and Horowitz 25 upon reinvestigation found that by suitably treating extracts to free them of endogenous amino acids, it was possible to demonstrate the presence of both the D- and L-amino acid oxidases in all strains of Neurospora tested. It was further observed that L-amino acid oxidase activity of Neurospora manifests properties of an adaptive enzyme. Burton, 26 who also found that both D- and L-amino acid oxidases were present in seventeen different strains, succeeded in obtaining a preparation of L-amino acid oxidase from Neurospora with a turnover number of 2100 moles phenylalanine per mole FAD per minute. This activity is of the same order as that reported by Singer and Kearney 6 for ophio-L-amino acid oxidase (3100) and by Warburg and Christian 29 for kidney D-amino acid oxidase (1440). The prosthetic group of the purified enzyme was shown to be FAD in a ratio of 1 g. mol. bound FAD per 11,000 g. nondialyzable nitrogen. 26 In confirmation of the findings of Thayer and Horowitz, 25 Burton also found that the yield of L-amino acid oxidase was highest when growth was limited by the biotin content of the medium. (a) Specificity. Although retaining a high substrate specificity for the L-amino acids, Neurospora L-amino acid oxidase appears to have a broader substrate specificity than the other L-amino acid oxidases studied. Thus, as can be seen from Table I, thirty-three of thirty-eight amino acids tested were active as substrates. In addition to these amino acids, the following amino acids were found to be active as substrates: canavanine, arginine, citrulline, cx,e-diaminopimelic acid, cystathionine, a-amino-hydroxy-ncaproic acid, glutamine, α-γ-diaminobutyric acid, and glycine.25,26 The different preparations used by the different investigators showed some 24

Bender, A. E., Krebs, H., and Horowitz, N. H., Biochem. J. {London) 45, xxi (1949). Thayer, P. S., and Horowitz, N. H., J. Biol. Chem,. 192, 755 (1951). 26 Burton, K., Biochem. (London) 60, 258 (1951). 25

10

P. P.

COHEN

striking variations in relative rates of oxidation of the different amino acids, although the discrepancy in part may be due to the fact that Bender and Krebs 8 and Burton'26 employed systems at pH 8.3-8.4, whereas Thayer and Horowitz 25 carried out their studies at pH 6.O. (b) Mechanism of Action. In general, the mechanism of action of Neurospora L-amino acid oxidase appears to be the same as that for ophio L-amino acid oxidase. As in the case of ophio L-amino acid oxidase, the rate of oxidation is three to five times greater in pure 0 2 than in air. 25 · 26 Ferricyanide and reducible dyes can replace 0 2 , but the reaction proceeds at a slower rate. Bender and Krebs, 8 Burton, 26 and Thayer and HoroAvitz25 all were able to demonstrate that the H2O2 formed during the course of the reaction was decomposed by the catalase present in their preparations. Coupling with ethanol according to reaction 11 was demonstrated to occur,25,26 with a resultant increase in O2 consumption. Burton 26 observed a broad plateau between pH 6 and 9.5 in the pH activity curve using phenylalanine as substrate, whereas Thayer and Horowitz, 25 using a narrower pH range of 5.6-7.6, found considerable variation in the pH optimum using different amino acids as substrates. The latter investiga­ tors interpreted their findings to mean that the state of ionization of the amino acid substrate was of importance for activity. Further support for this was pointed out 25 in the case of esterification of the carboxyl group of histidine with the resultant loss of activity. Substrate concentration studies reveal that an optimum is reached beyond which the enzyme is inhibited. A similar effect has been observed with ophio L-amino acid oxidase.16 This phenomenon has been interpreted on the basis that the substrate attaches at two points on the enzyme surface. (c) Inhibitors. As mentioned above, competitive inhibition by slowly reacting amino acids has been shown to occur.25 Purified Neurospora L-amino acid oxidase (free from catalase) was not affected by 0.01 M HCN, hydroxylamine, azide, or 0.001 M quinine sulfate; atabrine (0.01 M) inhibited 15%; crystal violet (0.001 M), 69%; and 0.002 M methylene blue, 30%. 26 In the absence of pyrophosphate buffer, CuS0 4 inhibited 6 3 % and 15% at 0.01 and 0.001 M concentrations, respectively. 26 Other mold L-amino acid oxidases have been studied using acetone powders of the mycelia of various species of Penicillium and of AspergiUus niger.27 These enzymes were for the most part insoluble and behaved in accordance with reaction 9, since they contained catalase. (4) Other L-Amino Acid Oxidases. Proteus vulgaris has been shown to contain an active L-amino acid oxidase (dehydrogenase) of the anaerobic type. 28 The enzyme is sensitive to cyanide and octyl alcohol.28 While the 27 28

Knight,, S. G., / . BacterioL 55, 401 (1948). Stumpf, P. K., and Green, D. E., / . Biol. Chem. 163, 387 (1944).

N I T K O G E N M E T A B O L I S M O F A M I N O ACIDS

11

enzyme has been prepared as a soluble cell-free preparation, the highest activity is obtained with the particulate fraction. Further purification has not been reported, and the nature of the coenzyme is not known. Since this enzyme represents an anaerobic dehydrogenase, no hydrogen peroxide is formed. The fresh bacterial suspension was capable of oxidizing twenty-two different amino acids, but the aged suspensions and cell-free extracts (prepared from the latter by ultrasonic disintegration) oxidized only eleven amino acids. The bacterial suspension oxidized all the unsubstituted monocarboxylicmonoamino, primary amino acids of the L- series with the exception of alanine and valine. b. D-Amino Acid Oxidases (Dehydrogenases) Although D-amino acids can no longer be considered "unnatural," they are, from a quantitative standpoint, nevertheless, of limited distribu­ tion in nature. This fact makes the explanation of the widespread occur­ rence of highly active D-amino acid oxidases somewhat difficult. The capriciousness of nature—or of the enzymologist—is clearly revealed in the fact that a great deal is known about the properties and mechanism of action of D-amino acid oxidases in the animal but very little about the role of D-amino acids in metabolism. (1) Mammalian-Ό-Amino Acid Oxidases (Dehydrogenases). Since the initial demonstration by Krebs 18 of the existence of a D-amino acid oxidase in mammalian liver and kidney, considerable progress has been made in the purification, mechanism of action, substrate specificity, etc., of this enzyme. The enzyme has been shown to be present in the liver and kidney of all vertebrates studied, but absent from other mammalian tissues. 18 The prosthetic group was shown to be FAD by Warburg and coworkers. 2930 Apoenzyme of approximately 70% purity has been prepared by Negelein and Brömel. 31 This degree of purity was arrived at on the basis that 1 g. mol. of pure FAD binds 100,000 g. of the purified apoenzyme. Negelein and Brömel 31 assumed by analogy with other flavoproteins that the pure protein had a molecular weight of 70,000. These investigators estimated the turnover number of their purified enzyme with D-alanine as substrate to be about 2,000, whereas Warburg and Christian 29 estimated the turnover number to be 1,440 per mole of flavin per minute. If the pro­ tein had a molecular weight of 100,000 and was assumed to be pure, the turnover number of the Negelein and Brömel preparation would be 1,400, 29 30 31

Warburg, O., and Christian, W., Biochem. Z. 298, 150 (1938). Warburg, O., Christian, W., and Griese, A., Biochem. Z. 297, 417 (1938). Negelein, E., and Brömel, H., Biochem. Z. 300, 225 (1939).

12

P. P.

COHEN

approximating that obtained by Warburg and Christian. If the value of 1,440 is accepted as correct, the protein would be approximately 97% pure. (a) Specificity. Substrate specificity studies of D-amino acid oxidase have been carried out by a number of different investigators. 4 ' 8 ' 9,32 A comparison of the relative rates of oxidation of different amino acids and by different amino acid oxidases is shown in Table I. (b) Mechanism of Action. Negelein and Brömel 31 established that under anaerobic conditions only the flavin equivalent to the added protein is reduced concomitantly with the oxidation of substrate, indicating that the prosthetic group does not dissociate and that there is no formation of hydrogen peroxide. Upon exposure of the reduced flavoprotein to oxygen, the flavin is reoxidized with the formation of hydrogen peroxide. The mechanism of action is that of other FAD oxidative enzymes and may be represented by reactions 2, 4, and 5. A theoretical treatment of the kinetics of coenzyme-linked reactions by Stadie and Zapp 33 revealed that D-amino acid oxidase has practically an identical affinity for the substrate (D-alanine) when the enzyme is in either the Zwitter or anion forms, the respective Km values being 8.7 X 10~3 and 9.2 X 10~3 M. Other estimates 34 - 3 of Ks are 5, and 6.1-6.6 X 10~3 M. The i£fiavin-protein value 34 is of the order of 2.5 X 10~7 M. Stadie and Zapp 33 found that the flavoprotein may be treated as an acid with a pK of 7.9, so at pH 7.9 and 9 X 10~3 M alanine, the enzyme is half dissociated as an acid and both forms half combined with the substrate. (c) Inhibitors. Benzoic acid is a remarkably potent competitive in­ hibitor of mammalian D-amino acid oxidase.35,36 A detailed study of benzoic acid derivatives as competitive inhibitors has been carried out by Bartlett. 36 The structural unit necessary for inhibition is the phenylcarboxylate ion. Ring substitution has a less marked effect, although in general meta substitution, particularly by a halogen, methyl, or nitro group, results in greater inhibitory activity than does substitution in other positions. Quinine, atabrine, and related substances were found by Hellerman et αΖ.34 to act as effective competitive inhibitors of FAD at low concentrations (1 X 10~3 M). More recent studies 360 showed that AMP and ADP were potent competitive inhibitors of FAD, being considerably more effective than quinine and a number of other compounds tested. Of 32

Klein, J. R., and Handler, P., / . Biol. Chem. 139, 103 (1941). Stadie, W. C , and Zapp, J. A., J. Biol Chem. 150, 165 (1943). 34 Hellerman, L., Lindsay, A., and Bovarnick, M. R., J. Biol. Chem. 163, 553 (1946). 35 Klein, J. R., and Kamin, H., J. Biol. Chem. 138, 507 (1941). 36 Bartlett, G. R., J. Am. Chem. Soc. 70, 1010 (1948). 36a Burton, K , Biochem. J. (London) 48, 458 (1951). 33

NITROGEN METABOLISM OF AMINO ACIDS

13

a series of compounds studied, it was found that AMP alone of the FAD competitors was able to protect the oxidase against thermal inactivation. Other protecting agents were FAD, substrates, and the competitive sub­ strate inhibitors, L-lecuine37 and sodium benzoate. According to Burton, 36a the site of protection by benzoate or by AMP is the same as the site of inhibition. A number of synthetic amino acid analogs, chiefly of the ß-arylalanine type, were studied by Frieden et al.9 as possible substrate inhibitors. No outstanding inhibitor was found, and in fact most of the compounds were found to be readily oxidized by the hog kidney D-amino acid oxidase preparation used. Urethane at 0.57 M inhibits 100 %.3 (#) Mold D-Amino Acid Oxidases (Dehydrogenases). Horowitz 38 ob­ served that extracts of Neurospora contained an active D-amino acid oxidase similar in its action to that of kidney. The enzyme was found to oxidize the D- forms of most of the amino acids tested. In general, activity for the different amino acids was similar to that reported later by Bender and Krebs, 8 with methionine being the most active. The Neurospora D-amino acid oxidase has a pH optimum of 8.0-8.5 and was not signifi­ cantly inhibited by cyanide (0.001 M) or iodoacetate (0.001 M). Benzoate (0.01 M), in contrast to its effect on kidney D-amino acid oxidase, did not inhibit Neurospora D-amino acid oxidase. DL-a-Amino-a-methylbutyric acid was observed to act as a competitive inhibitor, although it was not itself oxidized. According to Thayer and Horowitz, 25 the D-amino acid oxidase is present in young cultures of all strains of Neurospora examined. The presence of D-amino acid oxidase in the following molds has been reported by Emerson et αΖ.:39 Penicillium chrysogenum, notatum, and roqueforti (the latter particularly active), and Aspergillus niger. Penicil­ lium sanguineum was found to be practically free of the enzyme. These investigators were able to prepare soluble D-amino acid oxidase prepara­ tions from P. chrysogenum which showed a pH optimum at 8.5. The reac­ tion involved was that shown by 9, as deduced from balance studies. The enzyme was specific for D-amino acids and was highly active for leucine, methionine, alanine, α-aminobutyric acid, norleucine, phenylalanine, and valine. Less active as substrates were isoleucine, a-aminocaprylic acid, tryptophan, lysine, threonine, and glutamic acid. Aspartic acid and glycine were not oxidized. All attempts to purify the enzyme were unsuccessful. The authors make no mention of the nature of the prosthetic group, but it would appear that this enzyme is a flavoprotein. 37 38 39

Edlbacher, S., and Wiss, O., Helv. Chim. Ada 27, 1831 (1944). Horowitz, N . H., / . Biol. Chem. 154, 141 (1944). Emerson, R. L., Puziss, M., and Knight, S. G., Arch. Biochem. 25, 299 (1950).

14

P. P.

COHEN

c. Specific Amino Acid Oxidases (Dehydrogenases) The enzyme systems to be considered here are those which are rela­ tively specific for a single amino acid and which yield ammonia as a result of the primary oxidation. Only three systems have been investigated sufficiently to warrant inclusion at this time: glutamic acid dehydrogenase, glycine oxidase, and D-aspartic acid oxidase. (/) h-Glutamic Acid Dehydrogenase. Glutamic acid dehydrogenase is unique among the amino acid dehydrogenases because of its widespread occurrence, high specificity, and high activity. The enzyme has been re­ ported to be present in bacteria, 40,41 yeast, 42,43 plants, 43,44 and animal tissues. 45,46 The enzyme has been found in most mammalian tissues studied. The relative activity of different rat tissues for glutamic acid dehydrogenase is as follows:47 liver 100, kidney 50, brain 21, heart 10, spleen 10. The enzyme has been crystallized from beef liver by Olson and Anfinsen48 and by Strecker. 49 The former investigators estimated the molecular weight of the crystalline enzyme to be 1,000,000. (a) Specificity. Glutamic acid dehydrogenase is highly specific for glutamic acid, no other amino acid being known to react. (b) Mechanism of Action. Glutamic acid dehydrogenase is the only enzyme catalyzing the oxidation of amino acids which has a pyridine nucleotide as prosthetic group. While glutamic acid dehydrogenase prepa­ rations from yeast and E. coli are active only with TPN, 40,41,43 those from plant and animal tissues are active only with DPN, 45 with the exception of the liver enzyme, which is active with either.46-50 The ability of liver glutamic acid dehydrogenase to function equally well with either D P N or 40

Adler, E., Hellström, V., Günther, G., and von Euler, H., Hoppe-Seyler's Z. physiol. Chem. 255, 14 (1938). 41 Adler, E., Das, N . B., von Euler, H., and Heyman, U., Compt. rend. trav. lab. Carlsberg 22, 15 (1938). 42 von Euler, H., Adler, E., and Steenhoff-Eriksen, T., Hoppe-Seyler's Z. physiol. Chem. 248, 227 (1937). 43 Adler, E., Günther, G., and Everett, J. E., Hoppe-Seyler's Z. physiol. Chem. 255, 27 (1938). 44 Damodaran, M., and Nair, K. R., Biochem. J. (London) 32, 1064 (1938). 45 von Euler, H., Adler, E., Günther, G., and Das, N . B., Hoppe-Seyler's Z. physiol. Chem. 254, 61 (1938). 46 Dewan, J. G., Biochem. J. (London) 32, 1378 (1938). 47 Copenhaver, J. H., Jr., McShan, W. H., and Meyer, R. K., J. Biol. Chem. 183, 73 (1950). 48 Olson, J. A., and Anfinsen, C. B., J. Biol. Chem. 197, 67 (1952); ibid. 202, 841 (1953). 49 Strecker, H. J., Arch. Biochem. and Biophys. 32, 448 (1951); ibid. 46, 128 (1953). 50 Mehler, A. H., Kornberg, A., Grisolia, S., and Ochoa, S., J. Biol. Chem. 174, 961 (1948).

NITROGEN METABOLISM OF ΑΜΙΝΟ ACIDS

15

T P N can be demonstrated spectrophotometrically 50 but not manometrically. 47 Thus Copenhaver et αϋ.47 found that T P N was only one-tenth as active as D P N with fortified homogenates measuring 0 2 uptake. Thus, in the latter system there is a limited ability for reoxidation of T P N by TPN-cytochrome c reductase. The oxidation of glutamic acid by glutamic acid dehydrogenase may be represented as follows: COOH CH 2 | CH 2

I

CHNH2 COOH

COOH +

DPN or TPN DPNH + H CH 2 .or + | TPNH + H+ CH 2

I

C=NH COOH

COOH +H20 CH 2 + NH 3 | -H 2 0 CH 2

Ϊ = ±

I

(12)

CO COOH

With crystalline glutamic acid dehydrogenase the turnover numbers (moles of D P N reduced or of D P N H oxidized per minute per 100,000 g. of enzyme at 25°C.) were 290 and 3000 respectively. 48 Chance and Smith 51 have reported the following unpublished information about the crystalline enzyme of Olson and Anfinsen. The Km values for D P N and DPN.2H are of the same order (10~4 M) as those for glutamate and a-ketoglutarate (10~3 M). The equilibrium constant for the reaction L-glutamate- + DPN+ + H 2 0 —-=± DPNH + H+ + a-ketoglutarate= + NH 4 + (13)

is 1.4 X 10~13 at 0 ° C , neglecting the concentration of H 2 0 . Addition of glutamic dehydrogenase to D P N H under conditions similar to those em­ ployed by Theorell and Chance 52 resulted in no shift of the spectrum. The ratio of the specific extinction coefficient at 279 ηΐμ to that at 260 ηΐμ for ten different crystalline preparations was found to be 1.6, which indicates the absence of more than traces of nucleic acids or nucleotides. (c) Inhibitors. The presence of an SH— group in L-glutamic. acid dehydrogenase is indicated from the studies of Singer and Barron, 53 who observed complete inhibition with p-chloromercuribenzoate (5 X 10~4 M). Using approximately the same concentration of inhibitor, it was found that the crystalline enzyme was inhibited 50 %.51 Of the dicarboxylic acids which act as competitive inhibitors, aspartic acid46 was found to inhibit 55% at 0.06 M. Of a large number of glutamic acid analogs tested, no highly active competitive inhibitor was found with the crystalline glutamic acid dehydrogenase. (Personal communication from Dr. C. B. 51

Chance, B., and Smith, L., Ann. Rev. Biochem. 21, 716 (1952). Theorell, H., and Chance, B., Ada Chem. Scand. 5, 1127 (1951). 63 Singer, T. P., and Barron, E. S. G., J. Biol. Chem. 157, 241 (1945). 52

16

P. P.

COHEN

Anfinsen.) DL-a-Methylglutamic acid was found to be active neither as a substrate nor as an inhibitor with the crystalline enzyme. 54 (2) Glycine Oxidase. Ratner, Nocito, and Green55 have partially puri­ fied an apoenzyme from pig kidney which upon addition of FAD oxidizes glycine or sarcosine according to reaction 9 since their preparation con­ tained catalase. The preparation contained D-amino acid oxidase, but it was shown that the enzyme systems were different. The preparation had a Michaelis constant of the order of 0.04 M with a pH optimum of 8.3; at pH 7.0, the enzyme activity dropped to one-eighth that at the optimum pH. The enzyme was found to be present in the kidney and liver of all the following animals studied: cat, dog, lamb, ox, rat (liver only), pig, human beings, and rabbit. The enzyme does not attack iV-dimethylglycine, phenylglycine, p-aminophenylglycine, creatine, or peptides of glucine. (3) Ό-Aspartic Acid Oxidase. Still et al.b6 reported that rabbit kidney and liver contain a soluble enzyme which catalyzes the aerobic oxidation of D-aspartate to oxalacetate plus N H 3 with the formation of hydrogen peroxide. In a later study by Still and Sperling57 the D-aspartic acid oxidase was resolved and reactivated by the addition of FAD. The puri­ fied enzyme showed about one-sixth the activity with D-glutamate; this, according to these workers, is best explained by the presence of a D-glutamic acid oxidase. The activity of D-aspartic acid oxidase is higher than that of D-amino acid oxidase in rabbit kidney and liver, and they are of the same order of activity in pig kidney. In contrast to pig kidney D-amino acid oxidase, which is inhibited by benzoic acid, the D-aspartic acid oxidase was unaffected. These findings represent the only reported instances of a specific D-amino acid oxidase. d. Amine Oxidases (Dehydrogenases) The amine oxidases are usually considered under two categories, monoamine and diamine oxidases. (See Zeller58 for a recent review of this subject.) Both systems are considered to be represented by the following formulations: RCH2NH2 + 0 2 -> RCH^NH + H202 (14) RCH^NH + H20 -> RCHO + NH3 (15) In the presence of catalase, the over-all reaction would be represented as: RCH 2 NH 2 + H 0 2 - > RCHO + NH 3 (16) Lichtenstein, N., Ross, H. E., and Cohen, P. P., J. Biol. Chem. 201, 117 (1953). 55 Ratner, S., Nocito, V., and Green, D. E., J. Biol. Chem. 152, 119 (1944). 56 Still, J. L., Buell, M. V., Knox, W. E., and Green, D. E., J. Biol. Chem. 179, 831 (1949). 57 Still, J. L., and Sperling, E., J. Biol. Chem. 182, 585 (1950). 58 Zeller, E. A., Enzymes 2 (pt. 1), 536-558 (1951). 54

NITROGEN METABOLISM OF ΑΜΙΝΟ ACIDS

17

(1) Monoamine Oxidases (Dehydrogenases). Studies of monoamine oxidase distribution 58-61 reveal the enzyme to be present in all vertebrates and in many invertebrates. In all animals studied liver, kidney (except the rat), and intestine showed the highest activity. The following organs were also active: pancreas, heart, lungs, spleen, brain, thyroid, testicle, uterus, adrenal, and thymus. In the human being, placenta, 6263 prostate and seminal vesicles were found to have activity, but sperm plasma, blood plasma, and erythrocytes 64 are devoid of monoamine oxidase. Human normal and pathologic tissues have been studied for monoamine oxidase activity. 640 · 65 In rat liver, monoamine oxidase has been shown to be present pre­ dominantly in the mitochondrial fraction. 66 · 67 Recent studies 68 ' 69 reveal that plant tissues are active in oxidizing amines. In a study with pea and lupin seedlings and leaves of red clover,69 the reaction was shown to be that formulated in reactions 14 and 15. (a) Specificity. The substrate specificity has been studied by a num­ ber of investigators. 59 · 70-77 Blaschko, Richter, and Schlossmann59 pre­ sented strong evidence to support the view that the previously reported tyramine oxidase,78 epinephrine oxidase,59 and aliphatic amine oxidase79 were one and the same enzyme. Thus, the different amine oxidases were shown to be similar in respect to: 59

Blaschko, H., Richter, D., and Schlossmann, H., Biochem. J. (London) 31, 2187 (1937). 60 Bhagvat, K., Blaschko, H., and Richter, D., Biochem. J. (London) 33, 1338 (1939). 61 Pugh, C. E. M., and Quastel, J. H., Biochem. J. (London) 31, 286 (1937). 62 Luschinsky, H . L., and Singher, H . O., Arch. Biochem. 19, 95 (1945). 63 Thompson, R. H., and Tickner, A., Biochem. J. (London) 45, 125 (1949). 64 Zeller, E. A., and Joel, C. A., Helv. Chim. Ada 24, 968 (1941). 64a Birkhäuser, H., Helv. Chim. Ada 23, 1071 (1940). 65 Langemann, H., Helv. Physiol. et Pharmacol. Ada 2, 367 (1944). 66 Cotzias, G. C , and Dole, V. P., Proc. Soc. Exptl. Biol. Med. 78, 157 (1951). 67 Hawkins, J., Biochem. J. (London) 50, 577 (1952). 68 Werle, E., and Roewer, F., Biochem. Z. 320, 298 (1950). 69 Kenten, R. H., and Mann, P . J. G., Biochem. J. (London) 50, 360 (1952). 70 Richter, D., Biochem. J. (London) 31, 2022 (1937). 71 Kohn, H . I., Biochem. J. (London) 31, 1693 (1937). 72 Blaschko, H., Richter, D., and Schlossmann, H., J. Physiol. (London) 90, 1 (1937). 73 Beyer, K. H., J. Pharmacol. Exptl. Therap. 71, 151 (1941). 74 Alles, G. A., and Heegaard, E. V., / . Biol. Chem. 147, 487 (1943). 75 Snyder, F . H., Goetze, H., and Oberst, F . W., / . Pharmacol. Exptl. Therap. 86, 145 (1946). 76 Snyder, F . H., and Oberst, F . W., J. Pharmacol. Exptl. Therap. 87, 389 (1946). 77 Blaschko, H., and Duthie, R., Biochem. J. (London) 39, 478 (1945). 78 Hare, M. L. C., Biochem. J. (London) 22, 968 (1928). 79 Pugh, C. E . M., and Quastel, J. H., Biochem. J. (London) 31, 2306 (1937).

18

P. P. COHEN

1. The chemical reactions catalyzed. 2. Their distribution in the animal kingdom. 3. The effect of inhibitors. 4. The competition between the substrates. However, Alles and Heegaard, 74 using purified liver preparations from different species, have reported that their data, in addition to that of others, make it questionable that a single enzyme is involved (see Table II). TABLE II L I V E R E X T R A C T ; M A X I M U M OXIDATION R A T E S ( P E R C E N T ) R E L A T I V E TO PHENETHYLAMINE74

(Figures in parentheses are Michaelis constants (Km), M X 10 - 4 ) Guinea pig

Cat

Cattle

0 100 140 80 70 10 100 30 30 120 190 200 160 70 10 200 180 40

20 90 110 90 100 100 100 90 55 105 110 120 80 40 45 80 30 20

5 110 85 85 85 130 100 105 5 65 95 130 85 5 80 85 65 5

15

40

20

5

25

40

20

5

Rabbit Ethylamine Butylamine Amylamine Hexylamine Heptylamine Benzylamine Phenethylamine Phenpropylamine Phene thanolam ine Phenethylmethylamine 3-Hydroxyphenethylamine 4-Hydroxyphenethylamine (tyramine) 4-Hydroxyphenethylmethylamine 4-Hydroxyphenethanolmethylamine (synephrine) 4-Hydroxyphenethyldimethylamine (hordenine) 3,4-Dihydroxyphenethylamine (hydroxytyramine) 3,4-Dihydroxyphenethylmethylamine (epinine) 3,4-Dihydroxyphenethanolamine (arterenol) 3,4-Dihydroxy phenethanolmethylamine (DL-epinephrine) L-3,4-Dihydroxyphenethanolmethylamine (epinephrine)

0 50 110 120 130 30 100 110 30 105 70 90 65 25 30 65 65 25

(8) (10) (13) (6.4) (16) (2.6) (15)

(29)

The effect of chemical structure on reactivity of amines may be sum­ marized as follows:59,74 7 7 · 8 0 - 8 1 1. Secondary methylamines of the type R—CH 2 —NHCH 3 , including epinephrine, are oxidized to form methylamine in place of ammonia; tertiary amines, RCH 2 —N(CH 3 )2, such as hordenine, react more slowly to form dimethylamine. 80 81

Härtung, λΥ. Η., Ann. Rev. Biochem. 15, 593 (1946). Beyer, K. H., and Morrison, H. S., Ind. Eng. Chem. 37, 143 (1945).

NITROGEN METABOLISM OF ΑΜΙΝΟ ACIDS

19

2. Aliphatic amines are deaminated more rapidly as the hydrocarbon chain is lengthened; branched-chain compounds of the type RR1CHCH2NH 2 react more slowly than the straight-chain isomers. 3. Secondary carbinamines of the type RRi—CH NH 2 are completely inactive, but inhibit monoamine oxidase activity. 4. Diamines of the type NH 2 —(CH 2 ) n —NH 2 are not oxidized, except those of 14-, 16-, and 18-carbon chain length. 5. Substitution of the benzene ring in phenylethylamine results in slight to marked changes in deamination rate by monoamine oxidase, depending on the nature and position of the substituent group. A comparative study of the oxidation of a series of amines by liver preparations from different species and some Michaelis constants are shown in Table II. Kinetic and inhibitor specificity studies of monoamine oxidase systems have been reported. 74,82 (b) Mechanism of Action. Although diamine oxidase has been re­ ported to be a flavoprotein (see discussion below), there have been no suitable preparations of purified monoamine oxidase which would permit characterization of the prosthetic group. Hawkins 83 found that the mono­ amine oxidase activity of livers from rats deficient in riboflavin was only one-half that of livers from normal animals. However, giving riboflavin to the deficient animals resulted in no restoration of activity unless inositol was added in addition to riboflavin. (c) Inhibitors. Many amines which are not active as substrates for monoamine oxidase, such as the α-methylated amines, are effective inhibitors 59 · 82 (for example, ephedrine). Blaschko and Duthie 77 found that monoamidines, diamidines, mono- and diguanidines, and diisothiourea derivatives inhibit strongly, some in concentrations as low as 10 - 5 M. Monoisothiourea derivatives are also potent inhibitors. 84 The enzyme apparently contains active free —SH groups, since it is inhibited by reagents which combine with or oxidize such groups. 53,85 (2) Diamine Oxidases (Dehydrogenases). As in the case of monoamine oxidases, there probably exist several diamine oxidases. Although the term histaminase has appeared in the literature for a long time, 86 and this enzyme has been claimed to be different from diamine oxidase, particu­ larly by Kapeller-Adler,87 the term diamine oxidase will be used in this discussion to include histaminase. The subject has been reviewed recently by Zeller.58 82

Heegaard, E. V., and Alles, G. A., J. Biol. Chem. 147, 505 (1943). Hawkins, J., Biochem. J. (London) 51, 399 (1952). 84 Fastier, F. N., and Hawkins, J., Brit. J. Pharmacol. 6, 256 (1951). 85 Friedenwald, J. S., and Herrmann, H., J. Biol. Chem. 146, 411 (1942). 86 Best, C. H., and McHenry, E. W., / . Physiol. (London) 70, 349 (1930). 87 Kapeller-Adler, R., Biochem. J. (London) 48, 99 (1951). 83

20

P.

P.

COHEN

Diamine oxidase is widespread in nature, having been found in animal and plant tissues and microorganisms. 88 The distribution of diamine oxidase (histaminase) in tissues of animals is shown in Table III. In TABLE III D I S T R I B U T I O N OF D I A M I N E OXIDASE ( H I S T A M I N A S E ) IN T I S S U E S OF ANIMALS 8 9

(Units per gram wet tissue)

Rabbit Guinea pig Rat Mouse Pig Calf

Liver

Spleen

Intestine (mucosa)

Lung

Heart

6.53 1.88 0.30 0.82 2.30 0.97

1.34 0.44 0 0.74

3.35 1.54 3.00 3.06"

21.2 0.60 8.25 1.17

2.52 1.30 2.52 0.73

Kidneycortex Brain 4.80 0.73 0.93 1.30 5.00 0.62

0 0 0.72 0.18

° Whole small intestine analyzed.

animal tissues (liver) most of the activity appears to be in the large intracellular participate fraction (mitochondria). 89 Of interest is the finding that there is a marked increase in diamine oxidase activity of human plasma during pregnancy which falls off abruptly post partum. 8 7 , 9 0 - 9 3 This has been ascribed to a high diamine oxidase activity of placental tissue. Diamine oxidase has recently been purified some 250-fold from hog kidney acetone powder by Tabor. 94 Using the purified enzyme it was possible to establish the nature of the end products with several sub­ strates. The formulation of the reaction as in 14 and 15 was confirmed. The highly purified preparations were white or gray and only occasionally showed a faint yellow tinge. The author makes no mention of the nature of the prosthetic group, but the absence of a yellow color would suggest that the purified enzyme is not a flavoprotein, as has been previously 88

Zeller, E. A., Advances in Enzymol. 2, 93 (1942). Cotzias, G. C , and Dole, V. P., / . Biol. Chem. 196, 235 (1952). 90 Zeller, E. A., Helv. Chim. Ada 23, 1509 (1940). 91 Kapeller-Adler, R., Biochem. J. (London) 38, 270 (1944). 92 Ahlmark, A., Ada Physiol. Scand. 9, Suppl. 28 (1944). 93 Anrep, G. V., Barsoum, G. S., and Ibrahim, A., J. Physiol. (London) 106, 379 (1947). 94 Tabor, H., / . Biol. Chem. 188, 125 (1951). 95 Swedin, B., Ada Med. Scand. 114, 210 (1943). 96 Swedin, B., Nord. Med. 17, 513 (1943). 97 Swedin, B., Arkiv. Kemi. Mineralogi Geol. 17A, 1 (1944). 97a Zeller, E. A., Stem, R., and Wenk, M., Helv. Chim. Ada 23, 3 (1940). 89

NITROGEN METABOLISM OF AMINO ACIDS

21

suggested by Swedin, 95-97 and Zeller and collaborators 970 and KapellerAdler.98 (a) Specificity. A partial list of active substrates for diamine oxidase is given in Table IV. In general simple diamines are readily oxidized, the rate of oxidation increasing as the number of carbon atoms increased from 2 to 5 and then decreasing. Peculiarly enough, diamines with 14 to 18 carbon atoms are not oxidized by diamine oxidase but by monoamine oxidase. Blaschko and Duthie 77 interpret this finding as being due to the T A B L E IV SUBSTRATES ACTIVE W I T H D I A M I N E

Ethylenediamine Trimethylenediamine Putrescine Cadaverine Spermidine

Spermidine homologs

Spermine Agmatine Histamine

OXIDASE 8 8

H2N(CH2)2NH2 H2N(CH2)3NH2 H 2 N (CH 2 ) 4 N H 2 H 2 N (CH 2 ) 5 N H 2 NH2(CH2)3NH(CH2)4NH2 NH2(CH2)3NH(CH2)3NH2 NH2(CH2)3NH(CH2)6NH2 NH2(CH2)4NH(CH2)4NH2 NH2(CH2)4NH4(CH2)5NH2 NH2(CH2)6NH(CH2)6NH2 NH2(CH2)3NH(CH2)4NH(CH2)3NH2 NH2C(NH)NH2(CH2)4NH2 N C-CH2—CH2—NH2

II

HC

II

\ / NH

CH

interference of the second amino group of short-chain diamines (contain­ ing up to 8 carbon atoms) with the affinity for monoamine oxidase. On the other hand, with diamines of chain length of 14 carbon atoms and above, the distance of the second amino group is such that it cannot affect the monoamine oxidase. Conversely, diamine oxidase appears to require as active substrates diamines of relatively short chain length, that is, up to 8 carbon atoms. I t is apparent from Table IV that one of the amino groups may be replaced by other basic groups such as amidine, pyridine, imidazole, etc. As is the case with monoamine oxidases, substitution of the α-hydrogen, and to some extent the ß-hydrogen, results in an inactive substrate which may actually inhibit. Substrate specificity varies con­ siderably with the source of enzyme and in particular when plant and animal preparations are compared. Recent studies by Renten and Mann 69 98

Kapeller-Adler, R., Biochem. J. (London) 44, 70 (1949).

22

P. P.

COHEN

indicate that in plant tissues the same enzyme may be involved in the oxidation of mono- and diamines. (b) Inhibitors. A large number of compounds have been studied as possible inhibitors of diamine oxidase, as a result of the finding that the so-called antihistamine drugs inhibit diamine oxidase. Ammonia, 86 aliphatic monoamines," ephedrine, 97 and choline" are weak inhibitors, whereas guanidines, 100 imidazole, 101 basic dyes such as pyocyanine, 102 methylene blue, 103 and toluylene blue,102 and diamines such as thiamine, 104 pyridoxamine, 104 piperazine, 104 and diamidines 105 (such as 4,4 ; -diamidinostilbene and its dimethyl derivative) are potent competitive inhibitors. Streptomycin and dihydrostreptomycin and diguanidine derivates have been reported to be powerful inhibitors of bacterial diamine oxidase.106 In addition to the long list of competitive inhibitors, diamine oxidase is inhibited strongly by carbonyl reagents such as bisulfite, semicarbazide, hydroxylamine, etc., and by cyanide. The possibility exists that this type of inhibition is due to blocking of an essential carbonyl group in the prosthetic group of the enzyme. Zeller58 has depicted a diamine oxidase model which accounts in part for the specificity and inhibition of the enzyme. The possible biological significance of the amine oxidase systems has been discussed by Zeller.58 2. NONOXIDATIVE DEAMINATION

a. Amino Acid Deaminases (1) Serine, Threonine, and Homoserine Dehydrases. This group of enzymes catalyzes a nonoxidative deamination reaction resulting from a primary dehydration of the substrate. Serine, threonine, and homoserine have been shown to be deaminated by bacteria 107-109 and animal tissues diver) 99

10

7,109a,110

Danforth, D. N., Proc. Soc. Exptl. Biol. Med. 40, 319 (1939). Blaschko, H., J. Physiol. (London) 95, 30 P (1939). 101 Zeller, E. A., Helv. Chim. Ada 23, 1418 (1940). 102 Zeller, E. A., Helv. Chim. Ada 21, 1645 (1938). 103 McHenry, E. W., and Gavin, G., Biochem. J. (London) 29, 622 (1935). 104 Zeller, E. A., Helv. Chim. Ada 24, 539 (1941). 105 Blaschko, H., Fastier, F . N, and Wajda, I., Biochem. J. (London) 49, 250 (1951). 106 Zeller, E. A., Owen, C. A., Jr., and Karlson, A. G., / . Biol. Chem. 138, 623 (1951). 107 Chargaff, E., and Sprinson, D . B., J. Biol. Chem. 151, 273 (1943). 108 Wood, W. A., and Gunsalus, I. C., J. Biol. Chem. 181, 171 (1949). 109 Metzler, D. E., and Snell, E. E., Federation Proc. 11, 258 (1952). 109 CH3CH=CCOOH Ϊ = ± CH3CH2CCOOH I 2 I 2 IINH NH NH

(18)

Η2Ο

CH3CH2CCOOH > CH3CH2COCOOH + NH3 II NH HOCH2CH2CHCOOH U CH2=CHCHCOOH ^=± CH3CH=CCOOH I I I NH2 NH2 NH2 CH3CH=CCOOH ; = ± CH3CH2CCOOH Η2Ο> CH3CH2 CO COOH + NH3 NH2

(19)

NH

Replacement of the hydroxyl hydrogen atom prevents deamination. 107 The dehydrative deamination of serine and threonine by purified preparations from E. coli has been studied by Wood and Gunsalus. 108 Their preparation behaved as a single enzyme acting on both L-serine and L-threonine. The D-isomer of serine or threonine inhibited deamination approximately 50%. Cysteine was found to be inactive with this enzyme. This finding is of some interest in view of the similarity in the mechanism of the desulfhydrases (see below) and the suggestion by Binkley 111 that enolase in addition to its action in converting 2-phosphoglycerate to phosphoenolpyruvate also catalyzed the deamination of cysteine and serine with the formation of pyruvate. The purified preparation of Wood and Gunsalus was resolved and shown to require AMP and reduced glutathione for reactivation. A purified preparation of homoserine deaminase from rat liver was found by Binkley and Olson110 not to attack serine or threonine. The latter finding would appear to rule out the possibility that threonine is an intermediate in reaction 19. Although some activation by AMP and glutathione was observed, neither substance was considered to be essential. The enzyme did not attack D-homoserine nor the lactones of L-homoserine or DL-homoserine. Using extracts of B6-deficient cells of a B 6 -requiring mutant of E. coli, Metzler and Snell109 observed that pyridoxal phosphate caused a tenfold . m Binkley, F., / . Biol. Chem. 150, 261 (1943)

24

P. P.

COHEN

increase in activity in serine deamination. AMP and glutathione were without effect. Threonine was only slowly deaminated by the system. Reissig112 recently reported that partially purified preparations of serine and threonine deaminases from Neurospora required pyridoxal phosphate as a cofactor. The possibility that biotin as a nucleotide is required as a cofactor of serine and threonine deaminases in several bacterial species is suggested from the studies of Lichstein and Christman. 113 b. Cysteine and Homocysteine Desulfhydrases This subject has been recently reviewed by Fromageot. 114 These enzymes are characterized by a nonoxidative desulfhydration with either a simultaneous or subsequent deamination. Microorganisms and animal tissues (chiefly liver) have been reported to contain these enzymes. The reactions may be formulated as follows: HSCH2CHCOOH

I

► H 2 S + C H 2 = C C O O H ^ = ± CH3CCOOH

I

NH2

(20)

II

NH2

NH

CH3CCOOH — ^ CH3COCOOH + N H 3

II

NH HSCH2CH2CHCOOH

> H2S + C H 2 = C H C H C O O H i = ± C H 3 C H = C C O O H

I NH2 C H 3 C H = C C O O H i = ± CH3CH2CCOOH

I NH2

I

I

NH2

NH2

(21)

Η2Ο

> CH3CH2COCOOH + N H 3

II NH

Kallio 115 has partially purified the cysteine and homocysteine desulf­ hydrases from P. morganii and found that pyridoxal phosphate was active as cofactor. The possibility that threonine might be an intermediate in reaction 21 was ruled out by the finding that threonine was relatively inactive as a substrate. The role of pyridoxal phosphate as cofactor for animal desulfhydrases is suggested from the findings of Braunstein and Azarkh 116 that liver homogenates from pyridoxine-deficient animals had lower cysteine desulfhydrase activity than those from normal animals. Dietary supplementation with pyridoxine raised the activity to the nor­ mal level. Evidence for the existence of two desulfhydrases in animal tissues has been advanced by Fromageot and co-workers.114 The cysteine desulf112

Reissig, J. L., Arch. Biochem. and Biophys. 36, 234 (1952). Lichstein, H., and Christman, J. F., J. Biol. Chem. 175, 649 (1948). IU Fromageot, C , Enzymes 1 (pt. 2), 1237-1243 (1951). 115 Kallio, R. E., / . Biol. Chem. 192, 371 (1951). 116 Braunstein, A. E., and Azarkh, R. M., Doklady Akad. Nauk S.S.S. R. 71, 93 (1950). 113

NITROGEN METABOLISM OF AMINO ACIDS

25

hydrase activity of livers of different species is reported by Smythe 117 to be as follows: rat, 100; dog, 60; human being, 50; beef, 18; rabbit, 5; pig, 3; guinea pig, 1. On a similar basis, the relative activity of other rat tissues was as follows: kidney, 1; muscle, 1; brain, 0. Partially purified cysteine desulfhydrase from rat liver is inhibited by cyanide, arsenous oxide, and the carbonyl reagents phenylhydrazine, semicarbazide, hydroxylamine, and sodium bisulfite. 118119 An exocystine desulfhydrase has been found in rat liver by Greenstein and Leuthardt. 120 This enzyme acts on peptides containing cystine in terminal linkage. 121 c. Other Nonoxidative Deaminases Microorganisms and some plant tissues are capable of catalyzing a multiplicity of nonoxidative amino acid deamination reactions. It is beyond the scope of this chapter to review these systems, since for the most part they have not been studied with cell-free preparations. Two exceptions to the latter statement are the aspartase and tryptophanase systems. The latter is discussed in the chapter, Carbon Catabolism of Amino Acids. The aspartase reaction may be formulated as follows: HOOCCH2—CHCOOH ; = ± HOOCCH==CHCOOH + NH 3

(22)

NH 2

The system has been reviewed by Erkama and Virtanen. 122 A similar reaction (discussed in the chapter, Carbon Catabolism of Amino Acids) is carried out by histidine deaminase to yield urocanic acid. Other types of nonoxidative amino acid deamination are as follows: RCHNH2COOH + H 2 0 -► NH 3 + RCHOHCOOH RCHNH 2 COOH + 2H -^ NH 3 + RCH2COOH RCHNH 2 COOH + RxCHNH^OOH + H 2 0 -+ 2NH 3 + RCOCOOH + RiCH2COOH

Gale 117

123124

(23) (24) (25)

has reviewed this general topic.

Smythe, C. V., / . Biol. Chem. 142, 387 (1942). Lawrence, J. M., and Smythe, C. V., Arch. Biochem. 2, 225 (1943). 119 Fromageot, C , and Grand, R., Enzymologia 11, 6 (1942). 120 Greenstein, J. P., and Leuthardt, F. M., J. Natl. Cancer Inst. 6, 209 (1944). 121 Leuthardt, F. M., and Greenstein, J. P., Science 101, 19 (1945). 122 Erkama, J., and Virtanen, A. I., Enzymes 1 (pt. 2), 1244-1249 (1951). 123 Gale, E. F., Bacteriol. Revs. 4, 135 (1940). 124 Gale, E. F., In C. H. Werkman and P. W. Wilson, Bacterial Physiology, pp. 441 454. Academic Press, New York, 1951. 118

26

P. P.

COHEN

III. DEAMIDATION a. Amino Acid Amidases The enzymatic production of ammonia from amino acid amides is limited chiefly to the two naturally occurring amides, glutamine and asparagine. A review of this subject has recently appeared. 125 (1) Glutaminases. Glutaminases catalyze the following reaction: COOH

I

HCNH 2 I CH 2

COOH

I

HCNH 2 H2O I > CH2 + NH 3

(26)

I I CH2 CH2 CONH2 COOH The enzymes show a relatively high degree of specificity, 126-128 although the limited extent of purification of animal glutaminases prevents a clear definition of substrate specificity. Isoglutamine was found to have about 6% the activity of glutamine with brain glutaminase. 126 Carter and Greenstein, 129 on the other hand, observed that rat liver extracts cata­ lyzed the hydrolysis of isoglutamine more rapidly than glutamine. Benzoylglutamine, phenacetylglutamine, and glutamylglutamic acid were found by Krebs 126 to be inactive as substrates with brain glutaminase preparations. DL-iV-(7-glutamyl)-ethylamine and L-iV-(7-glutamyl)-methylamine were found to be inactive as substrates of dog kidney glutaminase. 54 Glutaminases are widespread in nature, having been found in animal and plant tissues, yeast, and bacteria. 125 Systematic studies of glutaminase in animal tissues have been carried out by Krebs, 126 Archibald, 127 and Errera and Greenstein. 130 (See Table V.) While Greenstein and co-workers had previously reported the existence of an a-keto-acid-activated glutaminase and asparaginase in liver,131 this reaction was later shown by Meister and co-workers 132133 to be due to a transamination reaction between glutamine and α-keto acids and either a simultaneous or sub­ sequent deamidation. This reaction will be discussed under Transaminases 125

Zittle, C. A., Enzymes 1 (pt. 2), 922-945 (1951). Krebs, H. A., Biochem. J. (London) 29, 1951 (1935). 127 Archibald, R. M., J. Biol. Chem. 154, 657 (1944). 128 Krebs, H. A., Biochem. J. (London) 43, 51 (1948). 129 Carter, C. E., and Greenstein, J. P., J. Natl. Cancer Inst. 7, 433 (1947). 130 Errera, M., and Greenstein, J. P., J. Biol. Chem. 178, 495 (1949). 131 Greenstein, J. P., and Price, V. E., J. Biol. Chem. 178, 695 (1949). 132 Meister, A., and Tice, S. V., J. Biol. Chem. 187, 173 (1950). 133 Meister, A., Sober, H. A., Tice, S. V., and Fräser, P. E., J. Biol. Chem. 197, 319 (1952). 126

NITROGEN

METABOLISM

OF ΑΜΙΝΟ

27

ACIDS

on p. 29. The existence of an additional phosphate-activated glutaminase in many animal tissues was shown by Greenstein and co-workers.134'136 Krebs 126 and Archibald 127 earlier recognized the existence of different glutaminases in animal tissues. Thus Krebs 126 found that liver glutaminase had a pH optimum of 7-8, while the enzymes from kidney, brain, and retina had a pH optimum of 8-9. Liver glutaminase was not inhibited by glutamic acid, but the others were.126 A similar observation was made by TABLE V GLUTAMINASE ACTIVITY IN T I S S U E S OF VARIOUS S P E C I E S 1 3 0

(Expressed as μΜ glutamine hydrolyzed per gram tissue per hour) Kidney

Liver

Brain

Spleen

Phosphate

Phosphate

Phosphate

Phosphate

Rat Mouse Rabbit Guinea pig

+

-

+

- +

150 600 100 600 50 50 100 150

6 9 6 9

36 39 9 12

9 9 3 3

36 36 39 33

-

+

9 9 12 6

36 39 42 36

Archibald 127 in the case of dog kidney glutaminase, and by Greenstein and Leuthardt 135 in the case of rat kidney. Errera 136 was able to show that in rat liver the phosphate-activated glutaminase (referred to as glutaminase I) was associated with the insoluble particles—an observation later con­ firmed by Shepherd and Kalnitsky, 137 who established that this gluta­ minase activity was largely associated with, and bound to, the mitochondrial fraction. The purification of animal glutaminases beyond 3- to 4-fold136 has not as yet been realized, and thus an adequate explanation of the effect of anions and other factors is difficult. While animal glutaminases have not been extensively purified, the glutaminase from Clostridium welchii has been purified some 40- to 60-fold by Hughes and Williamson.138 The Cl. welchii glutaminase resembles the animal glutaminases with respect to the nature of the end products, that is, glutamic acid and ammonia, and inhibition by bromosulfalein. It differs strikingly in having a pH optimum at 5, and its activation by monovalent anions in the following order of effectiveness: bromide > chlp134 135 136 137 138

Carter, C. E., and Greenstein, J. P., / . Natl. Cancer Inst. 7, 433 (1947). Greenstein, J. P., and Leuthardt, F . M., Arch. Biochem. 17, 105 (1948). Errera, M., J. Biol. Chem. 178, 483 (1949). Shepherd, J. A., and Kalnitsky, G., / . Biol. Chem. 192, 1 (1951). Hughes, D . E., a n d Williamson, D . H., Biochem. J. (London) 51, 45 (1952).

28

P. P.

COHEN

ride > iodide > cyanide. The divalent ions sulfate, phosphate, and arsenate, active with the animal glutaminase preparations, 135 were with­ out effect on the Cl. welchii glutaminase. Potent inhibitors of the bacterial glutaminase were (at approximately 10~3 M) selenite, sulfite, iodoacetate, and the phthalein dyes, bromocresol purple, bromocresol green, and bromosulfalein (disodium sulfonate of phenol tetrabromophthalein). While atabrine (9 X 10~3 M) and bromosulfalein (2 X 10"3 M) were found by Archibald 127 to inhibit dog kidney glutaminase 100%, the former was not inhibitory for the bacterial enzyme. Some of these differ­ ences may be related to the different pH at which the different enzymes were tested. The activation of glutaminase in Cl. welchii cells by cetyltrimethylammonium bromide previously observed128 was not observed with the purified preparation; rather it was found to inhibit. Hughes and Williamson138 and Hughes 139 ' 140 are of the opinion that in the intact cell and crude preparations a naturally occurring competitive inhibitor exists which is removed by the detergent. The purified preparation was free of this inhibitor. Dog kidney glutaminase has been found to be inhibited strongly by D- and L-glutamic acids and by DL-a-methylglutamic acid.54 (2) Asparaginase. Asparaginase is widely distributed in microorgan­ isms, plants, and animal tissues.125 It catalyzes the following reaction: COOH I

HCNH 2

I

CH2

I

CONH 2

H2O

COOH I

> HCNH 2 + NH 3

I

(27)

CH 2

I

COOH

Aside from a study of yeast asparaginase by Grassmann and Mayr 141 there has been no systematic study of purified preparations. Purified yeast asparaginase is relatively specific for asparagine. Of a series of asparagine derivatives and analogs, including isoasparagine, only the diamide of aspartic acid was found to be active, and that only one-tenth as active as asparagine. The purified yeast enzyme was found to be extremely unstable in the absence of substrate. Since highly purified glutaminase and aspara­ ginase are yet to be prepared, the absolute specificity with respect to glutamine and asparagine is difficult to determine. Krebs 126 pointed out that the ratio of glutaminase to asparaginase activity varied from tissue to tissue; this would indicate that two different enzymes existed. Thus, whereas rabbit liver splits glutamine five times more slowly than aspara­ gine, rabbit kidney splits glutamine one hundred times more rapidly. The 139

Hughes, D. E., Biochem. J. {London) 45, 325 (1949). Hughes, D. E., Biochem. J. {London) 46, 231 (1950). 141 Grassmann, W., and Mayr, O., Hoppe-Seyler's Z. physiol. Chem. 214, 185 (1933). 140

NITROGEN

METABOLISM

OF AMINO

ACIDS

29

relative rate of ammonia production from glutamine and asparagine by aqueous extracts of different tissues has been studied by Greenstein and co-workers.135,142·143 In rat tissues, phosphate has no activating effect on asparaginase comparable to that of glutaminase. 135 IV. TRANSAMINATION (1) Amino Acid Transaminases. Transaminases catalyze reactions involving the transfer of an amino group from an α-amino acid to an a-keto acid, the over-all process being called transamination. This subject has been extensively reviewed by Braunstein, 144 who discovered the reac­ tion, and by the author. 145 Transaminases are widely distributed in microorganisms, plant, and animal tissues (see reviews 144145 ). The type reaction catalyzed is the following: R Ri R Ri

I 2 + CI= 0 — ^ C=O I + HCNH I 2 HCNH I COOH

I COOH

I COOH

I COOH

(28)

Whereas early studies left some doubt as to the scope of the trans­ amination reaction, more recent studies clearly 146-154 indicate that there is a large number of transaminases, and it thus appears highly likely that all naturally occurring amino acids can participate in transamination reactions. A comparative quantitative assay of the rates of transamination of different amino acids in the different animal tissues using improved assay methods has not been reported. Unpublished studies from the author's laboratory reveal that the pattern of transaminase activity for different amino acids varies quantitatively as well as qualitatively. This difference also applies to the intracellular distribution of transminases. Glutamic142

Errera, M., and Greenstein, J. P., J. Natl. Cancer Inst. 7, 285 (1947). Goncalves, J. M., Price, V. E., and Greenstein, J. P., / . Natl. Cancer Inst. 7, 281 (1947). 144 Braunstein, A. E., Advances in Protein Chem. 3, 1 (1947). 145 Cohen, P. P., Enzymes 1 (pt. 2), 1040-1067 (1951). 146 Cammarata, P. S., and Cohen, P. P., / . Biol. Chem. 187, 439 (1950). 147 Feldman, L. I., and Gunsalus, I. C , J. Biol. Chem. 187, 821 (1950). 148 Rowsell, E. V., Nature 168, 104 (1951). 149 Heyns, K , and Koch, W., Hoppe-Seyler's Z. physiol. Chem. 288, 272 (1951). 150 Holden, J. T., Wildman, R. B., and Snell, E. E., J. Biol. Chem. 191, 559 (1951). 151 Rudman, D., and Meister, A., J. Biol. Chem. 200, 591 (1953). 152 Umbarger, H. E., and Magasanik, B., / . Am. Chem. Soc. 74, 4256 (1952). " 8 Meister, A., / . Biol. Chem. 195, 813 (1952). 154 Hird, F. J. R., and Rowsell, E. V., Nature 166, 517 (1950). 143

30

P. P.

COHEN

oxalacetic and glutamic-pyruvic transaminase are the more soluble and readily extractable of the different transaminases. 155 Some properties of highly purified preparations of glutamic-oxalacetic and glutamic-pyruvic transaminases have been reported. 155-159 In the ultracentrifuge, the preparations studied appeared to be homogeneous, but upon electrophoresis glutamic-pyruvic transaminase showed the presence of two components/ 55 and glutamic-oxalacetic transaminase, three com­ ponents. 155159 Activity, however, was found to be associated with a single component.155-159 A preparation of unresolved glutamic-oxalacetic trans­ aminase was found to have an activity eight to fifteen times 159160 that of the activated resolved transaminase, 158 suggesting that considerable inactivation occurs in the preparation of the latter. Glutamic-oxalacetic transaminase has an equilibrium constant K of the order of 3/55>161 and the glutamic-pyruvic enzyme a K value of the order of l. 155 · 161 Detailed kinetic studies with highly purified preparations have not as yet been reported, although studies have been carried out with partially purified but unfortified preparations. 161 ' 162 Recent studies by Krebs162cr indicate that the equilibrium constant for the glutamic-oxalacetic system is 6.74 whereas that of the glutamic-pyruvic system is 1.52. Nisonoff et al.162b found an equilibrium constant of 7.8 for the glutamic-oxalacetic system. The coenzyme dissociation constant for glutamic-oxalacetic trans­ aminase has been estimated to be 1.5 X 10~6 mole per liter. 158 The pH optimum for glutamic-oxalacetic transaminase from animal tissues is 7.5; 161 from plant seedlings, 8.6;163 green plants, 6.9;164 bacteria, 8-8.5; 158 ' 165 and yeast, 7.8.166 (a) Specificity. Studies with highly purified preparations clearly indi­ cate a high degree of substrate specificity in the case of glutamic-oxal­ acetic 155159 and glutamic-pyruvic transaminases. 155160167 Thus Green et al.nb reported that with their preparation of glutamic-oxalacetic trans­ aminase no amino acid other than aspartic acid was capable of reacting 155

Green, D . E., Leloir, L. F., and Nocito, V., / . Biol. Chem. 161, 559 (1945). Lenard, P., and Straub, F . B., Studies Inst. Med. Chem. Univ. Szeged 2, 59 (1942). 157 Schlenk, F., and Fisher, A., Arch. Biochem. 12, 69 (1947). "« O'Kane, D . E., and Gunsalus, I. C , / . Biol. Chem. 170, 425 (1947). "» Cammarata, P. S., and Cohen, P. P., / . Biol. Chem. 193, 53 (1951). 160 Ross, H . E., M. S. Thesis, University of Wisconsin, Madison, (1952). 16 * Cohen, P. P., J. Biol. Chem. 136, 585 (1940). 162 Nisonoff, A., and Barnes, F . W., Jr., J. Biol. Chem. 199, 713 (1952). i62a Krebs, H. A., Biochem. J (London) 54, 86 (1953). "M Nisonoff, A., Barnes, F. W. Jr., and Enns, T., J. Biol. Chem. 204, 957 (1953). 163 Albaum, H. G., and Cohen, P. P., / . Biol. Chem. 149, 19 (1943). 164 Rautanen, N., / . Biol. Chem. 163, 687 (1946). '«Lichstein, H . C , and Cohen, P. P., / . Biol. Chem. 157, 85 (1945). 166 Roine, P., Ann. Acad. Sei. Fennicae IIA, No. 26 (1947). 156

NITROGEN

METABOLISM

OF ΑΜΙΝΟ

ACIDS

31

with α-ketoglutaric acid with the exception of cysteic acid, which had only slight activity. Glutamine was found incapable of replacing glutamic acid, and mesoxalic acid showed only a slight activity when used in place of oxalacetic acid. Cammarata and Cohen159 found a similar high degree of specificity with their unresolved preparation, the following amino acids proving to be inactive: leucine, isoleucine, tyrosine, methionine, tryptophan, arginine, cysteine, glycine, ornithine, valine, phenylalanine, asparagine, α-amino-n-butyric acid, α-aminoisobutyric acid, α-aminoadipic acid, and glutamine. Alanine had slight activity. In the case of glutamicpyruvic transaminase, Green et αΖ.155 reported that while a-aminobutyric acid showed slight activity when used in place of alanine, the following were inactive: n-monomethylalanine, phenylalanine, valine, serine, methionine, leucine, α-aminovaleric acid, cysteic acid, and D-alanine. In the reaction between glutamic and pyruvic acid, these investigators found that the latter could be replaced by α-ketobutyrie acid and mesoxalic acid, but the former could not be replaced by cysteic acid, glycylcysteine, glutathione, pyrrolidonecarboxylic acid, acetylglutamic acid, leucine, methionine, glutamine, tyrosine, threonine, α-aminocaproic acid, lysine, phenylalanine, cystine, valine, and hydroxyproline. An even higher degree of specificity was found with a partially resolved (20 %) glutamic-pyruvic transaminase using twenty-one different amino acids and peptides. 167 If it is assumed that this degree of specificity holds for the other enzyme, it is clear that there exists a large number of transaminases which are yet to be purified and studied. It appears from studies to date that the glutamic-oxalacetic trans­ aminase is the most active and widely distributed of all the transaminases in all cells studied. 147163-165 · 168169 As a generalization it would appear that the reaction Amino acid + α-ketoglutaric acid ; = ± α-keto acid + glutamic acid

(29)

represents the more common type of transamination reaction in most plant and animal tissues and microorganisms (see Table VI). Oxalacetic acid, on the other hand, appears to be limited in its participation in trans­ amination to the glutamic-oxalacetic transaminase system,148*149155'170'171 contrary to the report of Kritzman and Samarina. 172 While previous 167 168

i69 170

171 172

Rand, M. C , M. S. Thesis, University of Wisconsin, Madison, (1950). Cohen, P. P., and Hekhuis, G. L., / . Biol. Chem. 140, 711 (1941). Leonard, M. J. K., and Burris, R. H., / . Biol. Chem. 170, 701 (1947). O'Kane, D. E., and Gunsalus, I. C , J. Biol. Chem. 170, 433 (1947). Cammarata, P. S., and Cohen, P . P., Biochim. et Biophys. Ada 10, 117 (1953). Kritzman, M. G., and Samarina, O. P., Doklady Akad. Nauk. S.S.S.R. 63, 171 (1948).

32

P.

P.

COHEN

studies seemed to establish the need for a dicarboxylic acid as one of the substrate pairs for transamination, 144145 recent reports indicate the possi­ bility of transamination between pyruvate and the amino acids, leucine and phenylalanine, 148 and between pyruvate and ornithine. 173 Confirma­ tion of these findings would of course suggest the possibility of a still larger number of transaminases. However, it should be pointed out that T A B L E VI GLUTAMIC-OXALACETIC TRANSAMINASE ACTIVITY OP ANIMAL AND P L A N T

TISSUES

AND MICROORGANISMS 1 4 5

Οτκ α E. coli A. vinelandii Cl. welchii Oat seedlings (96 hr.) P o t a t o root Potato stem Potato leaf Brain (rat) Liver (rat) Kidney (rat) Heart (rat) _

890 1575 1170 5650 3280 2280 650 2800 2200 1750 3330

m cr

i ° l i t e r s substrate transaminated mg. nitrogen X hour

trace amounts of glutamic acid could serve to couple transaminases in the following manner. 170 Pyruvic acid + glutamic acid ^==± alanine + α-ketoglutaric acid Amino acid + α-ketoglutaric acid ^ = ± α-keto acid + glutamic acid

(30) (29)

Experience in the author's laboratory and that of others 133 has shown that liver preparations in particular are apt to contain or form during incubation small but significant quantities of glutamic acid. Studies to date 144,145,153 indicate that transaminases are specific for L-amino acids and α-keto acids. Peptides 144-146 and other derivatives of amino acids, such as amides 155159 and iV-substituted amino acids,155 are not active. Recently it has been reported that E. coli extracts catalyze a transamination reaction between adenine and α-ketoglutaric acid.174 (b) Mechanism of Action. With the demonstration that pyridoxal phosphate is the cofactor for transaminases, 175,176 the mechanism shown 173

Quastel, J. H., and Witty, R., Nature 167, 556 (1951). Gunsalus, C. F., and Tonzetich, J., Nature 170, 162 (1952). 175 Schlenk, F., and Snell, E. E., J. Biol. Chem. 157, 425 (1945). 176 Lichstein, H. C , Gunsalus, I. C , and Umbreit, W. W., / . Biol. Chem. 161, 311 (1945). 174

33

NITROGEN METABOLISM OF ΑΜΙΝΟ ACIDS

in Fig. 1 has been proposed. Direct evidence for the participation of pyridoxamine phosphate has hitherto been lacking,145 but it was recently reported 177 that a resolved pig heart glutamic-oxalacetic transaminase was equally activated by pyridoxal phosphate and pyridoxamine phos­ phate. The role of the coenzyme in this reaction thus is that of an amino group acceptor and donor, and the intermediates represent SchifFs bases between pyridoxal phosphate and the amino acid, on the one hand, and

COOH 1 ■ N - CH2< 00 C -C + Η , Ν Ο Η ^ Ν | ° - : C =

0

I

Protein

vi

K

y^
CH3CHO + CH2NH2COOH

(4)

They found, also, that DL-allothreonine is more readily transformed than DL-threonine—a result which has been confirmed in the intact animal in the author's laboratory. 23 Employing isotopic threonine, Meltzer and Sprinson36 demonstrated its breakdown to glycine and acetic acid. The acetate, presumably, is formed by oxidation of the acetaldehyde. Lien and Greenberg 29 isolated glycine by ion exchange chromatography upon incubating DL-threonine-1, 2-C14 with rat liver mitochondria or after injecting the amino acid into the OH3 H CIO

+

CH3

I

CH2NH2 COOH

1 CHOH

- I

CH3 2

C H N H 2 -Η2Ο COOH

Glycine

HC

> II

C—NH2 COOH

Threonine CH3

CH3

/

I

5 ' \ Propionic acid

C—O

I

COOH

-

CH2

I

CH3

I

4

IC H

> I

2

C = N H +H 2 0 C = 0 + N H 3 COOH COOH a-Ketobutyric acid

5 / C H 2 + COo

I

CH

CH3

I3

ICOOH

\

CH3CH2CHNH2COOH α-Aminobutyric acid F I G . 3. Reactions of the catabolism of threonine.

intact animal. Formation of glycine in the intact animal was also shown by isolating hippuric acid from the urine after administration of isotopic threonine or allothreonine, along with benzoic acid.36-23 Additional evi­ dence is provided by the fact that on feeding N15-labeled threonine, gly­ cine exhibited the highest N 15 concentration of any of the derived amino acids.36 The anaerobic deamination of L-threonine with the formation of α-ketobutyric acid is induced by the same bacterial preparations that deaminate L-serine, most probably by the same enzyme.31·34α The formation of α-aminobutyric acid from threonine has been ob­ served by Lien and Greenberg. 29 This is probably formed from a-ketobutyric acid by reaction 5' (Fig. 3). That threonine is not reduced directly to α-aminobutyric acid is indicated by the results of an experiment with C14- and N15-labeled threonine. 38 Most of the N 15 was found to have been lost in the isolated α-aminobutyric acid; this would not have occurred if there was a direct reduction. 38

Lien, O. G., Jr., and Greenberg, D . M., J. Biol. Chem. 200, 367 (1953).

59

CARBON CATABOLISM OF AMINO ACIDS

The α-ketobutyric acid formed from threonine is decarboxylated to propionic acid,38° as shown in reaction 5. The latter is further metabolized as discussed under valine below. The amino group of threonine, like that of lysine, is not available for reversible transfer reactions. Thus, the N 15 from other amino acids is not found in threonine 36,39 and the C 14 :N 15 ratio of threonine, like that of serine, isolated from body tissues, shows only small differences in the dilution of the two labels.36 The explanation for this is that the catabolism of threonine by way of glycine does not involve loss of the amino group. 36 V. T H E ALIPHATIC BRANCHED-CHAIN AMINO ACIDS 1. VALINE

Valine is unique among the nutritionally essential amino acids in that its deficiency in the rat causes, in addition to loss of appetite and stunted growth, a syndrome in which the animal becomes extremely sensitive to touch and displays a severe incoordination of movement. 40 CH3 \

1 C H — C H N H 2 C O O H —=t

CH3

CH3 \

CH3 2 CHCO—COOH—■*

CH3 α-Ketoisovaleric acid

Valine > CH3—CH2COOH -CO2

>CH2=CH—COOH -2H

Propionic acid

\ CH—COOH

-

CH3 Isobutyric acid > CH3—CHOH—COOH

+Η2Ο

Acrylic acid 6

L-Lactic acid

- * D-Lactic acid

a-hydroxy "racemase"

F I G . 4. Reactions of the catabolism of valine.

7

> CH3CO—COOH Pyruvic acid

As with most other amino acids, the catabolism of valine is initiated by removal of its amino group, in this case to yield cx-ketoisovaleric acid. L-Valine is readily susceptible to transamination, 20 and it is slowly attacked by L-amino acid oxidase.41 The D-valine is readily deaminated by D-amino acid oxidase.42 Valine is glycogenic and antiketogenic. 42 ' 43 From the observation that α-ketoisovaleric acid and isobutyric acid, as well as valine, each con38a Kinnory, D . S . , Takeda, Y., and Greenberg, D . M., Unpublished data. 39 Elliott, D . F., and Neuberger, A., Biochem. J. {London) 46, 207 (1950). 40 Rose, W. C , and Eppstein, S. H., J. Biol. Chem. 127, 677 (1939). 41 Blanchard, M., Green, D . E., Nocito, V., and Ratner, S., / . Biol. Chem. 155,421, 1944; ibid. 161, 583 (1945). 42 Felix, K., and Dorn, K., Hoppe-Seyler's Z. physiol. Chem. 258, 16 (1939). 43 Cohen, P . P., / . Biol. Chem. 119, 333 (1937).

60

D. M.

GREENBERG

tributed 3 of their carbons to the formation of glucose, Rose and coworkers44 postulated the steps 1 and 2 shown in Fig. 4 in the catabolism of valine. Subsequently Atchley 45 was able to show that isobutyric acid is converted to propionic acid by the action of the fatty acid-oxidizing systems of liver and kidney. The oxidation of propionic acid has been studied extensively because the dissimilation of odd-numbered carbon acids leads to the production of this acid.46,47 Another reason for the interest in this acid is that the catabolism of propionate is an apparent example of the α-oxidation of a fatty acid, which certainly appears to be not found in connection with the catabolism of either the straight-chain or branched-chain carbon acids. It was demonstrated by Grafflin and Green46 that washed liver cyclo­ phorase preparations could oxidize propionate completely to C 0 2 and H 2 0 if the system was supplemented with the supernatant fluid of the original liver preparation. Further experiments by Huennekens et a/.47 established that the necessary conditions for the complete oxidation of propionate are: (1) An active mitochondrial system capable of maintain­ ing the citric acid cycle and oxidative phosphorylation; (2) a propionic acid oxidase (found only in liver); (3) a fraction from the supernatant fluid of centrifuged kidney or liver homogenates, believed to contain a "racemase"; (4) Mg + + , AMP, and Pi; and (5) oxygen. Kidney lacks the propionic acid oxidase, but contains the "racemase." Consequently, washed kidney cyclophorase can oxidize propionate and acrylate com­ pletely only when fortified with sources of propionate oxidase and "race­ mase." The oxidation of propionate is strongly inhibited by NH 4 + . The propionate oxidase can be obtained as a saline extract from ace­ tone-dried, washed liver cyclophorase preparation. This enzyme is very labile and loses its activity within several hours after its preparation even at 5°C. or in the frozen state. The material called α-hydroxy acid racemase is found in the first supernatant fluid of cyclophorase preparations of either liver or kidney. The active substance appears to be a protein fairly stable to elevated temperatures, repeated freezing and thawing, and precipitation by acetone and alcohol. The action of this enzyme as a racemase 48 offers a number of puzzling features. Because the products of its "racemic" activity, if that is its 44 45 46 47

48

49

Rose, W. C , Johnson, J. F., and Hanes, W. J., / . Biol. Chem. 145, 679 (1942). Atchley, W. A., / . Biol. Chem. 176, 123 (1948). Grafflin, A. L., and Green, D . E., / . Biol. Chem. 176, 195 (1948). Huennekens, F . M., Mahler, J. R., and Nordmann, J., Arch. Biochem. 30, 66, 77 (1951). Racemizing enzymes are known, e.g., an enzyme present in Streptococcus faecalis49 which specifically converts both D- and L-alanine to the racemic mixture. Wood, W. A., and Gunsalus, I. C., / . Biol. Chem. 190, 403 (1951).

CARBON CATABOLISM OF AMINO ACIDS

61

function, are subject to further oxidation in the systems in which it is found, it has not been possible to make a direct test by allowing the enzyme to act on one of the isomers of lactic acid and isolating the racemic mixture that should result. Favorable to the interpretation that it is a racemase is the fact that it is capable of promoting the complete oxidation of racemic mixtures of lactate, malate, and isocitrate, only half of which are oxidized in its absence. It also is able to promote the oxidation of both lactate isomers by baker's yeast and pigeon breast muscle, which, in con­ trast to liver or kidney, oxidize L- but not D-lactate. On the other hand, the racemase material was unable to promote the oxidation of D-lactate by the L-lactic acid oxidases of pig heart or tobacco leaves. A suggestion that the "racemase" material might contain an a-hydroxy acid oxidase for one stereoisomer of lactic acid that is lacking in the enzyme preparation used comes from the observation of Baker 50 that two α-hydroxy acid-oxidizing enzymes, one specific for the L- and the other for the D-isomer, occur in kidney and liver. Additional and more direct evidence on the postulated racemase activity of the enzyme and on the correctness of the catabolic pathway proposed for propionic acid shown in Fig. 4 was secured by observing that when propionate-1-C 14 was incubated with liver cyclophorase in the presence of an excess of unlabeled L-lactate, or pyruvate, the label appeared in each of these compounds. 51 Proof of the formation of L-lactate was obtained by oxidizing it with a specific soluble L-lactic acid oxidase from yeast and isolating the radio­ activity in the pyruvate formed as the dinitrophenylhydrazone. Evidence for racemase activity was obtained by carrying out the incubation in the presence of unlabeled D-lactic acid and determining the amount of L-lactate formed from the radioactivity of the pyruvyl dinitrophenyl­ hydrazone after treatment with the L-lactic acid oxidase, suitably cor­ rected for endogenously present pyruvate. The result, although favorable for racemase activity, was not wholly unambiguous. Evidence for the mechanism of the conversion step of isobutyric to propionic acid (reaction 3, Fig. 4) was sought by Atchley 45 on the premise that any compound which is an intermediate between isobutyrate and propionate should be completely oxidized by the fatty acid oxidizing sys­ tem of liver. Using as a guide in the selection of compounds the ß-oxidation theory of fatty acids, he found that ß-hydroxyisobutyrate and methylacrylate were completely oxidized. Methylacrylic acid could be expected to give rise to methylmalonic semialdehyde, which, on structural grounds, 60 51

Baker, C. G., Arch. Biochem. and Biophys. 41, 325 (1952). Mahler, H. R., and Huennekens, F. M., Abstracts of papers of 12th Intern. Congr. Pure Applied Chem., p. 109 (1951); Biochim. Biophys. Ada 11, 575 (1953).

62

D. M. GREENBERG

could be expected to decompose to propionaldehyde and C0 2 . Methylmalonic acid was eliminated as an intermediate because it was not oxidized. Propionaldehyde, on the other hand, was found to be readily oxidized by the liver system. This led to the scheme of isobutyric acid oxidation shown below (equation 5), which involves loss of the carboxyl group and the oxidation to carboxyl of a terminal methyl group of isobutyric acid. CH 3

CH 2

-2H

CH—COOH -

+ Η2Ο

C—COOH

HOCH 2 CH—COOH

CH 3

CH 3

CH3

0=CH \

HO2

CH—COOH

-CO2

> CH3CH2CHO

HÖ2

>CH3CH2COOH

(5)

CH 3

Definite proof that the carboxyl and not the methyl group of isobutyrate is lost has been obtained from incubation experiments with isotopic valine. 52 In brief, on the basis of the decarboxylation hypothesis of isobutyric acid, the catabolism of valine would proceed as follows: Valine-44'-CVi

Valine-2-C^ CH 3

CH 3 CHCHNHU—COOH

cm CH 3 C ^

'

CHC—COOH

CH3 X

II o

I 1 1 1

CHCOOH + C0 2

I I

CK/

/ OCH CH3

\

* i

\

* / CH 3 * CH 3 \

CHC—COOH * /' II o CH 3 * CH 3 \ ^CHCOOH + C0 2 * / CH 3 * OCH CH—COOH

CH—COOH

CH3CH2" -COOH + C0 2

CHCHNH 2 —COOH I

CH 3

*

I

iI

CHsCH2- -COOH + C0 2

These expectations were confirmed by the experimental results (Table I). The evolved C 0 2 was radioactive upon incubation with valine-2-C 14 , inert with valine-^^-C 1 4 . 52

Kinnory, D. S., and Greenberg, D. M., Federation Proc. 12, 320 (1953).

C A R B O N CATABOLISM O F A M I N O

63

ACIDS

Evidence for the pathway of the catabolism of valine 53-55 has also been sought from the nature of the distribution pattern of the label in glucose (of liver glycogen) or glucose excreted in the urine by phlorizinized rats. The isotope distribution pattern of liver glycogen upon administration of isotopic valine yielded the following results: L-Valine-4,4'-C13 gave rise to glucose labeled in all six carbons. 54 This observation led the authors to suggest that the catabolism of valine proceeds by the way of an intermedi­ ate formation of a three-carbon acid (propionic acid), labeled only in the TABLE I Expected Findings from Isotopic Valine Labeled at 2-C 14 4,4'-C 14

Propionic Acid Formed

non-radioactive radioactive (C 14 equally distributed in carbons 1 and 3)

Lactic Acid Formed

CO2 Evolved

non-radioactive radioactive

radioactive non-radioactive

Data from Ph. D. Thesis of D. S. Kinnory, University of California, 1952.

ß-position and that the excess C13 observed in carbons 3 and 4 of the glu­ cose resulted from fixation of isotopic C0 2 . The uniform distribution of the label in the glucose can, however, be equally well, or better, explained by the intermediate formation of 1,3-labeled propionate according to the oxidation scheme established by Kinnory and Greenberg. 52 With such a labeling, the methyl group, because of randomization at carbons 2 and 5, would give rise to an equal isotope concentration in glucose carbons 1, 2, 5, and 6 and the carboxyl group would also contribute (in addition to that from C 0 2 fixation) to the relatively high excess of the label in car­ bons 3 and 4. Administration of L-valine-3-C13, yielded glycogen having a relatively small content of C13 in carbons 3 and 4, and a relatively high isotope con­ centration in carbons 2,5 and 1,6 of the glucose.55 The average ratio of excess C13 in positions 2,5 to that in 1,6 was 1.13. This is in good agree­ ment with the corresponding ratio of 1.1 found in glycogen derived from 53

Fones, W. S., and White, J., Arch. Biochem. 28, 145 (1950). Fones, W. S., Waalkes, T. P., and White, J., Arch. Biochem. and Biophys. 33, 89 (1951). 55 Peterson, E . A., Fones, W. S., and White, J., Arch. Biochem. and Biophys. 36, 323 (1952).

54

64

D . M.

GREENBERG

C2-labeled propionate, 56 which is proposed as an intermediate in the oxidation of valine. The distribution of the labeled carbons of propionate in glucose and other metabolic products formed from it should help provide evidence of the pathways of the oxidation of valine. However, the results of the experiments with isotopic propionate have been difficult to interpret, because the distribution of the label was not the same as that found from lactate or pyruvate, which are postulated to be intermediates in the metabolism of propionate, and, thus, should be expected to lead to the same isotope distribution. 56-59 The direct α-oxidation of propionate to pyruvate has been questioned for some time. The degree of randomization found in glucose upon admin­ istration of propionate-2-C 14 or -3-C14 was found to be more complete than for the correspondingly labeled lactate. 56 · 57 Shreeve,58 in studying the formation of acetyl groups from propionate, observed that the acetyl radicals in N-acetyl phenylaminobutyric acid derived from propionate2-C14 was 100% randomized while that derived from the correspondingly labeled lactate or pyruvate was only about 20% randomized. Daus, Meinke and Calvin 58a found complete randomization between the a- and ß-carbons of the lactic acid formed from either propionate-2-C 14 or -3-C14 incubated with mouse liver slices. There is a noticeable disagreement between the results just cited and the reaction sequence advanced for the catabolism of propionate by Huennekens, et al.*7 (see Fig. 4, reactions 4-7). The catabolic pathway proposed for propionate should lead to the same distribution in glycogen and acetyl groups derived from it as from similarly labeled lactate, which, as has already been pointed out, is not the case. These discrepancies can, at the present time, only be explained by postulating two alternate mechanisms leading to randomization in the pathway of oxidation of propionate. One mechanism would be through the carboxylation of pro­ pionate to the symmetrical succinate. The succinate would then give rise to pyruvate with its original a- and ß-carbons equally distributed between these two positions. Evidence for metabolism of propionate through an initial carboxylation to succinate has recently been obtained in Lardy's laboratory. 59 There it was shown that C1402 was fixed in succinic acid 56

Lorber, V., Lifson, N., Wood, H. G., Sakami, W., and Shreeve, W. W., / . Biol. Chem. 183, 517 (1950). 57 Lorber, V., Lifson, N., Sakami, W., and Wood, H. G., / . Biol. Chem. 183, 531 (1950). 58 Shreeve, W. W., / . Biol. Chem. 195, 1 (1952). δ8α Daus, L., Meinke, M., and Calvin, M., / . Biol. Chem. 196, 77 (1952). 59 Lardy, H. A., Proc. Natl. Acad. Sei. U.S. 38, 1003 (1952); Lardy, H. A., and Peanasky, R., Physiol. Rev. 33, 560 (1953).

65

CARBON CATABOLISM OF AMINO ACIDS

upon incubating anaerobically propionate and radioactive bicarbonate with liver homogenates of biotin-fed rats. In biotin-deficient rats there was very little CO2 fixation. Another scheme that leads to randomization and that retains the α-oxidation pathway and the racemase action has been suggested by Mahler and Huennekens. 51 The mechanism involved is shown in Fig. 4A. O

Propionate

\

C - 2 e > H OOL/

+H2O

I H

c

/

O-

Ϊ = ± H O C/ ßC H 3 -H2O

c

I H

H

O

\ c/

+ H2O / τ==± H O C

\

/

O-

\ CH

"

c/

-H2O /f COH 5 = i C

I

I

\

I

0

-

0

\ c /

O-

+ H20 a\ß C—OH ^ = ± H 3 C — C O H

F I G . 4A. Randomization and racemase action. H H H

I H

According to the authors the formulation shown in Fig. 4A requires that the carboxylate ion be bound to the enzyme so that complete ran­ domization between all three carbons is prohibited. In the above scheme randomization between the a- and ß-carbons would be complete with propionate where racemization is obligatory. In the case of lactate, how­ ever, use of the DL-substrate would only result in a partial randomization since the withdrawal of the D-isomer via pyruvate and the citric acid cycle would compete with the route leading to its inversion. 2. LEUCINE

Interest in the catabolism of leucine has been associated with the effort to explain its conversion to ketone bodies, as it is one of the most strongly ketogenic of the amino acids and yields extra ketone bodies in the diabetic organism or in isolated liver tissue. Formation of ketone body from isovaleric acid was observed as early as 1906 by perfusion experi­ ments, and it was proposed that this acid might be an intermediate in the biological degradation of leucine.60-61 Later Ringer and co-workers62 found that administration of isovaleric acid to phlorizinized dogs caused the excretion of extra ketone bodies. These authors suggested that the 60

61

62

Embden, G., Solomon, H., and Schmidt, F., Beitr. Chem. Physiol. u. Path. 8, 129 (1906). Baer, J., and Blum, L., Naunyn-Schmiedeberg's Arch, exptl. Pathol. Pharmakol. 55, 89 (1906). Ringer, A. I., Frankel, E . M., and Jonas, L., / . Biol. Chem. 14, 525 (1913).

66

D . M.

GREENBERG

isovaleric acid undergoes demethylation to form butyric acid, which is then oxidized directly to acetoacetic acid. Later isotopic investigations to be discussed below have shown this hypothesis to be incorrect. The isotopic experiments have led to the scheme of catabolism shown in Fig. 5, which is a slight modification of the one published by Coon.63 CH3

\+

*

CH 3 * CH 3

Δ

C H - -CH2—CHNH2COOH Leucine / 1

Δ \ CHCH2—CO—COOH +

* / «-Ketoisocaproic acid CH 3 -CO2

2

* 3 CH

*

CH 3

*

CH 3

*

CH 3

i

Δ \ +CH-!-CH —COOH 2

Isovaleric acid

/

/

3 /

\ 3 '

\

1

4'ir

\ + / J

CH

Λ

+

Δ

CHsCOOH Δ

Jl

Δ

HCHg—COCH2COOH Acetoacetic acid

*

CH 3 —CO--CH 3 Aceton e C02

CH 3 —CO—CH 2 —COOH F I G . 5. Proposed scheme for the catabolism of leucine.

A clue to the catabolism of leucine was secured from the observation of Bloch64 that administration of deutero leucine or isovalerate yielded a high concentration of deuterium in cholesterol and in the acetyl group of N-acetyl phenylaminobutyric acid. This demonstrated that a 2-carbon unit is a normal breakdown product of leucine and of isovaleric acid. The equivalence obtained with respect to the formation of acetate from leucine and isovaleric acid offered support to the hypothesis that conversion to 63 64

Coon, M. J., J. Biol. Chem. 187, 71 (1950). Bloch, K , J. Biol. Chem. 155, 255 (1944).

CARBON CATABOLISM OF AMINO ACIDS

67

isovaleric acid is a normal pathway in the metabolism of leucine. The efficiency of acetyl formation by leucine was interpreted to mean that 1 mole of acetic acid or 3^ mole of acetoacetic acid was formed per mole of leucine, which is much lower than has been obtained. More precise information on the pathway of leucine catabolism was obtained from studies on the formation of ketone bodies in liver slices incubated with C13- and C14-labeled leucine and isovaleric acid.65'66 In these experiments it was found that leucine-3-C14 yielded acetoacetate in which the label was virtually all contained in the methyl and methylene carbons, and to approximately the same extent in each of these. Only a trace of radioactivity was found in the carboxyl carbon. On incubation with leucine-4-C14, the label occurred solely in the carbonyl group. This suggested that the isopropyl group of the amino acid had been directly converted to acetone. The over-all conclusion was that the isopropyl group forms acetone, and carbons 2 and 3 of the amino acid yield a 2-carbon fragment which can condense to acetoacetate. The acetoacetate formed from leucine-4-C14 was not symmetrically labeled, the isotope being present only in the carbonyl carbon. In experiments with isovaleric acid-4,4'-C 13 -l-C 14 , 50% to 8 5 % of the labeled methyl groups were present in the acetone from the ketone bodies, whereas only one-third to one-sixth was C14 from the carboxyl carbon. This, taken in conjunction with the fact that the respiratory C 0 2 con­ tained ten to twenty times as much C14 as C13, indicates that the carboxyl fragment is more rapidly oxidized than the isopropyl moiety and that the isopropyl moiety is more readily converted to ketone bodies. The question arises as to whether acetone is a primary product of the oxidation of isovalerate or is formed secondarily by decarboxylation of acetoacetate. The experimental results of Zabin and Bloch66 are in accord with a reaction mechanism in which isovaleric acid is oxidized initially at the carbon 2-position to yield a 3-carbon and a 2-carbon fragment. This conclusion is based on the high absolute C13 concentrations found in the methyl carbons of acetone and also in comparison to that in the methyl or methylene carbons of acetoacetate. Secondly, the C 13 :C 14 ratios were significantly greater in the acetone fractions than in the corresponding carbon atoms of acetoacetate. This finding can be explained only if acetone is formed directly from isovaleric acid, but is at variance with the assumption that acetone arose exclusively by decarboxylation of aceto­ acetate. The authors also point out that it is possible that the postulated 3-carbon intermediate is not acetone, but that acetone is formed in a side reaction from a more labile 3-carbon precursor. 65 66

Coon, M. J., and Gurin, S., / . Biol. Chem. 180, 1159 (1949). Zabin, I., and Bloch, K., J. Biol Chem. 185, 117 (1950).

68

D. M.

GREENBERG

Advances in the knowledge of the metabolic reactions of acetoacetate and the further studies of Coon63 on leucine metabolism solved the remain­ ing uncertainties in the interpretation of the previous experimental data. This author demonstrated that the 3 carbons of the isopropyl group of leucine are incorporated as a unit into the 2-, 3-, and 4-positions of acetoacetic acid. The carboxyl carbon probably arises by a C 0 2 fixation reac­ tion, which had not been recognized previously. According to this scheme the complete breakdown of a mole of leucine by liver leads to the forma­ tion of approximately 1.5 moles of ketone bodies. Coon studied the conversion of carboxyl, ß-carbon, and methyl-labeled isovalerate to acetone bodies. As had been found by previous workers, the carboxyl labeling yielded acetoacetate with tracer in the carboxyl and carbonyl carbons. This is in agreement with reactions 3' and 4' of Fig. 5. It is interesting that the ratios of carboxyl to carbonyl radioactivity in the acetoacetate was higher than unity. This parallels the situation found with the 2-carbon unit arising from the first 2-carbons of straight-chain fatty acids. It has been found that isovalerate-3-C 14 yields acetoacetate labeled primarily in the carbonyl position 63 and isovalerate 4,4'-C14-yields aceto­ acetate with radioactivity concentrated in the methylene and methyl carbons, and approximately to the same extent in each portion. This shows that all 3 carbons of the isopropyl group are involved in acetoace­ tate formation, and it eliminates the possibility that a highly acetylating 2-carbon fragment is formed from the isopropyl group. The origin of the carboxyl carbon of the acetoacetate by the reversible fixation of C 0 2 by acetone to yield acetoacetate has been established by a number of investigators. 67-69 The formation of acetoacetate from the isopropyl moiety of leucine, then, appears to follow the course depicted in reactions 3 to 5 of Fig. 5.* It has been noted that a greater amount of ketone bodies was produced from the D- or DL-leucine than from the L-isomer. Since both optical isomers yield the same keto acid upon oxidative deamination, their carbon chains would not be expected to meet with different catabolic fates. The differ­ ence, it would appear, is the result of a difference in the rates of deamina­ tion of the D- and L-isomers. 3. ISOLEUCINE

The information on the reactions of the catabolism of isoleucine is still quite scanty. The scheme given in Fig. 6 is based on the results of 67

Borek, E., and Rittenberg, D., / . Biol. Chem. 179, 843 (1949). Sakarni, W., Federation Proc. 9, 222 (1950); J. Biol. Chem. 187, 369 (1950). 69 Plaut, G. W. E., and Lardy, H. A., / . Biol. Chem. 192, 435 (1951). * See Addendum 1, p. 111.

68

C A R B O N CATABOLISM O F A M I N O

69

ACIDS

experiments with 2-methylbutyrate preparations 70 isotopically labeled in the different positions shown. Isoleucine, in contrast to leucine, is glycogenic and only weakly ketogenic. 43 Incubation of liver slices with the variously labeled 2-methylbutyrates led to the following results: The carboxyl-labeled 2-methylbutyrate CH3—CH2—CH—CHNH2COOH CH3 Isoleucine CH3—CH2—CH—CO—COOH CH 3 1 -Keto-2-methy 1 valer ate 2I + I CH3—CH2—CH--COOH 1 1

CH 3 2-Methylbutyrate [cH3CO—]

n

+ + CH3COCH2COOH

+

[CH3—CH2COOH]

n

[Symmetric Intermediate (s)] 6 j CH —COCOOH 3 7

Γ *

1

*

0

Ί

LCH3—CO— + CO2J

n

CH3—CO—CH2—COOH FIG. 6. Proposed scheme of catabolism of isoleucine.70

yielded acetoacetate in which the acetone moiety was nonradioactive and the carboxyl carbon had only a trace of C14. About 5 % of the label appeared in the respiratory C0 2 . 2-Methylbutyrate-3-C 14 yielded aceto­ acetate in which the label appeared only in the carbonyl and carboxyl carbons (Fig. 6, reaction 4). Of the C14 of this compound, about 9% was recovered as C 0 2 and 15% as acetoacetate. The ratio of C14 in COOH: CO in the acetoacetate was determined to be 0.82. This is exceptional, for the terminal 2-carbon unit of a straight-chain fatty acid equivalently labeled yields acetoacetate with a still greater proportion of the label in the car­ bonyl portion. Butyrate-3-C 14 , for example, gave a COOH:CO ratio of 0.30. The difference in labeling, it was shown, was not the result of any 70

Coon, M. J., and Abrahamsen, N. S. B., J. BioL Chem. 195, 805 (1952); Coon, M. J., Abrahamsen, N. S. B., and Greene, G. S., / . Biol. Chem. 199, 75 (1952).

70

D. M.

GREENBERG

influence of isoleucine, 2-methylbutyrate, or propionate on the distribu­ tion of the label in the 2-carbon units produced from butyrate. Upon incubation of 2-methyl-C 14 -butyrate, about 4 % of the radio­ activity appeared in the respiratory CO2 and about an equal amount in the acetone bodies. The major portion of the label was contained in pro­ pionate. This was isolated by the carrier technique and the identity of the compound demonstrated by conversion to the p-bromophenacyl ester. No labeled propionate is formed from the corresponding 2-methylbutyrate-3-C 14 . The isotopic experiments lead to the scheme of catabolism given in Fig. 6. The scheme is in agreement with the conclusion of Carter 71 that α-methyl fatty acids undergo ß-oxidation on the longer carbon chain only. It also is in agreement with the conclusions of Wick72 that 2-methylbutyrate neither undergoes reductive demethylation nor does it give rise to 2 carboxyl-butyraldehyde and butyrate. The experimental results indicate that butyrate is not an obligatory intermediate in the chain of catabolic reactions. To explain the labeling of all 4 carbons in the acetoacetate formed from 2-methyl-C 14 -butyrate (reactions 6, 7, and 8) it is proposed that randomization occurs by C 0 2 fixation to pyruvate to form oxalacetate, which is then converted to the symmetric succinate. The problem is the same as that discussed in connection with the oxidation of valine (see p. 64). The transamination of L-isoleucine to d-a-keto-ß-methylvalerate and of L-alloisoleucine to Ζ-α-keto-ß-methylvalerate by a hog heart preparation and the cell-free extracts of a number of microorganisms (e.g., Lactobacillus arabinosus) has been demonstrated by Meister. 73 VI. CERTAIN AMINO ACIDS OF UNCERTAIN BIOLOGICAL SIGNIFICANCE There is very little exact information about the catabolic pathways of the three amino acids, α-aminobutyric acid, norvaline, and norleucine. They have been termed "unnatural" amino acids because they have not been found to occur in proteins. However, from the general knowledge of the metabolic reactions of the amino acids and of the deaminated carbon residues, their metabolic fates can be predicted with a high degree of prob­ ability . \Tt' has been established that the animal organism can tolerate 71 72 73

Carter, H. E., Biol. Symposia 5, 47 (1941). Wick, A. N., / . Biol. Chem. 141, 897 (1941). Meister, A., / . Biol. Chem. 195, 813 (1952).

CARBON CATABOLISM OF AMINO ACIDS

71

and metabolize these amino acids 74,75 as well as most of the D-amino acids. 76 1. ALPHA AMINOBUTYRIC ACID

Small amounts of this amino acid are found to be widely distributed in the nonprotein extracts of tissues by paper chromatography. Dent 77 has reported its appearance in the urine of a patient with hepatic disease following the administration of large amounts of methionine. Lien and Greenberg 29 have shown that is formed from threonine in the animal body. a-Aminobutyric acid is transaminated by heart muscle at a compara­ tively slow rate. 20 The D-form is oxidized by D-amino acid oxidase.78 It has CH3CH2CHNH2COOH a-Aminobutyric acid CH3CH2—CO—COOH α-Ketobutyric acid

1

CH3CH2—COOH + C 0 2 F I G . 7. Proposed scheme of catabolism of α-aminobutyric acid. For the subsequent oxidation of propionic acid see Valine (p. 59).

also been demonstrated that it is antiketogenic. 43 In view of these facts the probable pathway for the catabolism of this amino acid can be written as shown in Fig. 7. 2.

NORVALINE

(CX-AMINOVALERIC

ACID)

43

Norvaline is strongly ketogenic. The L-form is attacked by L-amino acid oxidase41 and the D-form by D-amino acid oxidase.78 Its susceptibility to transamination has not been reported. Its ready oxidation to C 0 2 in the intact animal has been observed by Hassan and Greenberg. 74 More of this amino acid is excreted unchanged in the urine than is leucine. Evidence for the formation of a 2-carbon unit was also obtained in this work. This leads to the scheme for the catabolism of norvaline shown in Fig. 8. 3. NORLEUCINE («-AMINOCAPROIC ACID)

Norleucine also is readily oxidized to C 0 2 in the intact animal. 74 A few per cent is excreted unchanged in the urine. 74 The L-amino acid is trans­ aminated by heart muscle.20 The D-form is strongly attacked by D-amino acid oxidase.78 A plausible scheme for its catabolism is given in Fig. 9. 74 75 76 77 78

Hassan, M., and Greenberg, D . M., Arch. Biochem. and Biophys. 39, 129 (1952). Armstrong, M. D., and Binkley, F., J. Biol Chem. 180, 1059 (1949). Pilsum, J. F . V., and Berg, C. P., / . Biol. Chem. 183, 279 (1950). Dent, C. N., Science 105, 335 (1947). Binder, A. E., and Krebs, H. A., Biochem. J. (London) 46, 210 (1950).

72

D. M.

GREENBERG

CH3CH2CH2—CHNH2—COOH Norvaline

I

CH3CH2CH2CO—COOH α-Ketovaleric acid

I

CH3CH2CH2—COOH + C0 2 Butyric acid

I 2CH3COOH FIG. 8. Proposed scheme of catabolism of norvaline. CH3CH2CH2CH2—CHNH2—COOH Norleucine

i

CH3CH2CH2CH2COOH + C0 2 Valeric acid

1

CH3CH2CO—CH2—COOH ß-Ketovaleric acid

i

CH3CH2COOH + CH3COOH Propionic acid Acetic acid FIG. 9. Proposed scheme of catabolism of norleucine.

VII. T H E SULFUR AMINO ACIDS Although there is a fairly extensive knowledge of the metabolism of the sulfur moieties of these amino acids, which is discussed in the chapter, Metabolism of Sulfur-Containing Compounds, there is little definite information about the fate of their carbon residues. A few inferences can be drawn from the incomplete and scattered data at hand, and these are discussed below. 1. CYSTINE AND CYSTEINE

For lack of knowledge to the contrary we will assume that the carbon catabolism of these two amino acids is identical. An incomplete pathway of the carbon catabolism of cysteine with­ out loss of sulfur is to taurine (see the chapter, Metabolism of SulfurContaining Compounds). As a more general pathway of metabolism in which the compound is completely degraded, we may take as a model the reaction of the cysteine desulfhydrases,79 which form pyruvic acid from the carbon chain. The subsequent metabolism of the pyruvic acid should then be along the well79

Fromageot, C , Advances in Enzymol. 7, 369 (1947); Enzymes 1 (pt. 2), 1237 (1951).

C A R B O N CATABOLISM O F A M I N O

73

ACIDS

known pathways of carbohydrate metabolism. In support of this it has been found that cysteine yields increased glucose in the diabetic animal. 80 On the other hand several investigators have found that administration of cystine does not lead to the laying down of liver glycogen,81 nor does it have a ketolytic effect.82 This represents the sum total of our present information regarding the fate of the carbon moiety of cystine and cysteine. 2.

METHIONINE

The catabolic patterns shown in Fig. 10 are largely hypothetical. There is good evidence for relationships of homoserine, a-keto-7-methiolbutyric acid, and α-aminobutyric acid to the metabolism of methionine, CH2—SCHj

CH2OH

COOH

CH2

CH2

CH2

CHNH2

CHNH2

__

CHNH 2 ^~ COOH Aspartic acid

COOH Homoserine

COOH Methionine

COOH I CO I COOH

Oxalacetic acid

-

C—CH2—CH—COOH C—OH

NH2

C

ΊΙ

^

N " H ß-3-Oxindolylalanine

NHo

N

(10)

H

2-Hydroxytryptophan

When synthesis of this compound was accomplished,181»182 experi­ ments with it made it clear that it is not a normal tryptophan metabolite. The metabolism of oxindolylalanine was found to be quite different from that of tryptophan or kynurenine in rat liver slices,183 or in the intact animal. 184 The paper chromatographs of the urines from normal and pyridoxine-deficient rats fed oxindolylalanine were quite different from those obtained when tryptophan was fed. Furthermore, the tryptophan peroxidase-oxidase enzyme system does not act on this compound, nor was it metabolized by the bacillus, Pseudomonas fluorescens, which had been adapted to tryptophan or kynurenine. 185 The identity of the first intermediate of tryptophan oxidation, therefore, is still unknown. It has been suggested 177185 that H2O2 might add to the double bond of the indolyl ring to give either 2-hydroxy- or 2,3-dihydroxytryptophan. Since 2-hydroxytryptophan is the tautomer of ß-3-oxindolylalanine, the arguments against the latter also apply to it. 181

Kotake, M., Sakan, T., and Miwa, T., J. Am. Chem. Soc. 72, 5085 (1950). Cornforth, J. W., Cornforth, R. H., Dalgliesh, C. E., and Neuberger, A., Biochem. J. (London) 48, 591 (1951). 183 Mason, M., and Berg, C. P., / . Biol. Chem. 188, 783 (1951). 184 Dalgliesh, C. E., Knox, W. E., and Neuberger, A., Nature 168, 20 (1951). 1! 5 > Sakan, T., and Hayaishi, O., J. Biol. Chem. 186, 177 (1950).

182

96

D. M.

0 v

GREENBERG

OH

I

C—CH 2 —CH—COOH C—OH

NH2

2,3-Dihydroxytryptophan

If it is the first intermediate, it can be speculated that the 2,3-dihydroxytryptophan, by dehydrogenation with simultaneous ring opening, could yield formylkynurenine. This is a hypothesis which remains to be proved. After the oxidation of tryptophan to kynurenine, the side chain can be cleaved to yield anthranilic acid, or if prior oxidation to 3-hydroxykynurenine takes place, the product is 3-hydroxyanthranilic acid. Alter­ natively, probably after the removal of the α-amino group to form the a-keto acids corresponding to kynurenine and 3-hydroxykynurenine, ring closure of the side chain can occur to yield kynurenic and xanthurenic acids. These alternative reactions will be discussed in turn. 3. KYNURENINASE

This enzyme has been shown to occur in liver and kidney 186 and also in certain microorganisms. 172 It decomposes L-kynurenine to anthranilic acid and hydroxykynurenine to 3-hydroxyanthranilic acid. According to Wiss and Fuchs, 187 the enzyme is able to split a variety of compounds containing the —COCH 2 CH(NH 2 )COOH group. Braunstein et al18* observed that alanine was produced by the fission of the side chain and that a pyroxidine derivative was the coenzyme for the reaction. The formation of L-3-hydroxykynurenine as a tryptophan metabolite has been demonstrated in animals. 189 This compound appears in the urine only in pyridoxine-deficient animals. Braunstein and co-workers188 demonstrated that pyridoxine deficiency did not affect the conversion of tryptophan to kynurenine. They showed that the kynureuinase activity of the liver of pyridoxine-deficient animals was greatly reduced and could be restored in vitro by the addition of pyridoxal phosphate—a result that has been confirmed by Dalgliesh et αΖ.184 According to Knox, removal of the alanyl side chain becomes the limiting step in the metabolism of tryptophan in pyridoxine deficiency and permits the accumulation of kynurenine, hydroxykynurenine, and their conversion products. 1890 Administration of extra tryptophan to the 186

Kotake, Y., and N a k a y a m a , T., Hoppe-Seyler's Z. physiol. Chem. 270, 76 (1941). Wiss, 0., and Fuchs, H., Experientia 6, 1472 (1950). 188 Braunstein, A. E., Goryachenkova, E . V., and Pashkina, T. S., Biokhimiya 14, 163 (1949); cf. C. A. 43, 6264« (1949). 189 Dalgliesh, C. E., Biochem. J. (London) 52, 3 (1952). i89a Knox, W. E., Biochem. J. (London) 53, 379 (1953). 187

CARBON CATABOLISM OF AMINO ACIDS

97

deficient animals can produce an increase in the tryptophan peroxidase activity 179 and thus greatly increase the kynurenine-forming potential of the animals. Dalgliesh et al.1SA were able to dissociate the coenzyme from the enzyme prepared from the livers of normal animals and thereby verify the requirement for pyridoxal phosphate. They also observed that after prolonged pyridoxine deficiency, kynureninase activity could no longer be restored in vitro by the addition of pyridoxal phosphate. More recently Wiss190 found that kynureninase inactivated by dialysis could be reactivated by pyridoxal-5-phosphate, but not by pyridoxal-2phosphate. As pyridoxine deficiency has no observable effect on the con­ version of hydroxyanthranilic acid to nicotinic acid,188 the action of this vitamin appears to be connected solely with the removal of the alanyl side chain at the kynurenine level. 4. FORMATION OF KYNURENIC AND XANTHURENIC ACIDS

Kynurenic acid was discovered in 1853 by Liebig191 and has been isolated from the urine of many mammalian species. It was shown to be a metabolic product of tryptophan by Ellinger192 by feeding tryptophan to rats. He also proposed a structural formula for it which, however, was not quite correct. The correct structure was established by Homer. 193 Kynurenic acid is formed from L-tryptophan and from indolepyruvic acid, but not from the D-amino acid.194 Xanthurenic acid was isolated from urine by Musajo in 1935195 and shown to be 4,8-dihydroxyquinoline-2-carboxylic acid. Because of its 8-hydroxyquinoline structure it forms colored chelates with metal ions and, in particular, gives an intense green color with ferric salts. Xan­ thurenic acid is only excreted in pyridoxine deficiency, as was discovered by Lepkovsky and co-workers. 170196 It is found only in pyridoxine-deficient animals fed L-tryptophan or kynurenine. Addition of pyridoxine or the elimination of tryptophan from the diet causes it to disappear from the urine. 170 Xanthurenic acid fed to normal animals does not accumulate in the body and is not lost in the urine. This shows that a mechanism for the degradation of this compound must exist in the body, which is a further 190

Wiss, O., Z. Naturforsch. 7b, 133 (1952). Liebig, J., Ann. chem. Justus Liebigs 86, 125 (1853). 192 Ellinger, A., Hoppe-Seyler's Z. physiol. Chem. 43, 325 (1904). 193 Homer, A., / . Biol. Chem. 17, 509 (1914). 194 Borchers, R., Berg, C. P., and Whitman, N . E., / . Biol. Chem. 113, 125 (1936). 195 Musajo, L., Atti reale accad. nazl. Lincei 21, 368 (1935), Gazz. chim. ital. 67, 165, 171, 182 (1937). 196 Lepkovsky, S., Roboz, E., and Haagen-Smit, A. J., / . Biol. Chem. 149, 195 (1943). 191

98

D. M.

GREENBERG

pathway for the complete catabolism of tryptophan through the quinoline pathway. The activity of this mechanism depends upon the presence of pyridoxine. Thus pyridoxine plays still another role in connection with the metabolism of tryptophan, in addition to being a part of the coenzyme of kynureninase and kynurenine transaminase. A clue to the mechanism of formation of kynurenic and xanthurenic acids is contained in the observation that kynureninase preparations exhibit transaminase activity. In the animal, Wiss190 reported that kynurenine is converted to kynurenic acid by liver preparations only if pyruvic or α-ketoglutaric acid is present. The presence of a kynurenine transaminase in the microorganism Pseudomonas sp., str. Tr-7, has been established by Miller, Tsuchida, and Adelberg.197 The enzyme prepara­ tion was active only in the presence of α-ketoglutaric acid and caused the formation of kynurenic acid and glutamic acid. It was deduced that 2-aminobenzoylpyruvic acid (Fig. 13) is the primary transamination product, although it has not been isolated, and that ring closure to form kynurenic acid occurs spontaneously. Xanthurenic acid would be ex­ pected to be formed analogously by the transamination of 3-hydroxykynurenine. The spontaneous rather than an enzymatic ring closure of 2-aminobenzoylpyruvic acid is supported by the observation that kynurenic acid is formed from L-kynurenine when it is deaminated by the L-amino acid oxidase of Neurospora. The observation of Dalgliesh et αΖ.184 that kynurenic acid was formed in their incubation mixtures in the absence of α-ketoglutarate or pyruvate and could be increased by the addition of excess pyruvate can be explained by the presence of small amounts of transaminase and of the acceptor keto acid as impurities in their enzyme preparations. The suggestion of Dalgliesh et al.lM that 2-aminobenzoylpyruvic might be formed in the kynureninase reaction and this could, as an alter­ native to cyclization, be split to anthranilic acid and pyruvic acid and the latter then animated to alanine has been negated by the observation that kynurenine decomposed by purified bacterial kynureniase in the presence of C14-labeled pyruvate forms unlabeled alanine. 1970 This rules out free pyruvic acid as an intermediate. Hayaishi and Stanier 198 found no evidence of kynurenic acid formation by bacterial kynureninase, either crude or purified. Analysis of the path­ ways of tryptophan metabolism by many different bacterial strains 199 at first indicated that there was a sharp dichotomy of reaction sequence 197

Miller, I. L., Tsuchida, T., and Adelberg, E. A., / . Biol. Chem. 203, 205 (1953). i97a Miller, I. L., and Adelberg, E. A., J. Biol. Chem. 205, 691 (1953). 198 Hayaishi, O., and Stanier, R. Y., / . Biol. Chem. 195, 735 (1952). 199 Stanier, R. Y., Hayaishi, O., and Tsuchida, M., / . Bacteriol. 62, 355 (1951).

C A R B O N CATABOLISM O F A M I N O

ACIDS

99

below kynurenine, some strains metabolizing this amino acid exclusively via anthranilic acid and alanine and others exclusively via kynurenic acid. It appeared at first that only bacterial strains that metabolize tryptophan via anthranilic acid contained any kynureninase. Strains that form kynurenic acid were found to contain transaminase. 200 More recently at least one strain of Pseudomonas was found to contain enzymes for both the aromatic and quinohne pathway. 200 Because it was observed that tryptophan-ß-C 14 administered to rats produced acetyl groups and glucose symmetrically labeled, Sanadi and Greenberg 201 concluded that a 2-carbon unit equivalent to C 14 H 3 COOH was split off from the side chain of the tryptophan. In the light of more recent work it is apparent that the labeled unit could just as well have been methyl-labeled pyruvate or the alanine which, in turn, could be converted to pyruvate by transamination. The increased excretion of kynurenic and xanthurenic acids observed in pyridoxine deficiency is probably due to the preferential combination of the pyridoxal phosphate coenzyme with the transaminase. By prevent­ ing the loss of the side chain, as a result of a decreased activity of kynuren­ inase in pyridoxine deficiency, cyclization is favored leading to increased formation of the two acids above. 5. TRYPTOPHAN CATABOLISM IN BACTERIA

a. Tryptophanase Reaction The presence of indole and skatole derivatives in urine and fecal contents has long been known. It was demonstrated by Hopkins and Cole202 that indole was formed from tryptophan by E. coli. The mechanism of the over-all reaction of indole formation was established by Woods, Gunsalus, and Umbreit, 203 and was confirmed by Davis and Happold. 204 Employing partially purified tryptophanase preparations from extracts of E. coli, it was demonstrated that the reaction yielded indole, pyruvic acid, and N H 3 in approximately equimolar ratio, and that there was no uptake of oxygen.205 The enzyme preparation did not deaminate 200

Stanier, R. Y., and Adelberg, E . A., Personal communications. Sanadi, D . R., and Greenberg, D . M., Arch. Biochem. 25, 323 (1950). 202 Hopkins, F . G., and Cole, S. W., J. Physiol. (London) 29, 451 (1903). 203 Woods, W. A., Gunsalus, I. C., and Umbreit, W. W., / . Biol. Chem. 170, 313 (1947). 204 Davis, E . A., and Happold, F . C., Biochem. J. {London) 144, 349 (1949). 205 Previous investigations of this subject led to t h e misleading results t h a t oxygen was consumed and C 0 2 produced in the reaction; also t h a t riboflavin and D P N , as well as pyridoxal phosphate, were required for maximal activation. T h e confusion resulted from the fact t h a t oxidation of pyruvate was being measured in the crude bacterial preparations as well as the decomposition of t r y p t o p h a n to indole. For literature, see 206 . 206 Happold, F . C., Advances in Enzymol. 10, 51 (1950). 201

100

D. M.

GREENBERG

alanine or serine. The coenzyme for tryptophanase was shown to be pyridoxal phosphate. The over-all reaction is represented by equation 11.

S\ -C II χ / \ /N - OH H

/ \

CH2

I

+ H

CHNH■ T COOH

-CH

CH3

' v \ N /CH + COOH ?° + NHs

! 0

(11)

H

The specificity requirements for this enzyme have been shown to be a free —COOH group, an unsubstituted —NH 2 group, a ß-carbon atom capable of oxidative attack, and an unsubstituted indole nitrogen atom. 206 " Beerstecher and Edmonds 2066 claimed that pyruvate and indole accelerated the reaction autocatalytically. The authors speculate that indole and pyruvate function by regenerating the coenzyme (pyridoxal phosphate) from its binding with tryptophan or tryptophan analogs. b. Other Catabolic Pathways in Bacteria Two alternate pathways for the complete oxidation of tryptophan 206 have been uncovered in microorganisms, as mentioned above. 172 The analysis of the adaptive patterns of the Pseudomonas group has proved of great value for this purpose. Both of these pathways apparently are initiated by the peroxidase-oxidase system and formylase which leads to the formation of kynurenine from tryptophan. The presence of kynureninase leads to the aromatic pathway and the formation of anthranilic acid and catechol, whereas the quinoline pathway, it now appears, depends upon the presence of a transaminase which induces the formation of kynurenic and xanthurenic acids. It was found that every strain using the quinoline pathway oxidizes the D-isomer at a high rate, whereas strains using the aromatic pathway either do not act on the D-compound, or only at a very low rate. Two bacterial strains that utilize the aromatic pathway have an impaired ability to oxidize anthranilic acid and thus accumulate large quantities of this compound in the incubation medium, when fed either tryptophan or kynurenine. The chemical products formed are shown below: L-Kynurenine —> anthranilic acid —* catechol —> eis, m-muconic acid —> ß-ketoadipic acid (12) L-Tryptophan —> L-kynurenine y—»kynurenic acid —> ?

(13)

D-Tryptophan —> D-kynurenine 206a

Baker, J. W., Happold, F . C , and Walker, N., Biochem. J. {London) 40, 420 (1946.) 2°6b Beerstecher, E. R., Jr., and Edmonds, E . J., / . Biol Chem, 192, 497 (1951).

CARBON CATABOLISM OF AMINO ACIDS

101

By means of extracts of acetone-dried bacterial cells,207,208 the further steps of the aromatic pathway of catabolism have been established. The enzyme system in these extracts, named pyrocatechase, oxidized catechol to eis, m-muconic acid209 with an uptake of 2 atoms of oxygen. Another such enzyme preparation catalyzed the oxidation of anthranilic acid, with an oxygen uptake of 2 atoms, to ß-ketoadipic acid. This observation can be explained in terms of the reaction steps shown above (equation 12). Only the eis, eis isomer209 of muconic acid is an intermediate in the bac­ terial degradation of benzoic acid and phenol to ß-ketoadipic acid, whereas the eis, trans and the trans, trans isomers have been reported to be inac­ tive. 211 The cell extracts that follow the aromatic pathway show strong activity against catechol, but are inactive against tryptophan itself. Grinding the wet cells with alumina yielded extracts containing many additional enzymes, which catalyze the oxidation of tryptophan with the consumption of 8 atoms of oxygen per mole, and yields ß-ketoadipic acid as one of the end products. These bacterial extracts contain the peroxidase-oxidase enzymes which degrade tryptophan to formylkynurenine and also a powerful kynureninase which causes the nonoxidative cleavage of L-kynurenine to anthranilic acid and L-alanine. The observations that have been made permit the construction of a broad conceptual scheme that describes the oxidation of tryptophan by many bacteria, and that links up one of the possible alternate pathways for the metabolism of tryptophan with a series of other primary oxidative sequences previously known to occur in bacteria. The oxidative pattern of the vertebrates diverges from that of the microorganisms at the point involving the fission of the benzene ring. The animal body converts aromatic compounds to the trans-trans muconic acid.210 This acid is excreted in the urine on feeding benzene and other aromatic compounds, and it has been established by Parke and Wil­ liams210 that the trans4rans muconic acid is formed primarily in the cleavage of the benzene ring by the animal. XIII. HISTIDINE Histidine is of interest biologically for a variety of reasons in addition to the fact that it is a naturally occurring amino acid. In the young rat 207

Hayaishi, O., and Hashimoto, K , Med. J. Osaka Univ. 2, 33 (1950). Hayaishi, O., and Stanier, R. Y., J. Bacteriol. 62, 691 (1951). 209 Parke a n ( j Williams,210 however, point out that the eis, irans-mueonie acid is readily transformed to the eis, eis form on crystallization from water, and, there­ fore, they consider that it is probable that the eis, frans-mueonic acid is actually the true product. This is difficult to understand in view of the inactivity of the eis, trans acid.211 210 Parke, D. V., and Williams, R. T., Biochem. J. (London) 51, 339 (1952). 111 Evans, W. C , and Smith, B. S. W., Biochem. J. (London) 49, x (1951). 208

102

D. M.

GREENBERG

it is an essential nutrient for growth, but the adult man can be main­ tained in nitrogen balance even if it is omitted from the diet. The com­ pounds carnosine (ß-alanylhistidine) and anserine (ß-alanyl-1 -methyl histidine) occur in muscle. Their particular function remains an enigma. Another histidine derivative of biologically unknown function that occurs in mammalian red cells and in ergot is thioneine, the betaine of thiolhistidine. In addition, histidine is converted to histamine by many body tissues. The latter is a compound with very potent effects on smooth muscle. Structural formulas of these compounds are shown below: HC-

C—CH2—CHNH2

I

I

N

I

NH

\

I

HC==C—CH 2 —CH—NHCOCH 2 —CH 2 NH 2

I

COOH

N

/

!

NH

COOH

% /

c

c

H H CH=CH—CH Histidine Carnosine 2 —CH—NHCOCHa—CH2NH2

CH=CH—CHa—CH—N(CH 8 ) 3

N

N

I

I

\ c/

NCHs

I

COOH

I

I

\ c / NH

H Anserine

I

COO"

SH Thioneine CH=CH—CH2—CH2NH2

v

NI

I NH

H Histamine

The presence of similar structural groups in their molecules led to much speculation and experimental work to demonstrate whether histi­ dine could serve as a precursor for the synthesis of arginine, creatine, the purines, and the pyrimidines. These deductions have all been shown to be erroneous in the course of time. 1. HISTORICAL DEVELOPMENT

The decomposition of histidine by liver brei was observed independ­ ently by Edlbacher 212 and by György and Röthler, 213 Edlbacher and 212 213

Edlbacher, S., Hoppe-Seyler's Z. physiol. Chem. 157, 106 (1926). György, P., and Röthler, H., Biochem. Z. 173, 334 (1926).

CARBON CATABOLISM OF AMINO ACIDS

103

co-workers214 demonstrated that histidine was decomposed by liver extracts to NH3, formic acid, and a derivative which yields glutamic acid upon further treatment with alkali. The enzyme causing this reaction was named histidase, and was described as capable of liberating 1 mole of NH3 from histidine and producing a compound which yielded a second mole of N H 3 on treatment with strong alkali.214 Histidase activity was shown to occur only in liver and to be present in the livers of all verte­ brates tested. Only L-histidine was decomposed. Urocanic acid has been isolated from the urine of various animals fed L-histidine (see215 for older references). None was obtained from D-histidine.216 Reports that urocanic acid was excreted following subcutaneous injection of histidine could not be confirmed.215 Furthermore, the older work has been criticized because of inadequate isolation methods. Orally administered urocanic acid is promptly excreted. The urocanic acid obtained in histidine feeding experiments, it was suggested, could have been formed by intestinal bacteria, as certain species have the capacity to convert histidine into urocanic acid.217 The hydrolytic splitting of urocanic acid by liver extracts was first observed by Sera and Yada, 218 and confirmed by others. 219-222 The enzyme that causes this decomposition was named urocanase. Edlbacher and co-workers214·221 concluded that urocanic acid forma­ tion represented a minor pathway in the metabolism of histidine. It was their contention that histidase directly caused the opening of the imidazole ring of histidine to yield the compound with the properties men­ tioned above. Indirect support for this claim was provided by the obser­ vation that urocanic acid, when administered to rabbits, was not easily metabolized, 215 and upon injection into guinea pigs was excreted in the urine practically quantitatively. 223 The Japanese investigators 218_220 ' 224 proposed an alternate pathway, namely, that histidine was first deaminated to urocanic acid by an enzyme named by them histidine deaminase, and the urocanic acid, in 214

Edlbacher, S., Ergeh. Enzymforsch. 9, 131 (1943). Darby, W. J., and Lewis, H. B., J. Biol. Chem. 146, 225 (1942). 216 Konishi, M., Hoppe-Seyler's Z. physiol. Chem. 143, 189 (1925). 217 Raistrick, H., Biochem. J. (London) 11, 71 (1917). 218 Sera, K., and Yada, S., / . Osaka Med. Soc. 38, 1107 (1939). / . Japan. Biochem. Soc. 15, 3 (1940); J. Osaka Med. Soc. 41, 745 (1942). 219 Takeuchi, M., / . Biochem. (Japan) 34, 1 (1941). 220 Oyamada, V., J. Biochem. (Japan) 36, 227 (1944). 221 Edlbacher, S., and Bidder, H., Hoppe-Seyler's Z. physiol. Chem. 273, 165 (1942). 222 Hall, D . A., Biochem. J. (London) 51, 499 (1952). 223 Edlbacher, S., and Hertz, F., Hoppe-Seyler's Z. physiol. Chem. 276, 117 (1942). 224 K o t a k e , Y., Hoppe-Seyler's Z. physiol. Chem. 270, 138 (1941). 215

104

D. M. GREENBERG

turn, was converted to a product which yielded glutamic and formic acids. The over-all reaction regardless of the path by which it is reached can be represented by equation 14. Histidine + 4 H 2 0 —> 2 N H 3 + glutamic acid + formic acid

(14)

The reports of the Japanese investigators read convincingly, but the prestige of Edlbacher prevented their immediate acceptance. Recent investigations 225-227 suggest that, in spite of the poor utilization of uro­ canic acid by the body, the Japanese group were probably correct and Edlbacher wrong. The metabolic inertness of urocanic acid is a serious objection to accepting it as the key intermediate in the dissimilation of histidine. 2. CATABOLISM OF HISTIDINE BY BACTERIAL ENZYMES

The correct catabolic pattern of histidine was established first in bacterial preparations and for this reason wTill be discussed first. Cell-free extracts of Pseudomonas fluorescens were obtained which catalyze the quantitative conversion of L-histidine to L-glutamic acid, formic acid, and 2 moles of NH 3 , as required by equation 14. Proof of the formation of L-glutamate was obtained by isolating the crystalline hydrochloride from the incubation mixture and establishing its identity. 225 The demonstration that urocanic acid was an intermediate in the con­ version, and proof of the reaction pathway to glutamic and formic acids was made by employing preparations of histidine labeled with N 15 in the a or 7 position referred to the side chain or with C14 at C2 of the imidazole ring (see Fig. 14).226 On incubating these variously labeled histidines with the bacterial enzyme preparations and utilizing appropriate dilution techniques, urocanic acid was isolated which retained the label in the imidazole ring and in which the N 15 of the α-amino group was converted to NH 3 . The histidine labeled with N 15 in the y position yielded glutamic acid in which over 90% of the excess N 1 5 was found in the amino group. Incubation of histidine labeled with C14 at C2 of the imidazole ring resulted in the formation of formic acid containing all of the C14. Additional indirect evidence that this carbon yields formate or some other 1-carbon unit is that administration of histidine labeled in the 2-position to rats resulted in considerable amounts of C14 activity in 225 226

227

Tabor, H., and Hayaishi, O., / . Biol. Chem. 190, 171 (1952). Tabor, H., Mehler, A. H., Hayaishi, O., and White, J. W., J. Biol. Chem. 196, 121 (1952). Mehler, A. H., and Tabor, H., Federation Proc. 11, 374 (1952); / . Biol. Chem. 201, 775 (1953).

105

CARBON CATABOLISM OF AMINO ACIDS

compounds which are known to incorporate 1-carbon units including formate. 228-231 The results of the experiments with the variously labeled histidines lead to the scheme of catabolism shown in Fig. 14. 3. PATTERN OF CATABOLISM IN LIVER

Convincing evidence has been secured by Mehler and Tabor 227 that liver can convert histidine to glutamic acid and formic acid by the HC—N

I >

δΗ

1

1

1

φ histidase

CHNH 2 | COOH

I >

6 H

C—NH

C—NH CH2

HC—N

>

1 CH ||

CH

urocanase

COOH

c3 T3

u "d

COOH HC—NH 2

1

CH 2

NH 3 ♦

+

+ HCOOH

CH 2 COOH

+

Histidine

• NH 3 Urocanic Acid

Glutamic Acid

Formic Acid

F I G . 14. Scheme of catabolism established from experiments with isotopic histidine.

same reaction sequence as was found to occur with bacterial enzyme preparations. a. Formation of Urocanic Acid A new procedure for the determination of urocanic acid has been developed, based on the fact that this compound exhibits a strong absorption in the ultraviolet in the region of 240-280 πΐμ.222·227 This can be employed both to demonstrate the formation of urocanic acid and as a method of assay for the enzyme deaminating histidine to urocanic acid. With this test method it has been shown that urocanic acid accumulates when histidine is incubated with liver extract 227 or with extracts of acetonedried liver powder.222 Mehler and Tabor determined that the activity of the enzyme forming urocanic acid appeared to be sufficient to account for the total histidine degradation of liver extracts, as judged by the rate of formation of urocanic acid. 228 229 230 231

Soucy, R., and Bouthillier, L. P., Rev. can. biol. 10, 290 (1951). Reid, J. C , and Landefeld, M. O., Arch. Biochem. and Biophys. 34, 219 (1951). Sprinson, D . B., and Rittenberg, D., / . Biol. Chem. 198, 655 (1952). Toporek, M., Miller, L. L., and Bale, W. F., J. Biol. Chem. 198, 839 (1952).

106

D. M.

GREENBERG

b. Isotopic Tracer Experiments Additional evidence of the importance of urocanic acid as an inter­ mediate in the degradation of histidine was obtained by incubating histidine labeled with C14 in the C 2 position of the imidazole ring with crude liver homogenate in the presence of twenty times its concentration of inert urocanic acid. From samples taken at zero time and at the end of the incubation period it was determined that most of the radioactivity disappearing from the histidine fraction appeared as urocanic acid. To rule out the possibility that the isotope originally found in histidine could enter urocanic acid by an exchange mechanism while net degrada­ tion of histidine could occur by a different reaction, in which case the bulk of urocanic acid degradation would necessarily proceed through histidine, an experiment was performed in which liver homogenate was incubated with unlabeled histidine and isotopic urocanic acid. The histidine from this experiment, isolated by ion exchange chromatography, contained little of the radioactivity, an amount that indicated that less than 5% of the urocanic acid degraded could have been converted to histidine. The experimental work with liver extracts apparently shows that the same reaction sequence for the catabolism of histidine found in the Pseudomonas preparations and represented in Fig. 14, occurs in liver. That this sequence is correct is supported by the isolation of radioactive glutamic acid upon incubating histidine labeled with C14 in the carboxyl group with liver slices.232 The radioactivity was not in the a-carboxyl group and may have been in the δ-carboxyl group, as predicted by Fig. 14. Wolf233 isolated glutamic acid from the protein upon administration of a:-C14-DL-histidine to the intact rat, in which the radioactivity was mainly in the ß- or γ-carbon (or both). This finding agrees with the urocanic acid pathway for the metabolism of histidine. The principal objection of Edlbacher and co-workers to the hypothesis that histidine was catabolized by the way of urocanic acid was that their purified histidase preparations split the imidazole ring of histidine in the absence of urocanase and did not act on urocanic acid.234 Histidase pre­ pared according to the best purification procedure from Edlbacher's laboratory 235 by Mehler and Tabor 227 yielded a product which had an eightfold increase in the ability to produce urocanic acid from histidine but did not split the imidazole ring. This preparation did not attack 232 233 234 235

Abrams, A., and Borsook, H., J. Biol. Chem. 198, 205 (1952). Wolf, G., J. Biol Chem. 200, 637 (1953). Edlbacher, S., and Viollier, G., Hoppe-Seyler's Z. physiol. Chem. 276, 108 (1942). Morel, C. J., Helv. Chim. Ada 19, 905 (1946).

C A R B O N CATABOLISM O F A M I N O

ACIDS

107

urocanic acid. It produced only 1 mole N H 3 from bistidine, and no addi­ tional N H 3 was released by strong alkali. It would appear, therefore, that the enzyme preparations of Edlbacher and co-workers were somewhat contaminated with urocanase. c. Enzyme Converting Histidine to Urocanic Acid (1) Terminology. Edlbacher employed the term histidase to represent the catalytic activity responsible for histidine degradation to the extent of the cleavage of the imidazole ring. The Japanese investigators 218,220 referred to the histidine —> urocanic acid conversion activity as histidine deaminase. More recently Hall 222 suggested the name histidine a-deaminase. Because it appears that the histidine —> urocanic acid conversion represents the first step in the reaction originally described by Edlbacher, and another enzyme, urocanase, is required for the subsequent cleavage, Mehler and Tabor 227 propose retaining the name histidase for the enzyme catalyzing the former reaction. This name will be employed here. {2) Properties. Histidase and urocanase were separated from each other by Takeuchi 219 by taking advantage of the observation that heat­ ing liver extract at 55°C. for 30 minutes and then acidifying the solution caused the histidase to be thrown down in the voluminous precipitate formed, while the urocanase remained in the supernatant liquid. Edlbacher and Viollier234 found that the two enzymes could be sepa­ rated owing to the fact that histidase is selectively absorbed on Pb 3 (P04)2 and urocanase on Ca 3 (P04). In Pseudomonas fluorescens extract, histidase free from urocanase can be prepared by heating the extract at 85°C. for 15 minutes. 227 By this procedure all ability to destroy urocanic acid is lost, but the full capacity to deaminate histidine is retained when the product is supplemented with 10~3 M glutathione. Some of the reported purifications of histidase 236 can not be judged for their freedom from urocanase, because, apparently, this was not determined. The test method employed was merely the rate of liberation of N H 3 from bistidine. Histidase activity can be measured by determining the accumulation of urocanic acid, through its strong ultraviolet absorption. The optimum activity of histidase is at pH 7.8 ;222 of urocanase, at pH 7-8. 219 Histidase is inhibited by a considerable number of substances. Among these are D-histidine, pyruvic acid,214 urocanic acid,214,222 and by com­ pounds that bind divalent cations. 227 A particularly potent substance of this type is ethylenediamine tetraacetate. This caused 50% inhibition 236

Walker, A. C , and Schmidt, C. L. A., Arch. Biochem. 5, 445 (1944).

108

D. M. GREENBERG

at 10~6 M, and virtually complete inhibition at 10~5 M. The inhibition could be overcome by additions of the ions, Mn + + , Ca + +, and Mg++. The literature on the properties of histidase and urocanase is reviewed in greater detail by Leuthardt. 237 The histidase reaction probably is not reversible. This is supported by the lack of effect of urocanic acid on growth. It has been reported to have no growth-promoting effect238 on the rat, or only a slight effect239 on diets deficient in histidine. Feeding or injecting of urocanic acid was unable to check the loss of weight on a histidine-deficient diet.239" 4. REACTION INTERMEDIATES

The action of what now must be accepted as a mixture containing histidase and urocanase on histidine results in the following effects.236 There is no change in the acidity of the solution during the reaction, a new acid group is formed with a pK of 4.2, the glutamic acid retains its optical activity, the α-amino nitrogen of histidine no longer reacts, the intermediate compound formed is similar to formamide and glutamine with respect to its stability to alkali but is considerably more stable to acid, and, on being decomposed with NaOH (or enzymatically), yields 1 mole each of N H 3 and formic acid. The reaction for α-amino nitrogen in the substrate decreases during the incubation and the decrease is approximately equivalent to the amount of N H 3 liberated from the amino acid.214 This must signify either that the α-amino group is cleaved during the reaction or that it becomes combined, while at the same time there is an equivalent loss of a nitrogen from the imidazole ring. Edlbacher and Neber 240 assumed that the pri­ mary intermediate product was formyl glutamine, since according to their scheme the α-amino group of histidine remains intact and becomes the α-amino group of glutamic acid. This involved these authors in a complicated scheme of ring closure and rearrangement to account for the loss in reactivity of the α-amino nitrogen. The evidence that the reaction proceeds through urocanic acid elimi­ nates the need for such speculations, and discussion of the different proposals to explain the observed results now becomes only of historical interest. They are discussed in the review by Leuthardt. 237 The Japanese group reported the isolation of isoglutamine 218,219 and 237

Leuthardt, F., Enzymes 1, 1156 (1951). Cox, G. J., and Rose, W. C , J. Biol. Chem. 68, 781 (1926). 289 Harrow, B., and Sherwin, C. P., J. Biol. Chem. 70, 683 (1926). 239a Celander, D . R., and Berg, C. P., J. Biol. Chem. 202, 351 (1953). 240 Edlbacher, S., and Neber, M., Hoppe-Seyler's Z. physiol. Chem. 225, 261 (1934).

238

CARBON CATABOLISM OF AMINO ACIDS

109

formyl-DL-isoglutamine,220,241 as well as urocanic acid from the enzyme reaction mixtures. A compound that appears to be isoglutamine has also been isolated by Abrams and Borsook.232 Borek and Waelsch,242 however, have reported the isolation of α-f ormamidinoglutaric acid [HOOC—CH 2 — CH 2 —CH(NHCHNH)COOH] from enzymatic digests of L-histidine or urocanic acid with extracts of cat liver. This compound could be degraded by extracts of Pseudomonas fluorescens, but was not attacked by rat liver or kidney slices. Identification of the formamidinoglutaric acid was based largely on the titration curve of the compound and chemical differences from synthetic α-iV-formyl-L-isoglutamine. Because of the enzymatic inertness of their compound, the authors suggest that the true histidine intermediate may be a more labile compound which is converted to the f ormamidinoglutaric acid. They also suggest that the α-Ν-ϊormyl-isoglutamine of the Japanese investigators 220,241 may have been formed from formamidinoglutaric acid during the isolation. It has been observed that the excretion of a compound yielding one equivalent each of glutamic and formic acids and ammonia in the urine of folic-deficient rats is greatly enhanced upon feeding histidine. 243 The administration of L-histidine-7-N15 to folic-deficient rats led to the result that approximately 55% of the glutamic acid derivative was derived from the dietary N 15 -histidine. The isolated L-glutamic acid obtained from the compound contained essentially all of the N 15 . Since this demon­ strates that the amino group of glutamic acid is derived from the γ-nitrogen of histidine, it is in favor of the urocanic acid pathway of histidine metabolism in the folic-deficient rat. In attempting to explain the conversion of the formyl-DL-isogluta­ mine 244 to L-glutamic acid, Oyamada 220 reported that the former was oxidatively attacked by extracts of guinea pig livers. He suggested, therefore, that in the organism the formyl-DL-isoglutamine is not hydrolyzed to formylglutamic acid and NH 3 , but instead is oxidatively at­ tacked to yield L-isoglutamine, and subsequently L-glutamic acid. Oyamada proposed the following reaction sequence (Fig. 15) to explain the observations on the decomposition of histidine by the above enzymes. 241

Suda, M., Miyahara, I., Tomihata, K., and Kato, A., Med. J. Osaka Univ. 3, 115 (1952). 242 Borek, B. A., and Waelsch, H., J. Am. Chem. Soc. 75, 1772 (1953); / . Biol. Chem. 205, 459 (1953). 243 Tabor, H., Silverman, M., Mehler, A. H., Daft, F. S., and Bauer, H., J. Am. Chem. Soc. 75, 756 (1953). 244 With histidine adapted Pseudomonas the decomposition of DL-formylisoglutamine and DL-isoglutamine was found to be too slight for them to be considered as meta­ bolic intermediates of histidine according to Suda et al.241

110

D. M. GREENBERG

He suggests that the instability of imidazolonpropionic acid would readily lead to its racemization with the production of formyl-DL-isoglutamine. The reported isolation of racemic formyl-isoglutamine but of optically active L-glutamic acid as intermediate products of the metabolism of histidine is a serious discrepancy in the proposed pathway of metabolism. The racemization of the formyl-isoglutamine may possibly occur during its isolation, and in nature it is formyl-L-isoglutamine which is present. CH=C—CH2—CH—COOH I

CH=C—CH=CH—COOH

NH2

NH

1

N

C H L-Histidine

NH

histidase

N

C H Urocanic acid O

OH I

C===c- -CH 2 I CH2

urocanase N H

N

COOH

c

II c

CH—CH2

NH

N

I

Hydroxyimidazole propionic acid

I

CONH2

II

I

CH2 4 CH—NHCH 5 CHNH2 COOH CH CH2

C H

H

CONH2 O

Imidazolonpropionic acid

I

I

CH2

CH2

I COOH

ICOOH

α-Formyl-DL

L-ISO-

isoglutamine

glutamine

F I G . 15, Hypothetical intermediates of the catabolism of histidine.

The hypothesis of Oyamada that the resolution to L-isoglutamine results from the oxidative decomposition of formyl-DL-isoglutamine is not very convincing. The hydrolytic splitting of the formyl group is much more reasonable. The reactions of Fig. 15 show how the γ-nitrogen of the imidazole ring can become the α-amino group of glutamic acid. It has been pointed out by Mehler and Tabor that the number of enzymes involved in the over-all transformation is not completely known, and that the intermediate products of the reaction are still incompletely described. 5. OTHER METABOLIC PATHWAYS

A variety of evidence from nutritional, biological, and isotopic experi­ ments suggest that conversion to urocanic acid may not be the only manner in which histidine is metabolized in the vertebrates. No glycogen was found to be formed upon urocanic acid administration, in contrast to histidine.239«

CARBON CATABOLISM OF AMINO ACIDS

111

a. Biological Lability of a-Amino Group of Histidine The biological lability of the α-amino group of histidine has been shown in various ways. It is not nutritionally essential that histidine be fed intact. In the rat imidazolelactic acid238,239-239a and imidazolepyruvic acid238,239 can be substituted for it. D-Histidine can be converted to form the L-isomer.245 The histidine of the body tissues was found to be subject to a con­ tinuous process of deamination and reamination, as found with many of the other amino acids, which involves only the α-amino group. 246 The evidence for this is that the N 15 of administered N 1 5 H 3 was found in the α-amino group of the tissue histidine. It has been found, also, by feeding the isotopic compound, that D-histidine can be converted to L-histidine.247 The mechanism of the deamination and reamination of the a-amino group of histidine does not appear to be by the usual enzyme systems. This amino acid exhibits virtually no susceptibility to transamination by heart, liver, or kidney, 20 and the two isomers, also, are poorly attacked by their respective amino acid oxidases.78 In many bacterial species the decomposition of histidine may be mainly by a pathway involving first its decarboxylation to histamine. 245 246

247

Conrad, R. M., and Berg, C. P., / . Biol. Chem. 117, 351 (1937). Schoenheimer, R., Rittenberg, D., and Keston, A. S., / . Biol. Chem. 127, 385 (1939). Foster, G. L., Rittenberg, D., and Schoenheimer, R., J. Biol. Chem. 125, 13 (1938). ADDENDA

1. The reaction has been found to be more complex t h a n was assumed and is now represented b y the following equation (Robinson, W. G., Bachhawat, B. K., and Coon, M. J., Federation Proc. 13, 281 (1954)). CH3

CH3 \

\ CHCH2COSC0A ->

/

CH3 Isovaleryl Co A

'

C = C H — C O S C o A -> CH3

/ Senecioyl Co A

CH3 \ HO —C—CH 2 —COSCoA

/

CH3 /3-Hydroxyisovaleryl-CoA

+C02

► (Intermediate) —> Acetoacetate + acetyl CoA

2. Recent discoveries (Rothstein, M., and Miller, L. L., Federation Proc. 13, 286 (1954); J. Am. Chem. Soc. 76, 1459 (1954); Schweet, R. S., Holden, J. T., and Lowy, P. H., Federation Proc. 13, 293 (1954)) t h a t L-pipecolic acid is a metabolite of lysine requires modification of the view t h a t its metabolism is not initiated through the α-amino group. The new results require modification of Fig. 11 to the following reaction scheme.

112 CH 2 NH 2

1

(CH 2 ) 3 -> CHNH 2

1

COOH Lysine

D. M. GREENBERG CH 2 NH 2

u - w""

(CH 2 ) 3

,/\

COOH

COOH α-keto-eaminoadipic acid

+

Δ '-Dehydropipecolic acid

/ \

\^/"'

COO]

H Pipecolic acid

\

^/

COOH

COOH

CH (CH 2 ) 3 CHNH2 COOH

-

I

► CH2)3 CHNH2

I

COOH a-Aminoadipic acid 3. Mitoma C. and Leeper L. (Federation Proc. 13, 266 (1954)) separated the enzyme system into two enzymes which together hydroxylate phenylalanine in the presence of D P N and an aldehyde or alcohol. Effective compounds are acetaldehyde, propionaldehyde, butyraldehyde, benzaldehyde, crotonaldehyde, propanol and benzyl alcohol. Formaldehyde, ethanol, glucose, ribose, glyoxylate and xanthine were inactive.

CHAPTER

11

Synthetic Processes Involving Amino Acids D A V I D M. G R E E N B E R G Department

of Physiological

Chemistry, School of Medicine, Berkeley, California

University

I . Introduction I I . T h e Glycine-Serine Interconversion I I I . Formation of Phosphatide Bases 1. Ethanolamine 2. MethylMion of Choline 3. Demethylation of Choline IV. Interconversions of Glutamic Acid, Ornithine, and Proline V. Biosynthesis of Branched-Chain Amino Acids VI. Lysine Biosynthesis VII. Biosynthesis of Aromatic Amino Acids V I I I . Reactions Involving Tyrosine 1. Biosynthesis of Epinephrine Compounds 2. Thyroxine I X . Synthetic Reactions Involving Tryptophan 1. Biosynthesis of Tryptophan 2. Biosynthesis of Nicotinic Acid a. Probable Intermediates of Niacin X. Biosynthesis of Histidine Addenda

of

California, Page 113 114 116 116 119 122 123 127 130 131 134 134 137 138 138 140 140 145 147

I. INTRODUCTION Synthesis of the amino acids in the body of the vertebrates is limited to those that are not essential in the diet. The essential amino acids are the products of organisms with a more autotrophic metabolism, such as various plants and microorganisms. Three of the amino acids, alanine, aspartic acid, and glutamic acid are readily formed by transamination from products of the citric acid cycle. This has been explained in the chapter, Carbon Catabolism of Amino Acids. Glutamic acid is the probable precursor of a considerable number of the other nonessential amino acids, namely, proline, hydroxyproline, ornithine, and from it arginine. The carbon chain of cysteine and cystine is derived from serine by a mechanism discussed in the chapter, Metabolism of Sulfur-Containing Compounds. The sole source of tyrosine for the vertebrates is phenylalanine, as is explained in the chapter, Carbon Catabolism ofAmino Acids. 113

114

D. M.

GREENBERG

To give as complete an understanding of the chemical pathways of biosynthesis of all the different amino acids as is now possible, discussion of the mechanisms of the synthesis of the essential amino acids by autotrophic organisms is included in this chapter. In addition to the amino acids themselves, the formation of certain biologically important compounds which are derived from amino acids is also included in this chapter. Formation of other important compounds which also involve amino acids are contained in other chapters of the book. II. THE GLYCINE-SERINE INTERCONVERSION The formation of glycine from serine was conclusively demonstrated by Shemin. 1 By labeling with C 13 in the carboxyl group and N 15 in the amino group he showed that the conversion took place with utilization of the carbon chain and without loss of the «-amino group. The conversion was found to be specific for L-serine, very little if any glycine being formed from D-serine. The demonstration that the ratio of N 1 5 :C 1 3 in the glycine was almost the same as in the administered serine eliminated both ethanolamine and free aminomalonic acid as intermediates of the reaction. Otherwise the ratio could not have remained unchanged. It was con­ cluded, therefore, that the glycine could have been formed only by the splitting off of the ß-carbon of serine. Shemin suggested that that may take place according to equation 1. H HO—CH2—CHNH2—COOH Serine

-2H

|

> 0=C—CHNHr-COOH +H 2 0 > CH2NH2—COOH + HCOOH Glycine

(1)

The formation of glycine from serine has been verified by the manner of its utilization for purine synthesis. 2 The reverse of this reaction, the synthesis of serine from glycine, was observed by Goldsworthy, Winnick, and Greenberg in vivo,z by Winnick, Moring-Claesson, and Greenberg in rat liver homogenates, 4 and by Siekevitz and Greenberg in rat liver slices.5 The investigations cited above demonstrated the reversible nature of this reaction. 1 2 3

4

5

Shemin, D., / . Biol. Chem. 162, 297 (1946). Elwyn, D., and Sprinson, D . B., / . Biol. Chem. 184, 465 (1950). Goldsworthy, P. D., Winnick, T., and Greenberg, D . M., / . Biol. Chem. 180, 341 (1949). Winnick, T., Moring-Claesson, I., and Greenberg, D . M., J. Biol. Chem. 175, 127 (1948). Siekevitz, P., and Greenberg, D . M., J. Biol. Chem. 180, 845 (1949).

SYNTHETIC PROCESSES INVOLVING ΑΜΙΝΟ ACIDS

115

Subsequent work has given some insight into the precursors of the ß-carbon atom of serine and a hint on the mechanism of the condensation reaction with glycine, although knowledge of the latter is still quite incomplete. Substances that can serve as the precursors of the ß-carbon of serine are the α-carbon of glycine,5,6 formate, 6 formaldehyde, 7 and the methyl groups of methionine, 8 choline,8,9 acetone, 10 and sarcosine.11 The methyl group precursors were formerly believed to be first converted to formate before incorporation into the ß-carbon of serine took place. If this were true, synthesis of serine could be written as the reverse of the reaction proposed by Shemin for the degradation of this amino acid to glycine (see equation 1). However, experimental work with liver homogenates made it clear that while the carbon of formaldehyde was incorporated into serine, the carbon of formate was not so incorporated, 12,11 even though concurrently serine was being synthesized from glycine. A study of the biosynthesis of serine with mitochondrial preparations of rat liver showed that the best donor of the ß-carbon of serine was the methyl group of sarcosine. Formaldehyde followed sarcosine, and formate did not react at all in this system unless certain reducing agents (cysteine, ascorbic acid, hydroquinone) were added. Since the methyl group of sarcosine is readily oxidized to formaldehyde through the agency of sarcosine oxidase it appears highly probable that in the reaction with sarcosine, formaldehyde is an intermediate. This also appears to be the case for the methylene carbon of glycine. The evidence for this is that when the carbons of these groups are labeled with C14, the radioactivity in the ß-carbon of the serine is diluted by the addition of unlabeled formaldehyde to the system and the formaldehyde becomes radioactive. On the other hand, inert formaldehyde did not cause any dilution in the incorporation of formate into serine in the presence of reducing agents. This implies that, from the side of formate, the ß-carbon can be formed without directly going through formaldehyde. The observation that ATP, α-ketoglutarate, and citrate favor the conversion of both formate and formaldehyde to the ß-carbon of serine, and the comparatively low reac­ tivity of free formaldehyde, leads to the conclusion that an unknown "activated" 1-carbon compound, probably at the oxidation level of 6

Sakami, W., / . Biol. Chem. 176, 995 (1948); ibid. 178, 519 (1949). Siegel, I., and Lafaye, J., Proc. Soc. Exptl. Biol. Med. 74, 620 (1950). 8 Siekevitz, P., and Greenberg, D. M., J. Biol. Chem. 186, 275 (1950). 9 Sakami, W., / . Biol. Chem. 179, 495 (1949). 10 Sakami, W., J. Biol. Chem. 187, 369 (1950). 11 Mitoma, C , and Greenberg, D. M., J. Biol. Chem. 196, 599 (1952). 12 Kruh0ffer, P., Biochem. J. (London) 48, 604 (1951). 7

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formaldehyde, is required for condensation with glycine to form serine.12a These considerations are supported by the observation that the active oxidation of some member of the citric acid cycle (citrate) is necessary for the maximum incorporation of formate into serine by the cyclophorase system of Green. 13 III. FORMATION OF PHOSPHATIDE BASES The best known of the nitrogen constituents of the phosphatides are choline, found in lecithin (phosphatidyl choline); ethanolamine, in cephalin (phosphatidyl ethanolamine); and serine, in phosphatidyl serine. These three compounds are closely interrelated in the body and, in fact, appear to form part of a metabolic cycle which may be represented as shown in Fig. 1. The work on the interconversion of glycine and serine has been pre­ sented above. This section will be devoted mainly to a discussion of the reactions in Fig. 1 concerned with the formation of ethanolamine and choline. 1. ETHANOLAMINE 15

N was found in the ethanolamine of the phosphatides of the body upon feeding N 15 -labeled glycine.14 This appeared to indicate that ethanolamine was formed by reduction of glycine. However, in later experiments it was observed that N 15 -labeled serine also went to ethanol­ amine and to a lesser extent to choline.15 Consequently, Stetten now sug­ gested that serine was decarboxylated to form ethanolamine. Decarboxylation of serine by anaerobic bacteria had previously been observed by Nord. 16 That ethanolamine can be formed in the animal body by the decarboxylation of serine was demonstrated by the work of Levine and Tarver, 17 who showed that the label of serine-3-C14 appeared in liver 12a

I t has now been determined t h a t tetrahydrofolic acid added to pigeon liver extracts treated with Dowex-1 will induce the biosynthesis of serine from formate or formaldehyde and glycine, suggesting t h a t N 5 -hydroxymethyltetrahydrofolic acid is the coenzyme t h a t activates the 1-carbon compounds (R. L. Kisliuk, and W. Sakami, «/". Am. Chem. Soc. 76, 1456 (1954)). Alexander and Greenberg (un­ published data) found with a soluble r a t liver enzyme system t h a t leucovorin and A T P catalyzed the reaction of formaldehyde b u t not of formate with glycine to form serine. I t has also been demonstrated t h a t pyridoxal phosphate is required for serine formation by a Mannich-type reaction (J. Lascelles, and D. D . Woods, Nature 166, 649 (1950), S. Deodhar, and W. Sakami, Federation Proc. 12, 195 (1953)). 13 Sarkar, N . K., Beinert, H., Fuld, M., and Green, D . E., Arch. Biochem. and Biophys. 37, 140(1952). 14 Stetten, D., Jr., / . Biol. Chem. 140, 143 (1941). 15 Stetten, D., Jr., / . Biol. Chem. 144, 501 (1942). 16 Nord, F . F., Biochem. Z. 96, 281 (1919). 17 Levine, M., and Tarver, H., / . Biol. Chem. 184, 427 (1950).

SYNTHETIC PROCESSES INVOLVING AMINO ACIDS

117

ethanolamine. Since the label on the ß-carbon would have been lost if the serine was first converted to glycine, a decarboxylation to form ethanol­ amine appears to be obligatory. Furthermore, since glycine is readily converted to serine it appears plausible that ethanolamine is formed from glycine via serine and not directly.

HOCH 2 -COOH - H O C H 2 - C H 2 - N (CH3) 3 Choline

(CH 3 ) 2 NCH 2 -COOH Dimethylglycine

0=CH-CH2N(CH3)3 Betaine aldehyde -OOC-CH 2 N(CH 3 ) 3 Betaine

F I G . 1. Hypothetical cycle of metabolism involving glycine, serine, ethanolamine, choline, and betaine.

The pathway to ethanolamine through decarboxylation of serine, how­ ever, did not altogether eliminate the possibility that ethanolamine may also be formed by reduction of glycine. That this does not occur has now been shown, since ethanolamine isolated following administration of CH 2 NH 2 C 14 OOH contained no radioactivity. 18 · 19 18 19

Greenberg, D . M., and Harris, S. C , Proc. Soc. Exptl. Biol. Med. 76, 683 (1950). Arnstein, H. R. V., Biochem. J. (London) 48, 27 (1951).

118

D.

M.

GREENBERG

The magnitude of the conversion of serine to ethanolamine appears to be quite high. This is extremely interesting in view of the fact that it has not been possible to demonstrate the presence of an active serine decarboxylase in mammalian tissues. In the experiment of Stetten, 15 2.6% of the ethanolamine in the phosphatides of the liver of his animals was formed from the isotopic serine in eight days. Since the serine in the phosphatides showed a 6% incorporation of the label, he reasoned that if the serine decarboxylated had the same isotopic concentration, the actual ethanolamine synthesized was 100(2.6/6.0), or 4 3 % of the total phosphatide ethanolamine. This figure probably is high, as the dilution would not be expected to be complete throughout the decarboxylation process. TABLE I CONVERSION OF POSSIBLE PRECURSORS OF P H O S P H A T I D E B A S E S 1 4

Isotopic compound fed Source of compound

Compound isolated

Ethanolamine hydrochloride

Choline chloride

Glycine

Total phosphatides

Ethanolamine Choline

27.9 11.5

1.0 20.8

2.9 0.5

2.0 0.6

1.2 0.1

Organ protein

Glycine Glutamic acid

0.7 0.9

2.5

5.9 1.3

4.5 1.0

0.7 2.0

Urea NH4

3.4 4.8

1.9 2.1

5.1 6.1

3.5 3.6

8.7 5.9

Urine

Betaine chloride NH 4 C1

, ^ i i ^ , Atom % N 1 5 in compound isolated w 1Λ „ Values tabulated = — . .— ΓΎ~\— X 100 Atom M% XT1 N 15 in compound fed Isotope content computed on basis of 100 atom % in test substances fed. ΛΤ

Formation of ethanolamine from betaine and of choline from ethanol­ amine was also shown by the experiments of Stetten. 14 The data are reproduced in Table I. The observations support the main features of the cycle represented in Fig. 1, namely that ethanolamine is formed by the decarboxylation of serine, this in turn is methylated to choline, which is then oxidized to betaine, and the latter is demethylated to glycine. Further support for this scheme is supplied by the observations of Arnstein 19 that L-serine-3-C14 is converted to choline with about the same degree of efficiency as N l5 -glycine and that glycine- 1-C14 is not a precursor of choline. Having established the conversion pattern of the main carbon chains of the compounds of the cycle, consideration is required of the mecha­ nisms of methylation and demethylation.

SYNTHETIC PROCESSES INVOLVING AMINO ACIDS

119

First it needs to be pointed out that an alternate pathway exists for the metabolism of ethanolamine. It is readily deaminated, 19a as much as 40% of the N 1 5 of the nitrogen-labeled ethanolamine appearing in the urine as urea in 24 hours upon oral administration of the compound to rabbits. The first product of the dearnination is probably glycolaldehyde (Fig. 1). This, it is suggested, can undergo the sequence of reactions: 196 glycolaldehyde —> glycolic acid —> glyoxylic acid —> glycine. The extent of conversion to glycine by this route does not appear to be great. 19c In the experiments of Weissbach and Sprinson,19& the administered 14 C of the ethanolamine that appeared in C 4 of the excreted uric acid of a pigeon and the glycine of the hippuric acid from the urine of a rat was less than 0 . 1 % of the dose. It was determined in the author's laboratory 19 ^ that neither formalde­ hyde nor formate are formed appreciably from ethanolamine. 196 These might be expected to be formed through the reaction sequence proposed by Weissbach and Sprinson.196 2. METHYLATION OF CHOLINE

The hypothesis that certain of the methyl groups of choline or betaine could serve as donors for certain methylations occurring in the animal body was first suggested by Riesser20 and developed by Challenger and Higginbottom. 21 The transmethylation reaction from methionine to form choline has been established largely as a result of the work of du Vigneaud and co-workers. Recent reviews of the subject are given in references.22,23 The first evidence was that young animals fed a diet deficient in choline could be protected from the deleterious effects of the deficiency by methionine 24 but not by homocystine. 25 · 26 Subsequently it was estab19

« Abbott, L. A., Jr., and Klingman, J. D., Federation Proc. 12, 165 (1953). ' Weissbach, A., and Sprinson, D . B., J. Biol. Chem. 203, 1031 (1953). i9« Pilgeram, L. O., Gal, E. M., Sassenrath, E. N., and Greenberg, D . M., / . Biol. Chem. 204, 367 (1953). Pilgeram, L. O., P h . D . thesis, Univ. of Calif., Sept. 1953. i9d Pilgeram, L. O., and Greenberg, D . M. (unpublished d a t a ) . 19e It was observed t h a t formaldehyde was formed from dimethylaminoethanol, dimethylglycine, and sarcosine, b u t not from aminoethanol, monomethylaminoethanol, choline, betaine, and methionine upon incubation with liver homogenates [C. G. Mackenzie, J. M. Johnston, and W. R. Frisell, A-Acetylglutamic γ-semialdehyde —» Y«_Acetylornithine —» Ornithine —> Citrulline —> Arginine

The formation of hydroxyproline from proline has been demonstrated by feeding the latter labeled with D and N 15 . 56 Additional evidence for this is that in the experiments with ornithine-N 15 , the N 15 concentration of the isolated hydroxyproline was about one-third as high as that of the proline. Experiments with isotopic hydroxyproline indicated that it is not capable of being reconverted to proline. 61 Furthermore, it was concluded that the hydroxyproline of the body proteins is not derived from the 59

Vogel, II. J., and Davis, B. D., / . Am. Chem. Soc. 74, 109 (1952). Srb, A. M., Fincham, J. R. S., and Bonner, D., Am. ./. Botany 37, 533 (1950). ™a Abelson, P. H., J. Biol. Chem. 206, 335 (1954). 61 Stetten, M. R., / . Biol. Chem. 181, 31 (1949). eo

SYNTHETIC PORCESSES INVOLVING AMINO ACIDS

127

dietary hydroxyproline, but rather from the oxidation of proline already bound, presumably in peptide linkage. V. BIOSYNTHESIS OF BRANCHED-CHAIN AMINO ACIDS Work on the mechanism of the biosynthesis of isoleucine and valine began with the isolation of a mutant strain of Neurospora which required both isoleucine and valine for growth. 62 Bonner 63 suggested that the double requirement was due to the accumulation of an isoleucine pre­ cursor which then inhibited the conversion of an analogous intermediate in the scheme of valine synthesis. This explanation has been shown not to be true, as will be pointed out below. The steps in scheme for the bio­ synthesis of isoleucine and valine in microorganisms are given in Fig. 3. That the amino acids are formed by transamination of the correspond­ ing keto acids has been deduced from the following evidence: mutants of Escherichia coli are known which require isoleucine and valine for maximal growth, and which accumulate both keto acids.64 Neither the keto acids nor any other compounds can substitute for the growth requirement, except as noted below. Wild-type E. coli contains a transaminase which catalyzes amino transfer between any two of the following: isoleucine, valine, leucine, norleucine, norvaline, and glutamic acid.65 This enzyme is completely absent in the keto acid-accumulating mutants 65,66 and accounts for their inability to synthesize adequate amounts of isoleucine and valine. Both wild-type and the mutants, however, contain an enzymetransaminating valine with either alanine or α-aminobutyric acid; the presence of this enzyme permits a limited synthesis of valine in the mutants, so that these mutants grow slowly without added valine, and rapidly with either valine, alanine, or α-aminobutyric acid.65,66 The latter two compounds act by increasing the rate of transamination of a-ketoisovaleric acid to valine. From one such mutant, Adelberg and Umbarger 66 obtained a new strain capable of maximal growth on isoleucine alone and discovered that the organism had mutated so as to increase its valinealanine transaminase activity four- to fivefold. The existence of the dihydroxy acid precursors of isoleucine and valine was discovered by Adelberg and co-workers.67-68 Evidence that they are 62

Bonner, D., T a t u m , E. L., and Beadle, G. W., Arch. Biochem. 3, 71 (1943). Bonner, D., / . Biol. Chem. 166, 545 (1946). 64 Umbarger, H. E., and Magasanik, B., / . Biol. Chem. 189, 287 (1951). 65 Iludman, D., and Meister, A., / . Biol. Chem. 200, 591 (1953). 66 Adelberg, E. A., and Umbarger, H. E., J. Biol. Chem. 205, 475 (1953). 67 Adelberg, E. A., and T a t u m , E. L., Arch. Biochem. 29, 235 (1950). 68 Adelberg, E. A., Bonner, D., and T a t u m , E. L., J. Biol. Chem. 190, 837 (1951).

63

128

Τ). Μ.

GREENBERG

CH 3 CH 3

CH 2

I

CH-CHNH2COOH

CH 2 CH3-C-CH-COOH

CH 3 L-Isoleucine a-ketoglutarate

HO

OH III a,/3-Dihydroxy-/3-ethyl butvric acid ■

(10)

CH 3

(8)Jf

COOH

CH 2

I

I

CH-C-COOH

CH 2 CH3-C-CH-COOH HO

OH V

(4) I COOH I

I

-co 2

II

CH 3 O 1 α-Keto-jS-ethylbutyric acid

(5)

CH 3 CH3-C-CH-COOH

I

CH3 CH3-C-CHCOOH II I O OH VI α-Hydroxy-ß-ketobutyric acid (3)

CH3-CH2-C-COOH O VII α-Ketobutyric acid

HO

VIII L-Threonine

I

OH IV α,/3-Dihydroxyisovaleric acid -H 2 0

(6)

CH 3 CH-C-COOH I II CH 3 O II α-Ketoisovaleric acid r-glutamate (7)

(2)

CH3-CH2-CH-COOH I I OH NH 2

r ^-glutamate

>^a-ketoglutarate

CH 3 CH-CH-COOH I I CH 3 NH 2 L-Valine

(i) I HOCH2-CH2-CH-COOH I NH 2 IX Homoserine

F I G . 3. Scheme for biosynthesis of isoleucine and valine in microorganisms.

SYNTHETIC PROCESSES INVOLVING AMINO ACIDS

129

normal precursors may be summarized as follows: they are accumulated by Neurospora and E. coli mutants which require isoleucine and valine or their respective keto acids for growth; 69 they can be used in place of the corresponding amino acids by mutants blocked earlier in the biosynthetic pathways ;69a and the enzyme system dehydrating the dihydroxy acids to the keto acids has been extracted from wild-type E. coli and Neurospora.69 Mutants accumulating the dihydroxy acids lack this enzyme system; it is not yet established whether one or two enzymes are concerned, but the fact that both activities are lost as the consequence of a single gene mutation suggests that one enzyme functions for both biosyntheses. 69 Thus, in all cases of isoleucine-valine deficiency analyzed, the double requirement has been shown to result from the loss of two biosynthetic functions, and not from metabolic interactions as proposed by Bonner. The reactions leading to dihydroxy acid formation are still obscure. Much evidence has been published for the view that threonine and related four-carbon compounds (such as α-ketobutyric acid) are precursors of isoleucine, and it has been postulated that one of these compounds be­ comes the four-carbon chain of both isoleucine and valine. 69a This hy­ pothesis is untenable, however, since Ehrensvard 696 has shown that in several microorganisms the carbon skeletons of threonine, isoleucine, and valine are derived from acetate as follows, when the organisms are grown aerobically on C 13 H 3 C 14 OOH as the sole carbon source (Fig. 4): C(c) Isoleucine

C (m)

C(m) CI (m)

C (m)

COOH (c)

(m) = derived from methyl of acetate

C(m) Valine

C (m)

C (m)

C (m)

COOH (c)

Threonine

C (c)

C (m)

C (m)

COOH (c)

(c) = derived from carboxyl of acetate

F I G . 4. Labeling of isoleucine, valine, and threonine in microorganisms grown on C 1 3 H 3 C 1 4 OOH.

The derivation of the 7-carbon of threonine from acetate carboxyl is consistent with the recent finding690 that bacteria carry out the reactions: 69

Myers, J. W., and Adelberg, E . A., Proc. Natl. Acad. Sei. U.S. (in press). « Umbarger, H. E., and Adelberg, E. A., / . Biol. Chem. 192, 883 (1951). 69& Ehrensvard, G., Ada Chem. Scand. 5, 353 (1951); and private communication 69c Hirsch, M. L., and Cohen, G. N., Compt. rend. 236, 2338 (1953).

69

130

D. M.

GREENBERG

HOOC—Clio—CH—COOH

I

Aspartic acid 09 '

NHo

I

HOCH 2 —CH·,—CH—COOH

Homoserine

C H 3 — C H — C IH —NCHO2 O H

Threonine

I I

!

OH

I

NH2

The sequence of acetate derivations (c)-(m)-(m)-(c) is not found either in isoleucine or in valine; however, Adelberg and co-workers69d have found that a double mutant of Neurospora, which accumulates the dihydroxy acids and also is unable to synthesize threonine, converts added L-threonine to the dihydroxy acid precursor of isoleucine in 50% yield. When this mutant is provided with C 14 -l,2-threonine, it forms the iso­ leucine precursor labeled in the 1 and 2 positions.69d Thus, threonine sup­ plies at least carbons 1 and 2 of isoleucine (but not carbon 4) by reactions which have not yet been elucidated. The origin of the dihydroxy acid corresponding to valine is even more obscure. In the experiment with C 14 -l,2-threonine described above, no radioactivity was incorporated into the valine precursor, so that carbons 1 and 2 must have diverse origins in isoleucine and valine. However, the sequence of acetate derivations (m)-(m)-(m) is found in both amino acids and is unusual enough to suggest that this portion of the two com­ pounds may have a common precursor. It is interesting that this sequence is found in compounds of the citric acid cycle in Ehrensvard's experiments. 690 No information is available at present on the mechanism of the bio­ synthesis of leucine. VI. LYSINE BIOSYNTHESIS There appear to be two pathways for the biosynthesis of lysine among microorganisms. In E. coli there is strong evidence that the diamino dicarboxylic acid, a,e-diaminopimelic acid, is a precursor of lysine. 70,71 Diaminopimelic acid has been isolated from bacteria 72,73 and found to be decarboxylated by a specific decarboxylase occurring in wild-type E. coli. This enzyme has been found to be constitutive rather than adaptive, and it is absent from certain of the lysine auxotrophs. 73a 69d

Adelberg, E. A.7 private communication. See Addendum 1, p. 147. 70 Davis, B. D., Nature 169, 537 (1952). 71 Dewey, D . L., and Work, E., Nature 169, 533 (1952). 72 Work, E., Biochem. J. (London) 49, 17 (1951). 73 Asselineau, J., Choucroun, N., and Lederer, E., Biochim. et Biophys. Ada 5, 197 (1950). 7 3a Organisms t h a t exhibit a multiple requirement of analogous compounds for growth. r,9e

SYNTHETIC PROCESSES INVOLVING AMINO ACIDS

131

Among the lysine auxotrophs of E. colt, one mutant was found to have an absolute requirement for diaminopimelic acid together with a relative requirement for L-lysine.70 An examination of other lysine auxotrophs revealed one group that responded only to lysine and accumulated large amounts of diaminopimelic acid in the culture medium. A second group exhibited a relative requirement for the above two amino acids which was converted into an absolute requirement by the presence of L-aspartic acid in the culture medium. This group of organisms grew excellently on L-lysine and only slightly more slowly on diaminopimelic acid. Another strain was found which requires L-aspartic acid plus either diaminopimelic acid or lysine. Certain of these strains were found to accumulate L-threonine while others do not. These results establish a metabolic relationship between threonine, diaminopimelic acid, and lysine among E. coli strains. Additional evidence for such a relationship is evidenced by one mutant which grows rapidly on L-threonine and slowly on diaminopimelic acid or L-lysine. Other microorganisms, as evidenced by Neurospora, have a different pathway of biosynthesis of lysine. The lysine auxotrophs of this organ­ ism do not respond to diaminopimelic acid70 and do respond to such 6carbon compounds as DL-a-aminoadipic acid or DL-a-amino-e-hydroxycaproic acid to satisfy their lysine requirement. 74,75 The latter compounds are inactive for E. coli. Furthermore, diaminopimelic acid could not be detected in Neurospora. It is interesting that this is the first instance of the occurrence of two different pathways for the synthesis of the same amino acid among microorganisms. A speculative scheme for the biosynthesis of lysine has been proposed by Strassman and Weinhouse 76 on the basis of the distribution of the label in the lysine isolated from the yeast, Torulopsis utilis, grown on methyland carboxyl-labeled acetate. This scheme is that acetate may condense with a-ketoglutarate to yield a homolog of citric acid, which, by undergoing a series of reactions analogous to the citric acid cycle, should yield "homoisocitrate," oxaloglutarate, α-ketoadipate, and ultimately, α-aminoadipic acid. VII. BIOSYNTHESIS OF AROMATIC AMINO ACIDS The aromatic amino acids cannot be synthesized directly by the verte­ brate organism. The vertebrates can form tyrosine from phenylalanine. 77 74 75 76 77

Mitchell, H. K., and Houlahan, M. B., / . Biol. Chem. 174, 883 (1948). Good, N., Heilbronner, R., and Mitchell, H. K , Arch. Biochem. 28, 464 (1950). Strassman, M., and Weinhouse, S., / . Am. Chem. Soc. 74, 3457 (1952). Moss, A. R., and Schoenheimer, R., J. Biol. Chem. 135, 415 (1940).

132

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GREENBERG

Aromatization to benzenoid compounds can take place in the mam­ malian organism. One example is quinic acid (1,3,4,5-tetrahydroxycyclohexanecarboxylic acid), which is converted to benzoic acid and excreted as hippuric acid in the urine of man, sheep, and dog, but not of the rabbit, guinea pig, or rat 78,79 (see reference79 for literature). This substance occurs naturally in a number of plants, for example, prunes. Other compounds that have been found to be aromatized to benzoic acid are c^/cfohexanecarboxylic acid,79,80,80a and the several A-cyclo hexanecarboxylic acids79 which contain one double bond in the ring. Of the seven monohydroxy derivatives of c?/cfohexanecarboxylic acid, only the £raws-4-hydroxyc2/cfohexanecarboxylic acid was aromatized to any appreciable extent. The aromatization was found to take place in liver slices and to a lesser degree in kidney slices, but not in homogenates. 79 One scheme for the biosynthesis of the aromatic compounds from glucose has been developed, largely based on the work of Davis 81,82 on mutants of E. coli. This scheme is shown in Fig. 5. The scheme was deduced from studies on auxotrophic mutants of E. coli that require various combinations of phenylalanine, tyrosine, tryptophan, p-amiriobenzoic acid, and p-hydroxybenzoic acid for growth. These mutants fall into two main groups: (1) those which can satisfy part or all of their aromatic requirements with shikimic acid; and (2) those which cannot use this compound but accumulate it in their filtrates. Of fifty-five possible aromatic precursors tested, only shikimic acid was found to be active for group (1). Tatum has found that shikimic acid also supports growth of a Neurospora with a multiple requirement for aromatic compounds similar to that of the above E. coli mutants. Further studies by Davis on the E. coli mutants that can utilize shikimic acid indicated the presence of three different types: type 1 accumulates 5-dehydroshikimic acid; 83 type 2 can use this compound and accumulates 5-dehydroquinic acid; 83a type 3 can use all three aromatic precursors. There is some evidence that quinic acid may also be involved 78

Quick, A. J., / . Biol. Chem. 92, 65 (1931). Beer, C. T., Dickens, F., and Pearson, J., Biochem. J. (London) 48, 222 (1951). 80 Bernhard, K , Hoppe-Seyler's Z. physiol. Chem. 248, 256 (1937); ibid. 266, 49, 59 (1938). 80a Baltes, B. J., Elliott, W. H., Doisy, E. A., Jr., and Doisy, E. A., / . Biol. Chem. 194, 627 (1952). These authors found t h a t there was no aromatization of hexahydrophenylalanine, as determined b y the failure of this compound to promote growth in t h e absence of phenylalanine. 81 Davis, B. D., Experientia 6, 41 (1950); J. Biol. Chem. 191, 315 (1951). 82 Davis, B. D., / . Bacterial. 64, 729, 749 (1952). 83 Salainon, I. I., and Davies, B. D., / . Am. Chem. Soc. 75, 5567 (1953). 83 « Weiss, U., Davis, B. D., and Mingioli, E. S., ,/. Am. Chem. Soc. 75, 5572 (1953). 79

SYNTHETIC PROCESSES INVOLVING AMINO ACIDS

133

in aromatic biosynthesis as it has a low degree of growth-promoting activity for a Neurospora mutant 84 and for an Aerobactor aromatic polyauxotroph. The evidence for its normal participation in the sequence outlined in Fig. 5 is incomplete, however, since it has not been found by Davis to be accumulated by the mutant strains that accumulate the three other compounds of the sequence. The evidence is mounting that while glucose is the precursor of shikimic acid, the transformation is not a direct one. Shigeura, Sprinson,

r Tyrosine

Phenylalanine Anthranilic acid Dehydroshikimic acid

Ϊ \^

*

Indole Vi Tryptophan *

\

p-NH2-Benzoic acid p-HO-Benzoic acid F I G . 5. Scheme of biosynthesis of aromatic compounds from glucose. 84a

and Davis 85 showed that the radioactivity of glucose-1-C14 appeared in position 2 of shikimic acid with an activity half that of the Ci of the labeled glucose. The radioactivity of the Ce of glucose appeared predomi­ nantly in the Ce of shikimic acid with only a 15% dilution. With glucose3,4-C14, the carboxyl and C4 of shikimic acid each had one-third and C3 and C5 had each one-sixth of the total activity. The results suggest that two or more fragments of glucose are utilized for the synthesis of shikimic acid. Their nature is not known, but they do not reflect randomization reactions of glycolysis and the tricarboxylic acid cycle or loss of the Ci of glucose by the glucose-6-phosphate shunt. 84

Gordon, M., Haskins, F., and Mitchell, H., Proc. NatL Acad. Sei. U.S. 36, 427 (1950).

84a 85

See Addendum 2, p. 147. Shigeura, H., Sprinson, D . ; and Davis, B. D., Federation Proc. 12, 458 (1953).

134

D. M.

GREENBERG

In a study of the biosynthesis of phenylalanine and tyrosine by yeast, 86 it was determined that neither acetate nor intermediates derived from acetate (α-ketoglutarate, oxalacetate, etc.) were utilized. When the yeast was grown in the presence of glucose-1-C14, the labeling of the side chain was in the ß-carbon and the labeling in the ring was confined virtually to C 2 and/or the indistinguishable Ce of phenylalanine. The labeling in the side chain can be explained if it is formed from a triose derived from glu­ cose by glycolysis in which carbons Ci and Ce become the ß-carbon of the triose. The labelings observed in the ring of phenylalanine are incompatible with the following mechanisms of formation of the benzene ring: (1) con­ densation of three C2 units, (2) utilization of intermediates of the citric acid cycle, (3) condensation of two isotope-containing trioses, and (4) direct cyclization of glucose. Elimination of the last mechanism, direct cyclization of glucose, to which the authors were first inclined, requires further explanation. The direct cyclization hypothesis of glucose or a hexose derived from it to form a six-membered ring requires that virtually all of the label be con­ tained in only 1 carbon of the ring (which may be the case), and that the cyclization product should have the same specific activity as the precursor glucose. In the above experiments the radioactivity of the radioactive glucose was not significantly reduced or redistributed, whereas the specific activity of the ring moieties of phenylalanine and tyrosine was only 50 % to 70% of the glucose of the medium. For this reason direct cyclization of glucose is rejected. The authors point out that their data are not in­ compatible with a mechanism of ring synthesis involving the condensa­ tion of one singly labeled triose unit with an unlabeled tetrose unit (representing C2 to C5) to yield a heptose, which would be the direct precursor of the aromatic ring. Another hypothesis of aromatic amino acid synthesis, based on the distribution of the label in tyrosine of yeast grown on radioactive pyruvate or acetate, is that it involves the cyclic condensation of two unsymmetric 4-carbon acids, e.g., oxalacetate. 86a The side chain of tyrosine appears to be formed from pyruvate as an intact 3-carbon unit. VIII. REACTIONS INVOLVING TYROSINE 1. BIOSYNTHESIS OF EPINEPHRINE COMPOUNDS

Some of the possible pathways for the biosyntheses of the epinephrine bodies are shown in Fig. 6. That phenylalanine can serve as a precursor 86

Gilvarg, C , and Bloch, K , J. Am. Chem. Soc. 72, 5791 (1950). « Thomas, R. C , Cheldelin, V. H., Christensen, B. E., and Wang, C. H., J. Chem. Soc. 75, 5554 (1953).

86

Am.

SYNTHETIC PROCESSES INVOLVING AMINO ACIDS

135

of epinephrine has been reported by Gurin and Delluva. 87 These investi­ gators administered phenylalanine labeled with tritium and phenylalanine labeled with C14 in the a and carboxyl carbons. The isolated epinephrine from the animals fed the C14 phenylalanine contained essen­ tially all of the radioactivity in the terminal carbon of the side chain. Tyrosine

HO— ΗΟ

C H 0 H

HO

\

-> H O — < ^

\CH ?| |

|

2

Dihydroxyphenylethylamine HO \ -> HO— H O — /

CH 2 | NH2 %-CHOH

CH—NH2 CHNH, Norepinephrine C H 2 | | (Arterenol) | COOH COOH NH2 4-Hydroxyphenylserine 3,4-Dihydroxy phenylserine / (DOPS) / HO /

\ HO— H O O C — C = C H 2 + H 2 S NH2

NH2 -* H O O C — C — C H 3 + H 2 0 -> H O O C — C — C H 3 + H 2 0

II

II

NH

O

(4)

A similar but separate enzyme splits H 2 S from homocysteine. 50 This enzyme occurs in liver, pancreas, and kidney. Cystine and peptides of cysteine are attacked with the liberation of H 2 S by an enzyme named exocystine desulfhydrase. 51 The hydrogen sulfide produced by the desulfhydrase enzymes is capable of being oxidized to sulfate. 52,53 43

Virtue, R. W., and Doster-Virtue, M. E., / . BioL Chem. 119, 697 (1937); ibid. 128, 665 (1939). 44 Tarver, H., and Schmidt, C. L. A., J. BioL Chem. 146, 69 (1942). 45 Brand, E., Cahill, G. F., and Block, R. J., / . BioL Chem. 110, 399 (1935). 46 Fromageot, C , Wookey, E., and Chaix, P., Enzymologia 9, 198 (1940). 47 Lakowski, M., and Fromageot, C , / . BioL Chem. 140, 663 (1941). 48 Smythe, C. V., / . BioL Chem. 142, 387 (1942). 49 Lawrence, J. M., and Smythe, C. V., J. BioL Chem. 2, 225 (1943). 50 Fromageot, C , and Desnuelle, P., Bull. soc. chim. biol. 24, 1269 (1942). 51 Greenstein, J. P., and Leuthardt, F. M., / . Natl. Cancer Inst. 5, 209 (1944). 52 Denis, W., and Reed, L. L., J. Biol. Chem. 72, 385 (1927). 53 Dziewiatkowski, D . D., ,/. BioL Chem. 161, 723 (1945).

METABOLISM OF SULFUR-CONTAINING COMPOUNDS

163

V. THIOSULFATE AND THIOCYANATE Traces of thiosulfate are excreted in the urine. Fromageot and Royer 54 demonstrated that it is a product of the animaPs own metabolism. How­ ever, the precursors of the thiosulfate in the body have not been deter­ mined. The thiosulfate can be oxidized to sulfate both in the intact animal 55 and in tissue slices.54 A reaction in which thiosulfate participates that leads to another sulfur-containing compound which is excreted in the urine is the formation of thiocyanate. The reaction can be written as shown in equation 5. S203= + CN- -* SCN- + S03=

(5)

This reaction is catalyzed by the enzyme rhodanese. 56 Thiosulfate and colloidal sulfur are the only compounds so far discovered that react to form thiocyanate. 56 One useful function of this reaction is that it serves to detoxify cyanide, traces of which are also formed endogenously in the metabolism of the mammal. 57 The enzyme rhodanese is widely distributed in animal tissues, the activity in liver being particularly high. 58 Gal, Fung, and Greenberg 59 found that rhodanese activity in­ creases with fetal development up to the time of birth. The rhodanese activity of the mother was not influenced by pregnancy. The purification and crystallization of rhodanese from liver has been accomplished by Sörbo.60 The enzyme is slowly inhibited by CN~ and is protected by glutathione or cysteine. Sörbo has proposed that the active group in the enzyme is not an —SH group but a disulfide linkage. The protecting effect of sulfhydryl compounds against CN~ is explained by a combination with the rhodanese which gives a product not attacked by CN~. On the basis of the proposed active disulfide group Sörbo pro­ posed the reaction mechanism shown in equation 6 for the formation of thiocyanate.

,—s

EI

I+

S—S03= -> E

/

s—s—so3s

1—s

I

+ CN- -> E1—

+ S03= + SCN-

(6)

s VI. REACTIONS OF SULFUR-CONTAINING COENZYMES

L

V

L

1. GLUTATHIONE

Glutathione is widely distributed in nature and rapidly metabolized in the animal body, its rate of turnover being much faster than that of 54

Fromageot, C , and Roy er, A., Enzymologia 11, 361 (1945). Zörkendörfer, W., Biochem. Z. 278, 191 (1935). 56 Lang, K , Biochem. Z. 259, 243 (1933). 57 Boxer, G. E., and Rickards, J. C , Arch. Biochem. and Biophys. 39, 287 (1952). 58 Rosenthal, O., Federation Proc. 7, 181 (1948). 59 Gal, E. M., Fung, F. H., and Greenberg, D. M., Cancer Research 12, 574 (1952). 60 Sörbo, B. H., Ada Chem. Scand. 5, 724, 1218 (1951); ibid. 7, 238 (1953).

55

164

DAVID M.

GREENBERG

the metabolically active liver proteins. Since its discovery by Hopkins in 1921,61 there has been much interest in its metabolic function. Until recently, investigations into the functions of glutathione have yielded very few tangible results. One of the early discoveries in this field was that the enzyme glyoxalase required reduced glutathione for its action. 62 The mode of action of glutathione in the glyoxalase reaction has recently been satisfactorily explained. It has been shown that glyoxalase consists of two enzymes and that there are two separable enzymatic steps in the conversion of methylglyoxal to lactic acid.63,64 The first step consists in the condensation of methylglyoxal and glutathione, catalyzed by glyoxalase I. This condensa­ tion product is cleaved into glutathione and lactic acid through the agency of glyoxalase II. 6 5 When glyoxalase I is added to a solution of glutathione and methyl glyoxal, a condensation product is formed which has a marked light absorption in the ultraviolet at 240 ιημ. When the formation of the inter­ mediate is complete, if glyoxalase II is added the characteristic absorp­ tion disappears. 65 A mixture of glyoxalase I and glyoxalase II catalyzes the formation of lactic acid from methylglyoxal in the presence of cata­ lytic amounts of reduced glutathione. It was determined by Racker that the fractionation of Hopkins and Morgan 66 into purified glyoxalase and accelerating protein factor was essentially a fractionation of glyoxalase I and glyoxalase II. The intermediate condensation product was first described by Yamazoye. 67 The reaction steps catalyzed by the glyoxalase enzymes as proposed by Racker 65 are shown in equation 7. Methylglyoxal Enol Form CH3 CH3

I

I

C = 0 ^ = ± C—OH

I

\f

O

c

||

C=0

+

H

GSH

Glyoxalase I

Intermediate CII.3

I

Lactic Acid CH3

CH3

I

> C—OH —— H C — O H + H 2 0

||

C—OH

i

I

C=0

I

> HCOH

I

(7)

COOH

i

+

GS

GSH Glyoxalase I I

Another enzymatic reaction in which glutathione functions as a coenzyme in a manner similar to the glyoxalase reaction described above, 61

Hopkins, F . G., Biochem. J. {London) 15, 286 (1921). Lohmann, K., Biochem. Z. 254, 332 (1932). 63 Racker, E., Federation Proc. 9, 217 (1950). 64 Crook, E. M., and Law, K., Biochem. J. (London) 46, P X X X V I I (1950). «5 Racker, E., J. Biol. Chem. 190, 685 (1951). 6(5 Hopkins, F . G., and Morgan, E. J., Biochem. J. (London) 42, 23 (1948). 67 Yamazoye, S., J. Biochem. (Japan) 23, 319 (1936). 62

METABOLISM OF SULFUR-CONTAINING COMPOUNDS

165

is in the oxidation of glyceraldehyde-3-phosphate to 3-phosphoglycera t e 68-70 Qlutathione was found to be firmly bound to glyceraldehyde-3phosphate dehydrogenase, and the bound material did not exchange with free C14-labeled glutathione. From this it was deduced that it is a coenzyme for this enzyme. Glutathione also protected and restored the glycolytic activity of mouse brain homogenates. The observation that glutathione is firmly bound to the enzyme sug­ gested a reaction mechanism for the oxidation of glyceraldehyde-3-phosphate in which the SH group takes an active part. This is shown in equation 8. R O R _2H R R O \^ C

I HO

DPNox I +HOPO3- \f OH 1 ~C ^ O" ^ —C "" , DPNred

+ H SH

I

S

I

+ S

I

(8) OP03

SH

I

In the above equation R represents the balance of the 3-glyceraldehyde phosphate and E, the balance of the enzyme. The evidence for the above two-step reaction is that: (1) the formation of a thiol ester can be demonstrated when the reaction is carried out in the absence of phosphate and in the presence of glutathione, (2) the utilization of various thiol esters by the enzyme, and (3) by the observa­ tion that it is possible to separate the oxidative from the phosphorolytic step by treatment of the enzyme with iodoacetate. In the latter the oxidation of the aldehyde is completely blocked, while an appreciable rate of arsenolysis remains, but only upon addition of reduced glutathione as an SH acceptor for the reaction. 2. COENZYME A

Coenzyme A comes within the purview of this chapter by the dis­ covery that 2-mercaptoethaneamine is a component of CoA, and, in fact, is the reactive part of the coenzyme. It would be repetitive to describe the different functions of CoA as these are discussed in Chapters 2, 4, and 7. The structural formula of the compound is shown on page 149. The reaction of the SH group of coenzyme to form acyl CoA derivatives is illustrated in Scheme 7, page 151. According to Lipmann 71 "CoA is a general acetyl carrier, shuttling 68

69 70 71

Krimsky, I., and Racker, E., / . Biol. Chem. 198, 721 (1952); Federation Proc. 13, 245 (1954). Racker, E., and Krimsky, I., J. Biol. Chem. 198, 731 (1952). Harting, J., and Velick, S., Federation Proc. 11, 226 (1952). Lipmann, F., Bacteriol. Revs. 17, 1 (1953).

166

DAVID M.

GREENBERG

between the donor systems, the i transferases/ and the acceptor-specific enzymes, 'acetokinases.'" A representation of the different acetyl transfer reactions is contained in Fig. 3. DONOR SYSTEMS

ACCEPTOR SYSTEMS

TRANSACETYLASES:

ACETOKINASES: Acetyl SAM, choline

A c~Ph ACf'VPh

Histamine Glucosamine Amino acids Hydroxylamine

Acetylformatc (pyruvate) ----~

+ - - ) Citrate

+Oxaloacetate

Acetoacetate

Acetate+ CoA + ATP Acetoacetate Acetoacetate F I G . 3. Map of acetyl transfer ' ' t e r r i t o r y " (Lipmann 7 1 ).

A number of excellent reviews on the structural chemistry and func­ tions of CoA have recently appeared. 71-73 3. THIAMINE PYROPHOSPHATE

The structure of cocarboxylase (see Fig. 1 for formula) was estab­ lished by Lohmann and Schuster. 74 A long-established function of cocar­ boxylase (thiamine pyrophosphate, aneurin) is as the coenzyme of α-ketoacid carboxylase. M g + + is also required. The reaction involved is the nonoxidative decarboxylation of an α-keto acid to CO2 and an aldehyde with one less carbon atom. The most important example is the splitting of pyruvic acid to acetaldehyde and CO2 (equation 9). This H

I

CH 3 —CO—COOH -> C H 3 — C = 0 + C 0 2

(9)

reaction is one of the key steps in the fermentation of sugars to ethanol. 72 73 74

Novelli, G. D., J. Cellular Comp. Physiol. 41, Supplement 1, 67 (1953). Barker, H. A., Phosphorus Metabolism 1, 204 (1951). Lohmann, K , and Schuster, A., Biochem. Z. 294, 188 (1937).

METABOLISM OF SULFUR-CONTAINING COMPOUNDS

167

Another, more recently discovered function of thiamine pyrophos­ phate is in the oxidative decarboxylation of pyruvic acid in association with lipoic acid. This is discussed in the next section (Section IV, 4) of this chapter. The mechanism of action of cocarboxylase is still very obscure. The suggestion has been offered that the thiol and disulfide forms of thiamine might constitute an oxidation-reduction system. 75 Subsequently, it has been shown in enzyme experiments with yeast that the isomeric thiazole pyrophosphate exhibited full cocarboxylase activity, whereas the disulfide

—N /

CH3

CH3

I I c=c—SH

I I II c=c—s—s—c=c

\ H CForm = 0 of Thiol Thiamine

—N/

\

CH3

\ N—

/

H C = 0 Form of Thiamine 0=CH Disulfide

form was completely inactive. 76 The disulfide form can be reduced by cysteine; therefore, it may be assumed that in biological systems the inactive disulfide form can readily be converted to the active thiol form. There is no evidence to indicate that a cycle of oxidation-reduction of the sulfur atom or of any other portion of the thiamine molecule is concerned in the cleavage of the CO2 from α-keto acids. Thiamine pyrophosphate has also recently been shown to exert a coenzyme function in the metabolism of pentose phosphate. 77,78 The formation of sedoheptulose phosphate from ribulose-5-phosphate by a highly purified enzyme preparation from spinach, which loses activity upon precipitation of the protein with ammonium sulfate at a low pH, was found to be almost completely reactivated by the addition of thiamine pyrophosphate. 77 The transketolase enzyme of Racker et al.,78 obtained in crystalline form from baker's yeast, catalyzes the cleavage of ribulose-5-phosphate, with the formation of D-glyceraldehyde-3-phosphate upon the addition of an acceptor aldehyde, such as ribose-5-phosphate or glycolaldehyde. The reaction of hydroxypyruvate with D-glyceraldehyde-3-phosphate as acceptor aldehyde leads to the decarboxylation of the hydroxypyruvate with the formation of ribulose-5-phosphate. The transketolase enzyme was demonstrated to have a requirement for thiamine pyrophosphate. 78 75

Zima, O., Ritsert, K , and Moll, T., Hoppe-Seyler's Z. physiol. Chem. 267, 210 (1941). Karrer, P., and Viscontini, M., Helv. Chim. Ada 29, 711, 1981 (1946). 77 Horecker, B. L., and Smyrniotes, P. Z., J. Am. Chem. Soc. 75, 1009 (1953). 78 Racker, E., De La Haba, G., and Leder, I. G., / . Am. Chem. Soc. 75, 1010 (1953).

76

168

DAVID M.

GREENBERG

In the cleavage of pentose phosphate a 2-carbon fragment, presum­ ably glycolaldehyde, is expected to be formed and also to react in the reverse reaction. Glycolaldehyde neither accumulates nor does it react with these enzyme preparations. It is now postulated that an " active glycolaldehyde" is formed in the reactions of pentose-phosphate metabo­ lism. This may be conjugated with thiamine pyrophosphate. 4. LIPOIC ACID (THIOCTIC ACID)

Study of the mechanism of the oxidation of pyruvic acid by certain bacteria led to the discovery of lipoic acid (thioctic acid) as a nutrient metabolite essential for the oxidative decarboxylation of α-keto acids.79 It subsequently was determined that the acetate-replacing factor80 for lactic acid bacteria and protogen, 81 · 82 a growth factor for the protozoan, Tetrahymena gelii, were also identical with lipoic acid. The occurrence of considerable quantities of lipoic acid in mammalian preparations of pyruvate and α-ketoglutarate oxidases suggests that it has the same function in animal tissues as in microorganisms. 83 A number of different chemical forms of lipoic acid are known to occur. Those with biological activity are the disulfide, dimercapto, mono-5-acyl, and the mono-mercapto forms. 84 The most important chemically established compound is α-lipoic acid, the cyclic disulfide of 6, 8-dithiooctanoic acid. This has been isolated in crystalline form from acid-hydrolyzed liver,85 and also prepared syn­ thetically. 86 Thioctic acids with the —SH groups in different positions than the 6, 8 carbons have also been prepared. 87 Because of the asym­ metry of ΟΘ of the octanoate chain, the compound is optically active; the dextro form is believed to be the one biologically active. The synthetic DL-form has only half of the activity of the natural product in certain enzyme reactions. 79 80 81

82 83 84

85

86

87

O'Kane, D. J., and Gunsalus, I. C , / . Bacterial. 56, 499 (1948). Guirard, B. M., Snell, E. E., and Williams, R. J., Arch. Biochem. 9, 381 (1946). Stokstad, E. L. R., Hoffmann, C. E., Regan, M. A., Fordham, D., and Jukes, T. H., Arch. Biochem. 20, 75 (1949). Snell, E. E., and Broquist, H. P., Arch. Biochem. 23, 326 (1949). Green, D. E., Science 115, 661 (1953). Gunsalus, I. C., in W. D. McElroy and B. Glass, Mechanisms of Enzyme Action, Johns Hopkins University Press, Baltimore, 1954, p. 545. Reed, L. J., DeBusk, B. G., Gunsalus, I. C., and Hornberger, C. S., Jr., Science 114, 93 (1951). Hornberger, C. S., Jr., Heitmiller, R. F., Gunsalus, I. C., Schnakenberg, G. H. F., and Reed, L. J., / . Am. Chem. Soc. 75, 1273 (1953). Bullock, M. W., Brockman, J. A., Patterson, E. L., Pierce, J. V., and Stokstad, E. L. R., / . Am. Chem. Soc. 74, 3455 (1952).

METABOLISM OF SULFUR-CONTAINING COMPOUNDS

169

If the theory of the mode of action of lipoic acid is correct, the reduced dithiol form of α-lipoic acid is equally as important as the disulfide ring form and, in fact, the two are believed to form a reversible oxidationreduction couple,84 as is shown in equation 10. CH2—CH2—CH— (CH2)4—COOH ; = ± CH2—CH2—CH—(CH2)4—COOH

I SH

I

SH

S

I

SI

+ 2H+ + 2e-

(10)

In addition to the biologically active forms, a compound which is the sulfoxide of α-lipoic acid, probably formed as a chemical oxidation prod­ uct of the cyclic disulfide, has been isolated and named ß-lipoic acid. 88 It is biologically inactive. Calvin and Barltrop 89 have proposed the theory that a possible primary quantum conversion act of photosynthesis consists of the lightinduced dissociation of the disulfide bond from a strained five-membered disulfide ring, leading to a biradical, thereby converting the light energy to chemical energy. It is interesting that α-lipoic acid is colored yellow, which is evidence of strain in the disulfide ring. The biradical is suggested as the chemical species in which the quantum absorbed by the plant pigments and stored as electronic excitation in chlorophyll appears first as chemical bond potential energy. Subsequently, the thiyl free radicals take up two 2H atoms from a suitable donor, with the formation of the dithiol. This ultimately is reoxidized to the disulfide ring-containing form by C0 2 . The lipoic acids are believed to have a general function in the oxidative decarboxylation of α-keto acids. 84,90 The outstanding example is pyruvic acid, but it also functions in the oxidation of a-ketoglutaric and α-ketobutyric acids. The latter acid is employed as a substrate to study the characteristics of the enzyme system to avoid the complicating effect of acetoin formation which occurs with pyruvate. 90 According to Gunsalus, 84 lipoic acid functions between thiamine pyrophosphate and CoA as an acyl-generating and transfer catalyst, and as a hydrogen-transfer catalyst. A reaction sequence showing the opera­ tion of lipoic acid is given in Fig. 4. As shown in the figure, the function of lipoic acid in the oxidative acyl-generating keto-acid reaction appears to be preceded by a decarboxylase reaction mediated by thiamine pyro­ phosphate to generate a thiamine-pyrosphosphate-aldehyde. Of the four 88

89 90

Reed, L. J., De Busk, B. G., Hornberger, C. S., Jr., and Gunsalus, I. C., / . Am. Chem. Soc. 75, 1271 (1953). Calvin, M., and Barltrop, I. A., / . Am. Chem. Soc. 74, 6153 (1952). Gunsalus, I. C., J. Cellular Comp. Physiol. 41, 113, Supplement (1953).

170

DAVID M.

GREENBERG

reactions shown in the figure, 3 and 4 are considerably clarified, and the enzyme catalyzing 4 has been obtained separately. "Each of the 4 reactions from α-keto acid, (1) decarboxylation, (2) acyl generation, (3) acyl transfer, and (4) hydrogen transfer, constitutes a single step reaction." 84 Each may be catalyzed by a separate enzyme. In reaction 1, the heterolytic cleavage of the substrate to yield CO2 and a carbanion coordinated with thiamine pyrophosphate is deduced from the multiple reactions which the carbanion can undergo in biological systems. 90 O

0

O

II II

Ri—C—C—O- + DPT+

- ^ R

O + ··

Ri—C : D P T

>

Ί

R i — C : D P T J + CO,

^ = ± R i — C : S—< : S—

R-

9 R i — C : S—< > : S—/

- \ : S—/

+ DPT+

(2)

/

R-

9

HS—/ + CoA—SH ^ = ± R x —C : S CoA + > ' : S—/

RH S

(1)

(3)

R + D P N +

^ .

S

- \ S—χ

+DPNH

R x = CH 3 —, HOOC(CH 2 ) 2 — etc.

R =

(4) -(CH2)4COOH

D P T + = Diphosphothiamine F I G . 4. Reaction sequence of oxidative decarboxylation of α-keto acids, illustrating the coenzyme roles of diphosphothiamine and lipoic acid (after Gunsalus 8 4 ).

Lipoic acid presumably functions only when acyl generation and transfer occur and not in decarboxylation or aceton formation. The mechanism of acyl generation suggested in reaction 2, Fig. 4, is the carbanion cleavage of the disulfide bond to form the free thioester with CoA and the free sulfhydryl of lipoic acid. Reaction 3 shows the transfer of an acyl (acetyl) group to CoA. In the presence of Pi and phosphotransacetylase, the acyl group would accumu­ late as acetyl phosphate. The transacetylase reaction of lipoic acid has been determined by employing the reverse of the phosphotransacetylase reaction, mentioned above, and reaction 3 of Fig. 4. In the presence of catalytic amounts of CoA, lipoic transacetylase activity can be demon­ strated by measuring the accumulation of a heat-stable hydroxamic acid derived from the acyl-lipoate or by measuring the disappearance of —-SH groups.

METABOLISM OF SULFUR-CONTAINING COMPOUNDS

171

Reaction 4 depends upon the operation of a DPN-dependent, lipoic acid dehydrogenase. A protein fraction has been obtained from E. coli which exhibits this property. Measurement of lipoic acid dehydrogenase activity directly with DPN+ and reduced lipoic acid as reductant or reoxidation of D P N H with α-lipoic acid as oxidant was not particularly successful. However, lipoic acid dehydrogenase activity could readily be determined by linking it with lactic acid dehydrogenase in the presence of pyruvate and catalytic amounts of DPN, by measuring the residual —SH groups. Reed and De Busk have suggested that the true coenzyme form of lipoic acid is a conjugate of α-lipoic acid and thiamine pyrophosphate, given the name lipothiamide. 91 · 92 The bond between the two is presumed to be a peptide bond between the amino group on Ci of the pyrimidine moiety of thiamine and the carboxyl group of α-lipoic acid. The true coenzyme is reported to be the lipothiamide pyrophosphate. The evidence for the coenzyme function of lipothiamide pyrophos­ phate is that it supports the growth and activates enzyme preparations of a mutant of E. coli. Reed and De Busk also report the formation of a substance with coenzyme activity by treating thiamine pyrophosphate with α-lipoyl chloride. The above authors have reported the activation of a cell-free preparation of pyruvate dehydroganase from the mutant of E. coli, but Gunsalus 84 was unable to activate the pyruvate dehydro­ genase of S. faecalis, nor of certain purified enzyme fractions from E. coli which are thiamine pyrophosphate-dependent. Gunsalus 84 also cites other evidence, too lengthy and involved to reproduce here, against the proposals of Reed and De Busk. 91 92

Reed, L. J., and De Busk, B. G., J. Am. Chem. Soc. 74, 3457, 3964 (1952). Reed, L. J., and De Busk, B. G., / . Biol. Chem. 199, 873 881 (1952).

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CHAPTER

13

Enzymatic Syntheses of Peptide Bonds H. BORSOOK* Kerckhoff

Laboratories

of Biology,

California California

Institute

of Technology,

Pasadena,

Page I. Thermodynamic Considerations 174 1. The Free Energy of Formation of Peptide Bonds 174 2. The Effect of Concentration of Reactants and Products on the Degree of Synthesis 175 3. The Influence of the Amino Acid Side Chain on the Degree of Synthesis 177 4. The Effect of Hydrogen Ion Concentration on the Free Energy Change in Peptide Formation 178 I I . Classification of Enzymatic Peptide Syntheses According to the Sign and Magnitude of the Free Energy Change ( - Δ ^ ) 182 1. Peptide Syntheses Where — AF I S Positive and Large 183 2. Peptide Syntheses Where — AF Is Small and the Peptide Is Relatively Insoluble 184 3. Plastein Formation 185 4. Peptide Synthesis in an Exchange Reaction during Hydrolysis (Transamidation and Transpeptidation) 187 5. Peptide Synthesis from Amino Acid Esters 194 6. Glutamo- and Aspartotransferases 195 I I I . Peptide Syntheses Where — AF Is Negative and Large: Coupled with HighEnergy Phosphate 198 1. Synthesis of Glutamine 198 2. Synthesis of Hippuric Acid 199 3. Synthesis of p-Aminohippuric Acid 200 4. Synthesis of Ornithuric Acids 201 5. Synthesis of Glutathione 202 IV. Mechanism of Amino Acid Incorporation into Proteins 203 1. Effect of Inhibitors 203 2. Comparison of Transfer and Synthetic Reactions 207 3. Amino Acid Incorporation and Phosphorylation 208 4. Is Amino Acid Incorporation Synthesis of Protein De Novo or an Ex­ change? 210 * The writing of this article and the studies by the author and his colleagues referred to were aided by a contract between the Office of Naval Research, United States N a v y Department, and the California Institute of Technology, Division of Biology (NR-164-304). They were also supported (in part) by a research grant from the National Institutes of Health, United States Public Health Service, and by a grant from the American Cancer Society. 173

174

H.

BORSOOK

5. The Possibility of Peptides as Intermediates in Protein Synthesis 6. The Mode of Linkage of Incorporated Amino Acids V. Addendum (David M. Greenberg) 1. Incorporation of Amino Acids as an " E x c h a n g e " Reaction 2. Mechanism of Activation of Amino Acids for Protein Synthesis 3. Relation of Nucleic Acids to Protein Synthesis

Page 212 216 219 219 220 221

I. THERMODYNAMIC CONSIDERATIONS 1. T H E F R E E ENERGY OF FORMATION OF PEPTIDE BONDS

Until recently few enzymatic syntheses of peptides had been achieved in vitro. In the last few years a number of different types of such syntheses have been discovered. In the early exploration of this field thermodynamic considerations guided experimentation and thinking. Now the same con­ siderations are useful in distinguishing fundamental differences in the mechanism of these syntheses. Practically all of Biochemistry may be considered as operating in an isothermal system. Here the central energetic quantity is the change in free energy. 1 In the reaction AB± + H 2 0 -> A± + B±, where AB±, A±, and B± are a zwitterionic dipeptide and its constituent amino acids, respectively, — AF is positive, i.e., there is a loss of free energy on hydrolysis. And the loss of free energy is so large that at equilibrium hydrolysis is very nearly complete. Accordingly if one begins with the free amino acids there is practically no spontaneous formation of the dipeptide. The equilibrium constant and thence the equilibrium position of a reaction can be computed by means of the equation — Δ^ = RT 2.303 log K. Here — AF refers to the reaction where all reactants and products are considered to be at 1 molar activity except water which is assumed to have the molar activity of pure liquid water. Table I contains the free energies and heats of formation of some amino acids and peptides. 1

The sign convention we shall use is t h a t the free energy change, — AF, is (free energy content of the system in its initial state) — (free energy content of the system in its final state). In this convention the relation between the free energy change and the equilibrium constant is — AF = RT In K, where — Δ ^ is the free energy change where reactants and products are in their standard states, and K is the equilibrium constant. — AF is positive where K is greater t h a n 1. This is a case where, at equilib­ rium, the product formed in the reaction exceeds the reactants, i.e., the reaction, as written, goes largely to the " r i g h t . " — AF is negative where K is less than 1, i.e., in those cases where the reaction goes spontaneously to only a small extent.

ENZYMATIC SYNTHESES OF PEPTIDE BONDS

175

TABLE I THE

F R E E E N E R G I E S AND H E A T S OF FORMATION (IN GRAM C A L O R I E S ) OF SOME AMINO ACIDS AND P E P T I D E S 1

Compound DL-Alanine D L-Alany lgly cine Benzoic acid Benzoate ion Glycine Glycylglycine Hippuric acid Hippurate ion Hippurylglycine DL-Leucine D L-Leucy lgly cine Water

Δ^° 3 10.6

ΔΗ298.1

Δ^298.1

Δ^310.6

- 86,830 -113,190 - 57,660

-132,370 -186,410

- 89,110 -117,570

- 87,300 -114,680

- 86,920 -115,200 - 86,160

-122,500 -175,960

- 51,175 - 89,140 -118,060

- 49,710 - 87,710 -115,630

-114,710 - 80,350 -108,225 - 56,200

-191,700 -150,900 -203,620 - 68,320

- 81,000 -115,580 - 81,760 -110,900 - 56,690

- 78,590 -112,380 - 78,870 -107,065 - 56,200

AP0 refers to the pure substances; AH, AF refer to the substance in the standard state in aqueous solution, which is 1 M activity, except in t h e case of water where it is mole-fraction = 1. Temperatures in degrees absolute. 2. T H E EFFECT

OF CONCENTRATION OF REACTANTS AND PRODUCTS ON THE D E G R E E OF SYNTHESIS

The following approximate calculation shows the relation of the equilibrium constant to the equilibrium degree of synthesis of a dipeptide from its amino acids. Where a is the initial concentration of each of the two amino acids, and a the degree of synthesis, the equilibrium relation is aa a 2 (l - OLY

=

K

Since a is very small with respect to 1, this expression is simplified to a = Ka. It follows then that the degree, as well as the absolute amount of synthesis, is dependent on the initial concentration of the amino acids. Table I I contains the free energy change in the synthesis of some small peptides in the reactions as written, the equilibrium constant, and cal­ culations of the degree of synthesis by mass action at different initial concentrations of reactants. The data show first: that at the concentra­ tions of free amino acids in the tissues—the concentration is on the average about 0.001 M for any one amino acid—there can be very little synthesis by mass action, the di- and tripeptides listed in the table being more than 99% hydrolyzed. Of course it is possible, and there is no infor­ mation on this point, that at certain loci in cells, for example, within 1

Borsook, H., Unpublished data.



TABLE II F R E E E N E R G Y OF FORMATION OF SOME SMALL P E P T I D E S , THE EQUILIBRIUM CONSTANT AND D E G R E E OF SYNTHESIS BY M A S S ACTION AT D I F F E R E N T I N I T I A L CONCENTRATIONS OF REACTANTS

Reaction DL-Alanine + glycine—> DL-alanylglycine + H 2 0 2 Glycine -> glycylglycine + H 2 0 DL-Leucine + glycine -> DL-leucylglycine + H 2 0 Benzoate + glycine -> hippuric acid + H 2 0 Benzoate + glycylglycine —> benzoylglycylglycine + H 2 0 iV-benzoyltyrosine + glycineamide —> iVbenzoyltyrosylglycineamide + H 2 0 2 3

-AF (calories) —4130 -3590 -3315 -2630

at at at at

37.5°C. 37.5°C. 37.5°C. 37.5°C.

- 1 1 0 0 at 25°C. -361 at 37.5°C.

Per cent synthesis at equilibrium a t initial concentrations of

Equilib­ rium constant K

0.11

0.00125 0.00299 0.00467 0.0142

X X X X

0.1564 0.5582

Borsook, H., and Dubnoff, J. W., J. Biol. Chem. 132, 307 (1940). Dobry, A., Fruton, J. S., and Sturtevant, J. M., / . Biol Chem. 195, 149 (1952).

10- 2 lO" 2 lO" 2 lO" 1

Reactants 0.01 M

0.001 M

Reference

10" 4 10~ 3 10~ 3 10~2

+

ENZYMATIC SYNTHESES OF* PEPTIDE BONDS

179

where hi and k2 are ionization constants.

uwi

and

ri_-, _

mh

Mlu [I J - [ H + ] ki [I±][H+] , [I±]/c 2 _ rT±1 l[H+P + HK+] + frfe,) Σ[Ι] = — — + [I±] + T T F f - [I±] ( kjjj+j j, [I±] = ΣΙ

and

fcl[H+]

[H+]2 + HH+] + ki.kt

Substituting this general relation into the equilibrium expression for the zwitterions the form of the latter equation is

yrA1 S

lAJ

l[H+]2 + fciAB[H+] + /dAB/c2AB *iA[H+] . m] fc^H+J S LB J 22 , 7, ΑΓΤΤ+1 A . / . AA Τ τ τ I. A L J [H+]2 + fciB[H+] + A A * + Ί [H+] + /ciA[H+] +, /cx · /c2 Σ[ΑΒ] ί (VB) Α Σ[Α]· Σ[Β] 1(Κ1 ·Α:1Β[Η+]) ([Η+]2 + / V [ H + ] + /dAÄ:2A)([H +]2 + /V[H+] + AB AB ([Η+]2 + fc!AB[H+] -f+ /d /c2 )

kfkfXj /)'

The equilibrium constant is seen to be equal to the ratio of the total con­ centration of the dipeptide (i.e., in all its ionic forms) divided by the product of the total concentrations of each of the free amino acids, this ratio multiplied by an ionization term consisting of ionization constants and the hydrogen ion concentration. Simply stated this relation is total dipeptide . rr w . : K = (total T—— ammo -J A) Aw+ + i ammo · i ρB) acid (total acid : λ X ionization term. Hence total dipeptide (total amino acid A) (total amino acid B)

K ionization term

K is the equilibrium constant for the zwitterionic forms; if it is calculated from free energy data, those pertaining to the zwitterionic forms must be used. The same numerical result for the degree of total synthesis will be obtained whatever mechanism is formulated. If ions instead of zwitterions are considered to be the reactants, K and — AF will be different from those for the zwitterion mechanism, but there will be a change in the ionization term such that it will compensate exactly for the change in K. We shall apply the above equations to two examples. The first is the

180

H.

BORSOOK

synthesis of alanylglycine: alanine is designated as A, glycine as B, and alanylglycine as AB. Here fcxA is 4.6 X 10~3; k2A is 1.35 X 10~10; kf is 4.5 X 10- 3 ; /c2B is 1.7 X lO" 10 ; /cxAB is 6.8 X 10~4; and k2AB is 3.8 X 10~9 (reference5-6). From these data the values of the ionization term from pH 3 to 9 are computed and given in Table III. It is seen that, given the low TABLE III VARIATION OF THE IONIZATION T E R M WITH P H I N THE E Q U I L I B R I U M E X P R E S S I O N IN THE SYNTHESIS OF ALANYGLYCINE FROM A L A N I N E AND G L Y C I N E

pH

Value of ionization term

3 4 5 6 7 8 9

0.60 0.91 0.99 1.00 1.00 0.99 0.28

equilibrium constant for the synthesis from the zwitterions, the ioniza­ tion term does not decrease significantly from 1, and hence the increase in the over-all degree of synthesis that can be obtained by changing the pH over this range is always very small. The second example is the synthesis of benzoyltyrosylglycineamide from benzoyltyrosine and glycineamide. In this reaction one of the reactants is an anion, the other a cation, and the product has no charge. We shall designate benzoyltyrosine as 0 C O O , glycineamide as + H 3 NR, and benzoyltyrosylglycineamide as 0 C O . H N R . The equilibrium ex­ pression is [ 0 C O - HNR] = K [0COÖ] [ + H 3 NR] K refers specifically to the reaction between the two ionized forms of the reactants. As in the first example we shall restate the equilibrium expres­ sion in terms of the total concentrations of the reactants, the ionization constants, and the pH. 5

Borsook, H., and Huffman, H. M., Some Thermodynamical Considerations of Amino Acids, Peptides and Related Substances. In C.L.A. Schmidt, Chemistry of the Amino Acids and Proteins, p. 822. C. C Thomas, Springfield, Illinois, 1938. 6 Hitchcock, D., Amphoteric Properties of Amino Acids and Proteins. In C.L.A. Schmidt, Chemistry of the Amino Acids and Proteins, p. 596. C. C Thomas, Springfield, Illinois; 1938.

181

ENZYMATIC SYNTHESES OF PEPTIDE BONDS

10COOKH+] _ [0COOH] - ^ '

r t f p o o H l - Γ1 [0COOH] - [0COO]

[H+]

- ^

S[0COOH] = [0COOH] + [0COÜ]

= [0COÖ] · [ ^ + [0COÜ] [0ΟΟΟ](^-) [0COÖ] = S[0COOH] ( Ϊ Ϊ Ϊ + ^ Γ Χ Λ ) Similarly

w I F ^

[H.NR] = [+H3NR] ^

S[H 2 NR] = [+H 3 NR] + [H 2 NR] = [+H3NR] + [+H3NR] - p ^

= [+H,NR](lM^} .·. [*H,NR] = 2[H,NR1 (,„1"^ ,..)■ The equilibrium expression is, then, K

=

[0COHNR] *[0COOH] ( j g ^ )

[H 2 NR]

[0COHNR] S[0COOH] · 2[H 2 NR]

([H+] + /cA)([H+] + fcB) /cA[H+]

( j H ^ i )

As in the first example the equilibrium expression consists of two terms, one containing the total concentrations of product and reactant, and a second the ionization constants and hydrogen ion concentration. Table IV lists the values of the ionization term over the pH range 3-9. Here kA is 2 X 10"4, and kB is 1.18 X 10~8 (reference3). The minimum value of the ionization term is in the neighborhood of pH 5.8. This, then, is the pH of maximum synthesis, or conversely of minimum hydrolysis, if one begins with the peptide. The effect of changing pH is different in the two examples given. With alanylglycine the degree of synthesis increases progressively on either side of pH 6, whereas with benzoyltyrosylglycineamide it decreases. The reason is that in the first case the product is a zwitterion whose acid ionization constant is less, and whose basic ionization constant is about twenty-five times stronger, than that of either of its constituent amino acids.

182

H. BORSOOK T A B L E IV

VARIATION OF THE IONIZATION T E R M WITH P H IN THE E Q U I L I B R I U M E X P R E S S I O N OF BENZOYLTYROSYLGLYCINEAMIDE

FROM BENZOYLTYROSINE AND GLYCINEAMIDE

pH

Value of ionization term

3.0 4.0 5.0 5.8 6.0 7.0 8.0 9.0

6.0 1.5 1 05 J 02 1.02 1.22 2.18 12.8

II. CLASSIFICATION OF ENZYMATIC P E P T I D E SYNTHESES ACCORDING TO T H E SIGN AND MAGNITUDE OF T H E F R E E E N E R G Y CHANGE (-AF) Notwithstanding that the free energy of formation of peptide bonds from free amino acids corresponds to an equilibrium position in dilute solution beyond 99% hydrolysis, a number of authors have inclined to the view that peptide and protein synthesis may be catalyzed by proteases and peptidases. The free energy data indicate that, whether or not this is the case, the synthesis of small peptides from amino acids cannot, under physiological conditions, be a simple mass action reversal of hydrolysis. On the other hand, the condensation of large peptides may be promoted by proteases alone. M. Bergmann has been the most notable proponent in recent times of the view that proteases and peptidases participate in protein and peptide synthesis tn vivo. He and his collaborators considered that the differences among the reactions they studied arose from differences in enzyme-substrate specificity. Except in so far as the formation of a rela­ tively insoluble product tended to drive the reaction in the direction of the latter, the authors took no account of the free energy change of the reaction, i.e., the magnitude of the equilibrium constant. It is this char­ acteristic, however, which provides a basis of classification. The categories of reactions studied can be distinguished as follows: (1) where — AF for the condensation is positive in sign and so large that the reaction proceeds nearly to completion; (2) where — AF for the condensation is positive in sign but the order of magnitude is small, and as a result, the direction of the reaction is affected by the concentrations of reactants and products; (3) transfer reactions; (4) where — AF is negative in sign for the con­ densation, i.e., the reaction is an hydrolysis, and can be reversed only by

ENZYMATIC SYNTHESES OF PEPTIDE BONDS

183

coupling with another reaction system in which — AF is large enough to reverse the reaction, i.e., to effect synthesis. 1. PEPTIDE SYNTHESES W H E R E

— AF

Is POSITIVE AND LARGE

An example of this category is as follows: a solution containing 4.2% of carbobenzoxyglycine and 3.7% aniline was incubated with activated papain at 40°C. and at pH 4.6. Carbobenzoxyglycine anilide was formed in 80% yield. The optimum pH and the necessity for activation by cysteine, glutathione, or HCN were the same as for the hydrolytic action of the enzyme. Analogously, hippuric acid and aniline gave hippuric acid anilide. Acetyl, benzoyl, and carbobenzoxy derivatives of alanine, leucine, and phenylalanine yielded with aniline or phenylhydrazine the corresponding anilides or phenylhydrazides; acetyl-L-phenylalanyl-Lglutamic acid and p-toluenesulfonyl-glycine gave with aniline the corre­ sponding anilides. Similar reactions were catalyzed by bromelin and cathepsin, the proteolytic enzymes, respectively, of pineapple and pig liver. Under the conditions that promoted the above syntheses hippurylamide was completely hydrolyzed; there was no synthesis of the amide from hippuric acid and ammonia. 7 · 8 Waldschmidt-Leitz and Kühn 9 studied in detail the synthesis of hippurylanilide—the type of synthesis first found by Bergmann and associates. Using nearly equivalent amounts of hippuric acid and aniline, with papain as enzyme, there was 94% synthesis of the anilide. The equilibrium was approached from both sides. They could not obtain synthesis of hippurylamide from hippuric acid and ammonia; the amide was completely and rapidly hydrolyzed. The condensation with hippuric acid occurred with aniline, o- m-, and p-toluidine, o- and p-aminophenol, o-anisidine, p-aminobenzoic acid, sulfanilamide, and o- and p-phenylenediamine. The following compounds were inactive: iV-methylaniline, o-aminobenzoic acid, sulfanilic acid, α-aminopyridine, adenine, benzylamine, cyclohexamine, and ammonia. From the equilibrium data it is possible to calculate the free energy of formation of hippurylanilide under their conditions. The value is approxi­ mately 5000 cal. at 37°C, whereas the free energy of formation of small peptides and of amides is of the order of magnitude of —3500 cal. In other words, the formation of the anilide proceeds spontaneously, as they found, but the formation of analogous peptides and amides does not. This is the explanation of the failure to observe condensation of hippuric 7

Bergmann, M., and Fraenkel-Conrat, H., / . Biol. Chem. 119, 707 (1937). Bergmann, M., and Fraenkel-Conrat, H., / . Biol. Chem. 124, 1 (1938). 9 Waldschmidt-Leitz, E., and Kühn, K., Hoppe-Seyler's Z. physiol. Chem. 285, 22 (1950).

8

184

II.

BORSOOK

acid and ammonia. Specificity of the enzyme cannot be invoked because the enzyme hydrolyzed hippurylamide. The foregoing "peptide" syntheses can, at best, be only analogs of peptide synthesis in vivo from amino acids. Even if one grants that they are analogs of the in vivo process, it would still be necessary to find the physiological analogs of the acetyl, benzoyl, and carbobenzoxy deriva­ tives of the amino acids, and of aniline and phenylhydrazine. This leaves the problem where it was before: we would need to find the reactive inter­ mediates and to learn the mechanisms by which they are formed. Almost certainly there would have to be coupling with energy-yielding reactions in such a manner that reactive intermediates with higher chemical poten­ tial than free amino acids were formed. And it might very well then turn out that the enzyme promoting the coupling is not a protease or peptidase as, for example, in glycogen or sucrose synthesis, where glucose-1-phosphate is synthesized into glycogen or sucrose not by an amylase or disaccharase but by specific phosphorylases. 2. PEPTIDE SYNTHESES W H E R E

— AF

Is

SMALL AND THE PEPTIDE I S

RELATIVELY INSOLUBLE

Examples of the second category of condensations catalyzed by proteolytic enzymes are the following: by papain, benzoyl-L-leucine + L-leucine anilide to benzoyl-L-leucyl-L-leucine anilide, benzoylphenylalanine + leucine anilide to benzoylphenylalanylleucine anilide, 8 carbobenzoxyphenylalanylglycine + tyrosineamide to carbobenzoxyphenylalanylglycyltyrosineamide; 10 by chymotrypsin, benzoyl-L-tyrosine + glycine anilide to benzoyl-L-tyrosylglycine anilide, 11 benzoyl-L-tyrosine + leucine anilide to benzoyl-L-tyrosylleucine anilide. 10 One may include in this category the synthesis of benzoyl-L-tyrosylglycineamide in low yield from 0.02 M solutions of benzoyl-L-tyrosine and glycineamide. (The amide radical of benzoyl-L-tyrosylglycineamide was eventually hydrolyzed off by chymotrypsin.) In these reactions the enzyme was specific for the L-f orm of the amino acids whose derivatives participated in the reactions; even in the reaction of acetyl-DL-phenylalanylglycine with aniline, only acetyl-L-phenylalanylglycine anilide was formed, although glycine, with which the aniline combined, has no asymmetric carbon.12 In the above reactions two amino acids were combined in peptide linkage. There was no coupling with an energy-yielding reaction, nor was there an hydrolytic step in which part of the energy that might have been released was used for formation of the new peptide. It is certain, 10 11 12

Bergmann, M., and Fruton, J. S., Ann. N.Y. Acad. Sei. 45, 409 (1944). Bergmann, M., and Fruton, J. S., J. Biol. Chem. 124, 321 (1938). Bergmann, M., and Behrens, O. K , J. Biol. Chem. 124, 7 (1938).

ENZYMATIC SYNTHESES OF PEPTIDE BONDS

185

therefore, in view of the data in Table II, that benzoyl, carbobenzoxy, or phenylalanyl substitution on one of the amino acids and aniline or amide on the other, so increased the free energy content (i.e., the chemical potential) of the substituted amino acids that they could be condensed in peptide linkage by the enzyme without coupling with another free energy-yielding reaction. To some extent the formation of measurable amounts of peptides such as the above is promoted by their relative insolubility. They appear to be intermediates in hydrolysis. The following is an example of such an intermediate. 10 Glycylleucine is not hydrolyzed by papain-HCN. It is hydrolyzed when acetylphenylalanylglycine is added to the reaction mixture; and the final products are glycine, leucine, and acetylphenyl­ alanylglycine. Acetylphenylalanylglycylglycylleucine was postulated as an intermediate on the basis of the following evidence by analogy. Carbobenzoxy-L-phenylalanylglycine + glycine anilide give carbobenzoxy-L-phenylalanylglycylglycine anilide, which is so insoluble that it precipitates out soon after it is formed and so is not hydrolyzed further. It was argued that replacing the carbobenzoxy residue by acetyl rendered the intermediate peptide too soluble, and hence the reaction proceeded to hydrolysis. The difference between glycylleucine and glycine anilide was not taken into account in this argument, and was, presumably, con­ sidered as unimportant here. The difference may very well be significant, not only with respect to enzyme specificity and solubility of the inter­ mediate but also with respect to the free energy change in the subsequent formation of the peptide bond. 3. P L A S T E I N

FORMATION

Plastein formation appears to belong in the foregoing category of peptide syntheses. Plastein formation is a phenomenon that has been known for a long time. It consists in the enzymatic formation of an insoluble, high-molecular peptide, or mixture of such, from a concen­ trated partial (peptic) hydrolysate of protein. The enzyme usually used for this reversal of hydrolysis has been pepsin. 13,14 Folley 15 questioned whether peptide bonds were reconstituted in plastein formation (see also Ecker 16 ). Salter and Pearson 17 and Collier18 confirmed the original find­ ings: Collier used papain as well as crystalline pepsin. Northrop 19 was 13 14 15 16 17 18 19

Wasteneys, J., and Borsook, EL, Physiol. Revs. 10, 110 (1930). Borsook, H., Physiol. Revs. 30, 206 (1950). Folley, S. J., Biochem. J. (London) 27, 51 (1933). Ecker, P . G. E., / . Gen. Physiol. 30, 339 (1946-1947). Salter, W. T., and Pearson, C. M., / . Biol. Chem. 112, 579 (1935-1936). Collier, H. B., Can. J. Research 18B, 255, 272 (1940). Northrop, J. H., J. Gen. Physiol. 30, 377 (1946-1947).

186

H.

BORSOOK

inclined to the view that peptide bonds were synthesized, although the protein formed was obviously different from the original source of the partial hydrolysate. Virtanen and co-workers 20-23 found that in plasteins the free amino nitrogen was 2 % to 3 % of the total nitrogen, and estimated that, on the average, the plastein contained forty amino acid residues. Cryoscopic and viscosity measurements gave molecular weights that varied from 2500 to 10,000. This and the low free amino nitrogen, Virtanen and associates interpreted as indicating that plastein consists of cyclic peptides. No plastein was obtained when the initial reaction mixture con­ sisted of either free amino acids or a mixture of di- and tripeptides. Beginning with peptides of greater complexity plastein formation occurred. Tauber 24-27 observed plastein formation in peptic digests of several proteins with chymotrypsin as enzyme (trypsin is inactive in the syn­ thesis). Hitherto plastein formation had always been carried out in the neighborhood of pH 4; with chymotrypsin the reaction proceeds rapidly at pH 7.3. " T h e average molecular weights of the synthetic products as determined in the analytical centrifuge are estimated to be in the range 250,000-400,000." As in the case of plasteins formed by pepsin, those formed by chymotrypsin are hydrolyzed in dilute suspension by pepsin, chymotrypsin, and trypsin. In plastein synthesis the free energy of formation of the peptide bonds is small, as attested to by the reversal of hydrolysis merely by concentrat­ ing certain enzymatic hydrolytic products; the synthetic product is insoluble, which tends to drive the reaction towards synthesis. In these two respects plastein formation resembles reactions of the type benzoylL-leucine + L-leucine anilide to benzoyl-L-leucylleucine anilide. Whatever the biological significance of plastein formation may be (and on this point there is at present no evidence pro or con), the phenomenon is interesting in that it indicates that the condensation of certain large peptides entails only a small free energy change. Doubtless enzymesubstrate specificity as well as energy relations is involved. Synthetic substrates were first introduced by Bergmann in the study of protease 20

Virtanen, A. I., and Kerkkonen, H. K., Acta Chem. Scand. 1, 40 (1947). Virtanen, A. I., and Kerkkonen, H. K., Nature 161, 888 (1948). 22 Virtanen, A. I., Kerkkonen, H. K., Hakala, M., and Laaksonen, T., Naturwissen­ schaften 37, 129 (1950). 23 Virtanen, A. I., Kerkkonen, H. K., Laaksonen, T., and Hakala, M., Ada Chem. Scand. 3, 520 (1949). 24 Tauber, H. T., J. Am. Chem. Soc. 71, 2952 (1949). 25 Tauber, H. T., Federation Proc. 9, 237 (1950). 26 Tauber, H. T., / . Am. Chem. Soc. 73, 1288 (1951). 27 Tauber, H. T., / . Am. Chem. Soc. 73, 4965 (1951). 21

ENZYMATIC SYNTHESES OF PEPTIDE BONDS

187

and peptidase action in general, and it is the use of these substrates that has elucidated the action mechanism of these enzymes. It may well be that the use of synthetic substrates may, similarly, elucidate not only plastein formation but also the configurational requirements for con­ densation by proteases of certain, not necessarily very large, peptides, in vivo and in vitro. 4. PEPTIDE SYNTHESIS IN AN EXCHANGE REACTION DURING HYDROLYSIS (TRANSAMIDATION AND TRANSPEPTIDATION)

The third category of reactions discovered by Bergmann involving proteases is an exchange during hydrolysis. The first example was found when hippurylamide was hydrolyzed by papain in the presence of aniline. 7 Under these conditions hippuric acid anilide was formed faster than from hippuric acid and aniline, indicating some direct replacement of the amide group by aniline. Another example was the formation of benzoylL-leucine anilide from benzoyl-L-leucine and glycine anilide; here the glycine residue in glycine anilide was replaced directly by benzoyl-L-leucine.8 This class of reactions has been investigated further and extended in a new direction by Fruton and his collaborators. Fruton 28 digested benzoylglycineamide with cysteine-activated papain in the presence of N 15 H 3 . At all stages before complete hydrolysis, the amide radical in the unhydrolyzed benzoylglycineamide was found to be partially replaced by the N 1 5 H 3 from the medium. Eventually the substrate is practically completely hydrolyzed by the enzyme, yet the amount of replacement was greater than the calculated equilibrium amount. The degree of replacement was always much less than the degree of hydrolysis. The highest degree of replacement reported in a later study, that of Johnston et aL,29 was 3.5% (theoretical maximum 50%) when 4 1 % hydrolysis had occurred. When there was no hydrolysis there was no replacement. It was found that the optimum pH for replacement was several units higher than that for hydrolysis. At pH 7.9 they found a rough propor­ tionality between hydrolysis and replacement, the degree of replacement being always less than that of hydrolysis. The data in Table I I I indicate that a high pH tends to favor peptide synthesis where the ionization con­ stant of the amino group of the peptide is stronger than that of the con­ stituent amino acids. This may account for the observation that inter­ change in the course of hydrolysis was greater at higher pH values. As far as the actual amount of synthesis is concerned, both the free energy data and the observations of Fruton and associates indicate that this effect is small. An analogous reaction was found to be catalyzed by activated papain 28 29

Fruton, J. S., Yale J. Biol. and Med. 22, 263 (1950). Johnston, R. B., Mycek, M. J., and Fruton, J. S., J. Biol. Chem. 185, 629 (1950).

188

H.

BORSOOK

between acylamino acid amides and hydroxylamine to form hydroxamic acids: RCONH 2 + NH 2 OH ; F = ± RCONHOH + NH 3 . Substrates with hydroxylamine were: benzoyl-L-arginine amide, carbobenzoxy-L-isoglutamine, carbobenzoxy-L-isoasparagine, benzoylglycineamide, carbobenzoxy-L-serineamide,, carbobenzoxy-L-methionineamide, and carbobenzoxy-D-methionineamide. The D-compound was neither hydrolyzed nor did it form hydroxamic acid. In the case of all the others there was a proportionality betAveen the degree of hydrolysis and hydroxamic acid formation. A lower pH favored hydrolysis; a higher pH, hydroxamic acid formation, except in the case of benzyoyl-L-arginineamide. The authors report that they have found similar results with cathepsin as enzyme. The hydroxamic acid was always a small fraction of the ammonia liber­ ated from the amide. Fruton 28 ' 30 proposed that the mechanism of hydrolysis of a peptide or amide bond involves "activation" of the C = 0 bond, in which step it is OH / converted to C , and that free ammonia, hydroxylamine, a free amino group of an amino acid peptide or amide, or water compete for the unsubstituted bond on the "activated" carbonyl group. When the ac­ tivated carbonyl addition is with water, there is hydrolysis; when it is with any of the other radicals or compounds, there is replacement. Evi­ dence in favor of this mechanism was found30 in the following reaction with chymotrypsin. Benzoyl-L-tyrosylglycineamide (BTGA) was in­ cubated with glycineamide containing N 15 in the glycine nitrogen. After some time, when 42% hydrolysis of BTGA had occurred, the remaining BTGA was isolated and N 15 was found in the tyrosylglycine peptide bond. The amount of N 15 indicated 17% replacement of this nitrogen. The reaction was formulated as in the diagram below, the postulated inter­ mediate being enclosed in brackets. C6H5CO.NH.CH.CO.NH.CH2.CONH2

I

+

NH2.CH2.CO.NH2

CH 2 .C 6 H 4 OH Benzoyltyrosylglycineamide NH.CH2.CONH2

Glycineamide

rCeH6.CO.NH.CH— C(OH)NH.CH2.CONH21

L

I

CH2.CeH4OH I

CeH 5 .C().NH.CH.CO.NH.CH*.CO.NHo

I

15

J

|

CH 2 .C 6 H 4 OH Benzoyltyrosylglycylamide

+

NH2.CH2.CONH2

* = N . Johnston, R. B., Mycek, M. J., and Fruton, J. S., J. Biol

30

Glveineamide Chem. 182, 205 (1950).

ENZYMATIC SYNTHESES OF PEPTIDE BONDS

189

This conversion was designated as a transpeptidation reaction, since the exchange occurred at the peptide bond. It might be argued that the degree of transpeptidation observed could be explained as a result of simple mass action recombination of benzoyltyrosine with glycineamide (see Table I I ) ; however, this is unlikely. In any event, Fruton's mechanism is supported by other instances of re­ placement (cited below) in which the mass action explanation of recom­ bination of hydrolytic products appears to be excluded. The principle underlying the mechanism proposed by Fruton has already been demon­ strated in disaccharide exchange reactions. To bring transpeptidation into the same category as disaccharide exchange, it may be considered as, first, enolization of the peptide (or amide) bond, followed by cleavage on the enzyme surface with the unsatisfied valence bond of the carbonyl bond attached to the enzyme. This is an alternative picture to figuring the existence of an activated carbonyl group as a free radical; the carbonyl-enzyme bond would contain all or nearly all of the free energy of the original peptide (or amide) bond. This bond can then be broken by water resulting in hydrolysis, by free ammonia or hydroxylamine as in transamidation, or by the amino group of a substituted amino acid as in transpeptidation. Further experiments with chymotrypsin indicated that there is not only a substrate specificity for hydrolysis but also for transfer (replacement) reactions. Under the conditions in which the above transpeptidation occurred, benzoyl-L-tyrosineamide incubated with N 1 5 H 4 N0 3 underwent a negligible replacement of the amide radical by N 15 H 2 from the medium, although 28% hydrolysis of the amide had occurred. Amino acid exchange during enzymatic hydrolysis appears to be a general reaction. When glycylglycine is hydrolyzed by a dipeptidase in the presence of C14-labeled glycine, at equilibrium the theoretical (as indicated by the free energy data) amount of the labeled glycine is found in the dipeptide; however, when the reaction is stopped short of equilib­ rium, the amount of labeled glycine in the dipeptide is greater than the calculated equilibrium amount. Similar findings were obtained with carboxypeptidase; the replacement in the unhydrolyzed dipeptide was low.31'32 When L-lysyl-L-tyrosyl-L-lysine was subjected to the action of a mixture of trypsin and chymotrypsin, there was found among the prod­ ucts, in addition to lysine, tyrosine, lysyltyrosine, and tyrosyllysine, also lysyllysine. As there was not a lysyllysine sequence in the original 31

Zamecnik, P. C , and Frantz, I. D., Jr., Cold Spring Harbor Symposia on Quant. Biol. 14, 199 (1949). 32 Frantz, I. D., Jr., and Loftfield, R. B., Federation Proc. 9, 172 (1950).

190

H. BORSOOK

tripeptide, recombination must have occurred during hydrolysis. 35 Similarly lysyltyrosylleucine gave lysyllysyl among the hydrolysis products. As a lysyllysyl sequence in the condensation of two lysyl­ tyrosylleucine molecules is excluded, the appearance of lysyllysine after the hydrolysis of the latter tripeptide also argues against formation of lysyltyrosyllysyllysyltyrosyllysine as an intermediate in the hydroly­ sis of lysyltyrosyllysine. The findings are in accord with the mechanism proposed by Fruton of carboxyl activation during the hydrolysis of peptide-bound lysine: lysine freed in prior hydrolysis then competes with water for the activated lysine carboxyl, and the product is lysyllysine. In their latest studies Fruton and his collaborators have reported 34 extension of a peptide chain by chymotrypsin at the expense of an amide linkage. Benzoyltyrosineamide incubated with glycineamide gave benzoyltyrosylglycineamide and ammonia. The reaction was stopped when 40% of the benzoyltyrosineamide was hydrolyzed, and at this stage 14% of the benzoyltyrosineamide that had been hydrolyzed had been con­ verted to the longer peptide. The α-amino group of glycine was labeled with N 15 , the isotope appeared later in the peptide, and the free ammonia was unlabeled, thus proving that the extension of the benzoyltyrosine chain had occurred at the expense of its amide linkage. Analogous reactions were found with carbobenzoxyglycineamide and L-phenylalanineamide, lengthening of the chain at the glycine occurring at the expense of its amide linkage. In this case the reaction occurred also with D-phenylalanineamide. The enzyme, therefore, is not only not specific for the amino acid amide replacement agent, but it is not specific with respect to the isomeric form of the replacement agent. Fruton and collaborators suggest, however, that when the replacement agent is the D-form of the amino acid, further lengthening of the chain is blocked because the enzyme would be specific at least for the isomeric configura­ tion of the acceptor. This mode of lengthening a peptide chain at the expense of an amide radical is not restricted to chymotrypsin. Cysteine-activated papain catalyzed the reaction carbobenzoxy glycineamide + L-glutamyl-L-tyrosine —> carbobenzoxyglycyl-L-glutamyl-L-tyrosine + ΝΗ3Λ*Α similar re­ action occurred with carbobenzoxy-L-isoglutamine and phenylalanineamide, and here also D-phenylalanineamide could replace the L-isomer. Cathepsin synthesized glycyl-L-phenylalanyl-L-arginineamide from glycyl-L-phenylalanineamide and L-arginineamide, the elongation of the chain occurring at the expense of the former, the acceptor amide. In all the cases studied by Fruton and co-workers the optimum pH 33 34

Waley, S. G., and Watson, J., Nature 167, 361 (1951). Fruton, J. S., Johnston, R. B., and Fried, M. J., J. Biol. Chem. 190, 39 (1951).

ENZYMATIC SYNTHESES OF PEPTIDE BONDS

191

of the replacement reaction was at 7-8, whereas that of hydrolysis is at about 5. The authors suggest that at " n o r m a l " physiological pH values the catalysis of replacement may represent a major intracellular function of cathepsins and papainases. But what measurements there are in mammalian kidney, liver, and muscle of intracellular pH indicate it to be nearer to 6 than 7. The dependence on pH in these transfer reactions is related to the pK of the cation of the replacement agent. Two other types of amino acid transfer have been discovered by Hanes et al.Zb Enzyme extracts from kidney, ox, pig, or sheep, ox pan­ creas, and the lactating mammary gland of the cow catalyzed transfer of amino acids to form 7-glutamyl-peptides. The acceptor, i.e., the glutamyl conjugate, could be glutathione (reduced and oxidized forms), or 7-glutamyl-conjugates with aspartic acid, glutamic acid, phenylalanine, or tyrosine. The replacement amino acids tested and whose α-amino groups were found to condense with the 7-carboxyl of the glutamic acid were cysteine, glycine, glutamic acid, glutamine, histidine, leucine, phenyl­ alanine, tryptophan, 7-glutamylphenylalanine, 7-glutamylleucine, and 7-glutamyltyrosine. The authors have designated this enzyme as 7-glutamyltranspeptidase. There is a slow and ultimately complete breakdown of the newly formed 7-glutamylpeptide. Whether or not the hydrolyzing enzyme is the same as the transferring enzyme remains to be determined. This transfer enzyme is distinct from the glutamo-transferases (see be­ low). There is no transfer to glutamine, nor of ammonia to 7-glutamylpeptides. On the other hand glutathione and glutamine readily form 7-glut amy lglutamine. The same authors have found an enzyme in cabbage leaves that per­ forms analogous transfer reactions to form new glycine peptides. Thus glycylglycine, triglycine, glycyl-L-leucine, glycyl-L-phenylalanine, and glycyl-L-tryptophan could serve as acceptor. The following amino acids served as replacement agents: glycine, leucine, methionine, phenylala­ nine, and tryptophan. In a typical reaction glycylphenylalanine was formed from glycylglycine and phenylalanine. Triglycine and phenyl­ alanine also gave glycylphenylalanine. Preparations of this enzyme also hydrolyze glycylpeptides. The hy­ drolysis is markedly inhibited by various replacing amino acids, but eventually hydrolysis is complete. No peptide formation occurred with glycineamide, leucylglycine, or leucyltryptophan. This enzyme was, accordingly, designated as glycyltranspeptidase. 35

Hanes, C. S., Hird, F. J. R., and Isherwood, F. A., Nature 166, 288 (1950); Biochem. J. (London) 51, 25 (1952).

192

H.

BOKSOOK

Hanes and associates, like Fruton and co-workers, envisage that proteolytic enzymes may catalyze both hydrolysis and formation of new peptides from acceptor peptides and free amino acids. They cite the inhibitory effect of the 7-glutamyl linkage of glutathione on the widely distributed enzyme that hydrolyzes cysteinylglycine, and suggest that a function of glutathione is to serve as a reservoir of α-amino acyl peptide links, which by linkage to glutamic acid at its γ-carboxyl are resistant to hydrolytic cleavage and thus constitute a reserve available for transpeptidation rearrangements. Hanes and co-workers speculate further that according to this conception the significance of transpeptidation reactions lies in their providing metabolic pathways whereby certain primary peptide linkages synthesized in exergonic processes {e.g. in reactions involving the breakdown of ATP) could give rise secondarily to other peptides; by continuation of this process of group transfer, progressively longer chains could result, and these conceivably could contain amino acid residues in a particular order reflecting the specificity of transpeptidase enzymes concerned.

Hendler and Greenberg 36 tested the two basic mechanisms put forward by Hanes and associates: i.e., 7-glutamyl activation is an important early step intermediate in peptide synthesis; and the stabilization of 7-glutamyl peptide bonds by free amino acids thus facilitates amino acid incorpora­ tion into proteins. They compared the incorporation of labeled glycine in 7-glutamylglycine with that of free labeled glycine in the presence of glutamic acid, glutamine, or glutathione. They also tested the effect of the last three compounds on the incorporation of labeled phenylalanine and valine. Homogenates, whole cells, intracellular particles, and a lymphosarcoma were the tissues used. The authors reported (in a pre­ liminary note) that "in no case was there any appreciable activation of amino acid incorporation by 7-glutamyl compounds. The uptake of glycine from the peptide was always less efficient than from free glycine." Furthermore the 7-glutamyl peptide was found to be hydrolyzed very quickly in liver enzyme systems. It may be that the hydrolyzing enzyme is less active in the intact cell, but the findings, as they stand, argue against the stability of such peptides in vivo. There are some general objections to assigning to the transpeptidation so far found by Fruton and collaborators and Hanes and associates an important role in the lengthening of a peptide chain in a specific pattern. These transpeptidase enzymes are specific for the acceptor but not for the replacement amino acid or peptide; the proteases, cathepsin and chymotrypsin, are acceptor-specific and the acceptor is part of the lengthened chain; the 7-glutamyl and glycyl transpeptidases are also only acceptor36

Hendler, R. W., and Greenberg, D. M., Nature 170, 123 (1952).

ENZYMATIC S Y N T H E S E S

OF P E P T I D E

BONDS

193

specific, but the acceptor would not form part of a lengthened peptide chain, if its mode of action is that proposed by Hanes and co-workers, i.e., if it acts as a general reserve of α-amino acyl peptide links. Two types of enzymes operating together—7-glutamyl or glycyl transpeptidases and proteases—might provide the necessary specificity. It is pertinent to recall in this connection that the amino acid turn­ over of glutathione in liver is very active, 37 and yet the enzyme system used by Hanes and co-workers was present in kidney but absent from liver. In their studies on the enzymatic synthesis of glutathione, Johnston and Bloch38 found that the synthesizing enzyme was present in the liver of a number of animals, but was absent from rabbit kidney and rabbit intestine. Furthermore Johnston and Bloch found that, in their prepara­ tions, the enzyme activity which was responsible for the hydrolysis of glutathione was not involved in, and could be separated from, the enzyme system involved in the synthesis. So far transpeptidation reactions have been carried out with approxi­ mately 0.02 M amino acid and peptide, which is far greater than the con­ centration of free amino acids or peptides in the cell. The question arises to what extent the degree of transpeptidation depends on the concentra­ tions of reactants. In all cases so far studied, even with the above con­ centrations of reactants, the degree of hydrolysis exceeded that of trans­ peptidation, and the reaction was always stopped at an arbitrarily chosen stage of incomplete hydrolysis of the parent peptide; otherwise the newly formed peptide would have been completely hydrolyzed also. If the ratio of transpeptidation to hydrolysis decreases significantly with decreasing concentration of reactants, the importance of transpeptidation reactions in the economy of the cell would, then, appear to be as significantly lessened. There is a need, clearly, for kinetic data. Free amino acids are rapidly incorporated into proteins (see below). Is such incorporation a transpeptidation analogous to those discussed? That peptidases are not involved is indicated by the finding that metal ions, such as cobalt, copper, iron, manganese, and nickel, on which the activity of a number of peptidases has been shown to depend, 39 were found to be either inhibitory or without any effect.40 As far as peptidases and proteases promoting protein synthesis is concerned, it must be noted that in the above experiments the amount of new peptide formed was always less than the initial substrate hydrolyzed; and where a free 37

Waelsch, H., and Rittenberg, D., / . Biol. Chem. 144, 53 (1942). Johnston, R. B., and Bloch, K., / . Biol. Chem. 188, 221 (1951). 39 Smith, E. L., Federation Proc. 8, 581 (1949). 40 Borsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G., and Lowy, P. H., Federation Proc. 8, 589 (1949). 38

194

H.

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amino acid was incorporated into a new peptide, it was always terminal. On the other hand when glycine, histidine, leucine, or lysine were incor­ porated (in vitro) into the proteins (mainly hemoglobin) of rabbit reticulocytes, 41 none of the incorporated amino acid was found in an end position (at least the NH 2 group was not free); and further all four amino acids could be incorporated simultaneously without competing with each other. The fact that every amino acid which has been tested has been found to be incorporated into tissue proteins argues strongly against most of the incorporated amino acids being terminal. Similarly when aspartic or glutamic acids were incorporated into serum albumin from liver42 or alanine, and aspartic acid into ovalbumin,43-44 the incorporated (labeled) amino acids were found to be widely distributed in different loci in the protein. 5. PEPTIDE SYNTHESIS FROM AMINO ACID ESTERS

A new type of enzymatic synthesis of peptides has been found by Brenner et al.^~A7 Esters of DL-methionine or DL-threonine incubated with chymotrypsin give after one or two days incubation, in addition to the free amino acid, a large amount of a mixture of peptides of varying molecular weight. The reaction stops when the reaction product reaches so high a molecular weight that it becomes insoluble. No peptide forma­ tion occurred with only the free amino acid in the reaction mixture. Beginning with DL-methionine-isopropyl ester, L-methionyl-L-methionine was isolated from the reaction mixture. Higher peptides were left uniden­ tified.46 Similarly from DL-threonine-isopropyl ester, L-threonyl-L-threonine47 was obtained after partial hydrolysis of a mixture of more complex peptides. Evidently chymotrypsin acts only on the L-form of the ester and amino acids. In no case was peptide synthesis observed without simultaneous appearance of free amino acids. Beginning with methionine ester one part (by weight) of peptide was formed for every two parts (by weight) of methionine liberated as free amino acid. Threonine esters gave relatively more, phenylalanine, tyrosine, and tryptophan esters less peptide. Most of the work so far reported was done with methionine esterified with a wide variety of aliphatic and aromatic alcohols. Brenner and collaborators envisage two possible mechanisms of the 41

Borsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G., and Lowy, P. H. / . Biol. Chem. 196, 669 (1952). 42 Peters, T., Jr., and Anfinsen, C. B., / . Biol. Chem. 182, 171 (1950). 43 Anfinsen, C. B., and Steinberg, D., / . Biol. Chem. 189, 739 (1951). 44 Anfinsen, C. B., Science 114, 683 (1951). 45 Brenner, M., Müller, H. R., and Pfister, R. W., Helv. Chim. Ada 33, 568 (1950). 46 Brenner, M., and Pfister, R. W., Helv. Chim. Ada 34, 2085 (1951). 47 Brenner, M., Sailer, E., and Rüfenacht, K , Helv. Chim. Ada 34, 2096 (1951).

ENZYMATIC SYNTHESES OF PEPTIDE BONDS

195

reaction type they discovered. It may be a primary condensation of amino acid esters followed by degradation of the peptide ester thus formed to give the free peptide. Or the reaction is of the type alanine ester + glycine —> alanylglycine + alcohol. In both mechanisms the free energy of the original ester bond must be used to form the peptide. Neither mechanism explains the appearance of free amino acid contemporaneously with, and inseparably from, formation of the peptide. The simplest explanation appears to be an extension of that proposed for transpeptidation by chymotrypsin (chymotrypsin might be called an esterase). 48-52 It is immaterial to the present purpose, whether one considers that the first step, with Fruton, is a carbonyl activation at the ester linkage, or that a compound is formed between the enzyme and the carbonyl group of the amino acid detached from its bond with the alcohol and that most of the free energy of the original ester bond is retained in the carbony1-enzyme bond. The next step is condensation of an amino group with this carbonyl bond. The amino group may be that of an amino acid ester or of a peptide ester. It seems unlikely that the amino group of a free amino acid could compete so effectively with water for the carbonyl as to give the high yields of peptide obtained. More likely it is the amino acid (or peptide) ester which competes effectively against water and thus forms a new peptide bond. When the combination of the carbonyl is with water, there is hydrolysis, and this gives the free amino acid always found. All the features of the reaction discovered by Brenner are analogous to, and in accord with, the transpeptidation reaction mechanism proposed by Fruton. There is no information, at present, on the possible biological significance of this type of peptide synthesis via amino acid esters. 6. GLUTAMO- AND ASPARTOTRANSFERASES

The transamidation reactions involving either ammonia or hydroxylamine discussed above may be characterized as follows. The amino acid amide involved can be glycine and other amino acids, and the acceptor is not restricted to glutamic or aspartic acid; the transfer depends on, and its degree is less than, that of the concomitant hydrolysis; the reac­ tion does not depend on adenosinetriphosphate (ATP) or adenosinediphosphate (ADP), and it is not coupled with an energy or phosphate transfer system. Another type of transamidase reaction was discovered 48

Kaufman, S., and Neurath, H., Arch. Biochem. 21, 245 (1949). Kaufman, S., and Neurath, H., Arch. Biochem. 21, 437 (1949). 50 Kaufman, S., Neurath, H., and Schwert, G. W., J. Biol. Chem. 177, 793 (1949). 61 Schwert, G. W., Neurath, H., Kaufman, S., and Snoke, J. E., / . Biol. Chem. 172, 221 (1948). 52 Snoke, J. E.? and Neurath, H., Arch. Biochem. 21, 351 (1949). 49

196

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BOKSOOK

by Waelsch and co-workers, and shortly thereafter the field was broad­ ened by Stumpf and associates. The characteristics which distinguish it from the transamidation reactions studied by Fruton and co-workers appear to be as follows. The amide (or hydroxylamine) transfer is re­ stricted to the jö-carboxyl of aspartic acid or the 7-carboxyl of glutamic acid; the transfer is independent of hydrolysis of the amide grouping; it does depend on ATP or ADP, arsenate or phosphate, and Mn + + . It shares with the other type of transamidation its independence of energy of phosphate transfer, notwithstanding its dependence on ATP or ADP, arsenate or phosphate, and Mn + + . Recently Elliott has demonstrated that the enzyme effecting the synthesis of glutamine from glutamic acid and ammonia is the same as that in amide transfer to and from glutamine, and the difference between the two reactions is in the amount of ATP required (see below). Waelsch et α£.53-58 first found the enzyme in the washed cells and cell extracts of a number of microorganisms. The reactions catalyzed may be represented schematically as follows: (a)

G l u t a m o — N H 2 + N H 4 O H - + glutamo—NH 2 + N H 4 O H

and (b)

G l u t a m o — N H 2 + N H 2 O H -> g l u t a m o — N H O H + N H 3

Ammonia and a number of (but not all) amino acids in high concentra­ tions are inhibitory. The transfer of ammonia was proved by using N 1 5 H 3 in the medium and finding the labeled nitrogen, after incubation with the enzyme, in the amide radical. 54 The enzyme extract used effected the transfer between asparagine or glutamine and ammonia or hydroxamic acid, but with no other amides. The reaction was not inhibited by cyanide, fluoride, or iodoacetate. Acetone powders prepared from extracts of mammalian tissues also were effective; they required activation by Mn + + , and their activity was enhanced further by phosphate; they had no glutaminase activity. 56 Addition of ATP or ADP was without effect;58 but this was probably because the enzyme system was not sufficiently purified to show this dependence (see below). 53 54

55

56 57 58

Waelsch, H., Advances in Protein Chem. 6, 299 (1951). Waelsch, H., Owades, P., Borek, E., Grossowicz, N., and Schou, M., Arch. Biochem. 27, 237 (1950). Waelsch, H., Borek, E., Grossowicz, N., and Schou, M., Federation Proc. 9, 242 (1950). Waelsch, H., Federation Proc. 10, 266 (1951). Schou, M., Grossowicz, N., Lajtha, A., and Waelsch, H. ? Nature 167, 818 (1951). Grossowicz, N., Wainfan, E., Borek, E., and Waelsch, H., J. Biol. Chem. 187, 111 (1950).

ENZYMATIC SYNTHESES OF PEPTIDE BONDS

197

Stumpf et αΖ.69~61 found a similar enzyme in pumpkin seedlings. After the enzyme was purified, the following activating agents were found to be necessary: Mn++, ATP or ADP, and arsenate or phosphate. Insufficiently purified enzyme preparations did not show the dependence on ATP or ADP, resembling in this respect the enzyme preparations used by Waelsch. The latter's enzyme preparation resembles that of Stumpf so closely in all other respects that it seems highly probable that it too, when sufficiently purified, will show dependence on ATP or ADP. In the purified enzyme from pumpkin seedlings arsenate was two to three times as effective as phosphate; and addition of phosphate to a reaction medium containing arsenate reduced the activity, indicating a competition be­ tween arsenate and phosphate for the enzyme locus. Experiments de­ signed to detect phosphate transfer between the medium and ATP or ADP were negative, nor was any evidence found of a glutamylphosphate intermediate. Only glutamine was reactive with the pumpkin seedling enzyme; asparagine, a number of other amides including nicotinamide and coenzyme I, and also glutathione were inactive. As with the enzyme studied by Waelsch and co-workers, iodoacetate and cyanide were not inhibitory, nor were azide, diisopropylfluorophosphate, dinitrophenol, or malonate. Ammonia in concentrations of 10~2 M, and 5 X 10~2 M, and certain amino acids, inhibited 50% and 100%, respectively, resembling also in this respect the transferases of Waelsch and collaborators. Waelsch 53 in a recent review summarized his views on the possible biological significance of these transf erases as follows: I t is probable t h a t neither hydroxylamine nor ammonia is the biological substrate of the transfer reaction. Possible substrates appear to be those amino acids whose natural isomers have been found to inhibit the formation of glutamohydroxamic acid by glutamotransferase. Participation of amino acids in the exchange reaction would lead to the formation of 7-glutamyl or ß-aspartyl peptides. The wide distribution of the glutamo- and asparto-transferase potencies gives support to the hypothesis t h a t the naturally occurring amides, glutamine and asparagine, are implicated in the mechanism of peptide formation.

The finding that glycine in 7-glutamyl linkage is incorporated more slowly into proteins than free glycine36 tends to exclude the possibility put forward in the above quotation. And the general considerations which argue against the participation of the glycyl and 7-glutamyl transpeptidases in peptide and protein synthesis (see above) also argue against the function suggested for the asparto- and glutamo-amide transf erases. It is clear now that the former conception of the mechanism action of 59 60 61

Stumpf, P. K., and Loomis, W. D., Arch. Biochem. 25, 451 (1950). Stumpf, P. K., Loomis, W. D., and Michelson, C , Arch. Biochem. 30, 126 (1951). Delwiche, C. C., Loomis, W. D., and Stumpf, P. K., Arch. Biochem. 33, 333 (1951).

198

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of hydrolytic enzymes was too narrow. In those cases where hydrolysis goes practically to completion the reverse reaction, which is implicit in the Law of Mass Action, had been neglected; it was thought that the reverse reaction must be too slow to be detectable. The mistake was to infer a mechanism of reaction rates from thermodynamic equilibrium data which have nothing to say about rates. It is evident now that it will be more useful to consider all hydrolytic enzymes as transferases, with water as one, but not the only, replacement agent. Then, not only transfers but reactions such as peptide synthesis from amino acid esters can be included within one action mechanism. The glutamo- and asparto-amide transferases are in another category. They transfer the amide (or hydroxylamine) radical without hydrolysis, i.e., the enzyme's specificity does not include water as a replacement agent. And with sufficient ATP at least one glutamotransferase can effect the synthesis of glutamine from glutamic acid and ammonia. III. P E P T I D E SYNTHESES W H E R E -AF IS NEGATIVE AND LARGE: COUPLED W I T H HIGH-ENERGY PHOSPHATE 1. SYNTHESIS OF GLUTAMINE

We now come to the third category of enzymatic synthesis of gluta­ mine in which glutamine is synthesized from glutamic acid and ammonia. (The free energy of formation of the amide bond is of the same order of magnitude as that of simple peptides from amino acids.) This category is distinguished from the previous two as follows: it is not a transfer reac­ tion, but a synthesis; nor is it accompanied by any hydrolysis; the 7-carboxyl of glutamic acid is the only acceptor of the free base (ammonia, hydroxylamine, hydrazine, or methylamine); it depends on ATP and it is a coupled reaction in which inorganic phosphate is liberated from ATP in an amount equivalent to the amide synthesized; it is inhibited by fluoride in low concentration. Like the asparto- and glutamotransferases it requires either M g + + (optimum concentration 0.01 M) or M n + + (opti­ mum concentration 0.002 M); but the maximum activation with M n + + is only half that with Mg + + . This synthesis of glutamine was elucidated, and the enzyme some­ what purified, simultaneously and independently by Speck 62-64 and by Elliott. 65,66 The enzyme was found in the brain, liver, and kidney of a number of animals and in Staphylococcus aureus. Active cell-free extracts 62

Speck, J F., / . Biol. Chem. 168, 403 (1947). Speck, J. F., J Biol. Chem. 179, 1387 (1949). 64 Speck, J. F., / . Biol. Chem. 179, 1405 (1949). 65 Elliott, W. H., Nature 161, 128 (1948). 66 Elliott, W. H., Biochem. J. (London) 42, v (1948). 63

ENZYMATIC SYNTHESES OF PEPTIDE BONDS

199

and acetone powders of these were prepared. AMP and ADP could not replace ATP. Intermediates, such as glutamylphosphate were looked for, but none were detected. For maximum activity a reducing agent, e.g., cysteine, is required, presumably the activity of the enzyme is dependent on sulfhydryl groups. In addition to fluoride two other inhibitors were found, methioninesulfoxide and crystal violet. The methioninesulfoxide competes with glutamate for the locus of activity on the enzyme. The enzyme has been highly purified by Elliott, 67 who then found that the glutamotransferase and glutamine-synthesizing activities were not separable. The evidence indicates strongly that the two reactions are catalyzed by the same enzyme. This has raised an interesting problem of enzyme mechanism. In the transfer reaction only a catalytic amount of ATP is required, and the ATP is not cleaved or otherwise used up in the course of the reaction. In the synthetic reaction ATP is cleaved stoichiometrically with the synthesis. In short the activation of the enzyme by ATP is retained during the exchange reaction; it is lost in the synthetic reaction; and ATP is required to restore the enzyme. 2. SYNTHESIS OF HIPPURIC ACID

It has long been known that animals form hippuric acid from benzoic acid and glycine. Hippuric acid is only one of a number of such quasipeptide syntheses which are known to occur and which have been studied O / in vitro. The free energy of formation of the —C—NH— bond of hippuric acid2 is of the same order of magnitude as that of simple peptides (Table II). The synthesis of hippuric acid from benzoic acid and glycine was first attained in vitro with kidney and liver slices (dog, guinea pig, rabbit, and rat). 2 In the course of some hours 60% to 7 5 % of added benzoic acid and glycine were condensed to hippuric acid, although the thermodynamic equilibrium point was at less than 1% synthesis. Ob­ viously, so high a yield of hippuric acid could not have occurred by simple mass action; there must have been coupling with an energy-yielding reaction. Accordingly, as was to be expected, inhibition of respiration by 0.001 M KCN also completely inhibited the synthesis, as did also anaerobiosis. It was found possible later to carry out the reaction in a guinea pig liver homogenate; and indirect evidence was obtained that ATP was involved, i.e., that the free energy driving the reaction came from the high-energy phosphate bond of ATP, which in turn was formed by oxidative phosphorylation. 68 In the course of these studies ΛΓ-phosphorylated 67 68

Elliott, W. H., Seminar, California Institute of Technology. Borsook, H., and Dubnoff, J. W., / . Biol. Chem. 168, 397 (1947).

200

IT.

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glycine and benzoylphosphate were tested as possible intermediates, but were found to be no more active, or less active, than glycine and benzoic acid.69 The key to the mechanism of the reaction came from Chantrenne's discovery that Coenzyme A as well as ATP is needed for hippuric acid synthesis. 70 He also found that benzoylphosphate was unable to replace benzoic acid plus ATP, thus excluding benzoylphosphate as an inter­ mediate. On the basis of known reactions and interactions of ATP, Co A, and acyl groups 71 it appears that the reaction mechanism is (a) (b) (c)

A T P + Co A —> Co A—pyrophosphate or Co A-phosphate; Co A-pyrophosphate + benzoic acid —> benzoyl-Co A; or Co A-phosphate + benzoyl-Co A + glycine —> hippuric acid + Co A.

In this scheme the immediate high-energy intermediate is benzoyl-Co A and the action of ATP from which the energy is derived is indirect, in fact two steps removed. 3. SYNTHESIS OF ^-AMINOHIPPURIC ACID

A more searching study was begun independently at about the time ATP was being implicated in the synthesis of hippuric acid by Cohen and McGilvery 72-74 of an analogous reaction, the synthesis of p-aminohippuric acid (PAH) from p-aminobenzoic acid (PAB) and glycine. PAB is a constituent of naturally occurring peptides. 7576 Cohen and McGilvery first used slices of rat liver and kidney; their findings were, in general, similar to those in the synthesis of hippuric acid. Contributing toward insight into the coupled energy-yielding reaction they found the following inhibitions, expressed as per cent: arsenite (0.01 M), 95; azide (0.001 M), 44; fluoride (0.01 M), 90; HCN (0.001 M), 99; iodoacetate (0.01 M), 97; and malonate (0.001 M), 35. 69 70 71 72 73 74 75

76

Borsook, H., and Dubnoff, J. W., Unpublished observations. Chantrenne, H., J. Biol. Chem. 189, 227 (1951). Barker, H. A., Phosphorus Metabolism 1, 240 (1951). Cohen, P. P., and McGilvery, R. W., J. Biol. Chem. 166, 261 (1946). Cohen, P. P., and McGilvery, R. W., J. Biol. Chem. 169, 119 (1947). Cohen, P. P., and McGilvery, R. W., J. Biol. Chem. 171, 121 (1947). Angier, R. B., Boothe, J. H., Hutchings, B. L., Mowat, J. H., Semb., J., Stokstad, E. L. R., Subbarow, Y., Waller, C. W., Consulich, D . B., Fahrenbach, M. J., Hultquist, M. E., Kuh, E., Northey, E. H., Seeger, D. R., Sickels, J. P., and Smith, J. M., Jr., Science 103, 667 (1946). Ratner, S., Blanchard, M., Coburn, A. F., and Green, D . E., J. Biol. Chem. 156, 689 (1944).

ENZYMATIC SYNTHESES OF PEPTIDE BONDS

201

The authors then succeeded in carrying out the reaction in rat liver homogenates, 73 and in this enzyme system were able to add the following information regarding the mechanism: the optimum pH is at 7.5, replace­ ment of N a + by K + was stimulating, Ca + + was inhibitory, and M g + + was stimulating. Addition of cytochrome c was necessary for maximum synthesis. Addition of ATP (3 X 10~3 M) permitted some synthesis anaerobically, but much less than under oxygen without added ATP. A number of metabolites whose oxidation is known to lead to ATP forma­ tion increased the yield of PAH somewhat, or permitted the reaction to proceed for a longer time. Hexosediphosphate, coenzyme I, and cocarboxylase were without effect; nicotinamide was inhibitory. iV-Acetylglycine was inactive. Glyoxalate and ammonia could not substitute for glycine. One of the general mechanisms suggested 77 for protein synthesis is the formation of ΛΓ-acylated, specifically Λ^-acetylated, amino acids as intermediates; there is sufficient chemical potential as a consequence of the acylation to condense the nitrogen with the carboxyl of another amino acid. The inactivity of iV-acetylated glycine definitely excludes this mechanism in the synthesis of p-aminohippuric acid; it tends, also, to exclude condensation of a keto-acid derivative of an amino acid with the nitrogen of another amino acid78 as an intermediate in peptide synthesis. In a still later paper 74 Cohen and McGilvery purified the enzyme further and then found that ATP in the absence of oxidizable metabolite promoted p-aminohippuric acid synthesis anaerobically, that adenosinemonophosphate was active even aerobically only under conditions in which it was phosphorylated to ATP, that iV-phosphoglycine was inac­ tive, as were coenzymes I and II, cocarboxylase, and pyridoxalphosphate. 4. SYNTHESIS OF ORNITHURIC ACIDS

McGilvery and Cohen 79 later extended their studies to the formation of ornithuric acids from a- and δ-benzoyl-L-ornithine and p-aminobenzoic acid. They succeeded in obtaining enzymatic activity in an acetone powder of the sedimentable macroparticles of chicken liver homogenate, and found the conditions for the synthesis of the ornithuric acids, and the indications of the mechanisms involved, the same as in the synthesis of p-aminohippuric acid. Kielley and Schneider80 localized the enzyme for p-aminohippuric acid synthesis in mouse liver homogenates as almost entirely in the 77 78 79 80

Rittenberg, D., and Shemin, D., Ann. Rev. Biochem, 15, 247 (1946). Herbst, R. M., and Shemin, D., J. Biol. Chem. 147, 541 (1943). McGilvery, R. W., and Cohen, P. P., J. Biol. Chem. 183, 179 (1950). Kielly, R. K., and Schneider, W. C , / . Biol. Chem. 185, 869 (1950).

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mitochondria. And they adduced further evidence implicating oxidative phosphorylation leading to ATP in the synthesis of p-aminohippuric acid. The data show a relation between increase in PAH synthesis on the one hand, and increase in high-energy phosphate (ATP) on the other; but this relation, from the data reported, is hardly the "direct'' parallel the authors saw in their data. Sarkar et al.81 confirmed the localization of the hippuric acid-synthesiz­ ing enzyme in the mitochondria of rat liver homogenate, found that the synthesis was abolished by dinitrophenol (see below for the significance of this finding), that benzoic acid can be replaced by some other aromatic and heterocyclic carboxylic acids (phenylacetic and cholic acids were inactive), and that glycine could not be replaced by either ß-alanine or taurine. It has not yet been reported whether Co A, which is necessary for the synthesis of hippuric acid, is also necessary for the synthesis of such analogs as p-aminohippuric and ornithuric acids. Probably Co A is required because their synthesis resembles that of hippuric acid in all other respects. As mentioned above iV-acetylated (in general, iV-acylated) amino acids have been suggested as possible, even probable, intermediates in peptide and protein synthesis. The positive evidence cited in support is that animals do acetylate unnatural amino acids and amines. 77 This possibility, as a general mechanism, was excluded in the synthesis of p-aminohippuric acid. It was also excluded in protein synthesis in E. coli. Simmonds et al.s2 employed two mutants, one requiring L-phenylalanine, the other L-tyrosine, for growth. They found that acetyl-Lphenylalanine and acetyl-L-tyrosine, even in high concentrations, were unable to support growth of the respective mutants. There were similar negative or unfavorable results with dehydropeptides of phenylalanine, whereas the corresponding (normal) peptides were active: e.g., glycyldehydrophenylalanine did not support growth, glycylphenylalanine did. Thus dehydropeptides were excluded in this organism as intermediates in protein synthesis. 5. SYNTHESIS OF GLUTATHIONE

ATP is involved in the synthesis of hippuric acid and its analogs. The role of ATP is to form the aromatic acyl-Co A which then condenses with glycine to form the quasi-peptide. In the synthesis of glutamine from glutamic acid and ammonia Co A is not required, but ATP is. And in the synthesis of glutathione, the one case so far elucidated of an enzymatic 81 82

Sarkar, N. K , Fuld, M., and Green, D. E., Federation Proc. 10, 242 (1951). Simmonds, S., Tatum, E. L., and Fruton, J. S., J. Biol. Chem. 169, 91 (1947).

ENZYMATIC SYNTHESES OF PEPTIDE BONDS

203

peptide synthesis from amino acids, again Co A appears not to be re­ quired, whereas ATP is. The enzymatic synthesis of glutathione was first achieved by Bloch83~~8& in cell-free pigeon liver homogenates and in acetone powder prep­ arations of the homogenate. Bloch and his collaborators purified the en­ zymes involved and present the following mechanism of the synthesis. 83-90 (a) Glutamic acid + cysteine + ATP -> 7-glutamylcysteine + ADP + H3PO4 (b) 7-Glutamylcysteine + glycine + ATP —» glutathione + ADP + H3PO4

One pyrophosphate bond of ATP is used per peptide bond synthesized. The reaction is specific for ATP; ADP is inhibitory. Both reactions are accelerated by potassium, and both need Mg + +. The enzyme for reaction (b) is unable to condense ammonia or amino acids other than glycine with glutamylcysteine. Prolonged dialysis of this enzyme over a pH range 3.8-9.8 caused no loss in activity; therefore, the enzyme contains (probably) no dissociable cofactors. Evidence from experiments with hydroxylamine suggests that it is the cysteine carboxyl in glutamylcysteine which is activated by the enzyme with ATP in the course of its condensation with glycine. The enzyme for reaction (a) was shown not to be the same as that for the synthesis of glutamine from glutamic acid and ammonia. Neither did the purified enzymes for reactions (a) and (b) contain the enzyme that hydrolyzes glutathione. Therefore both transpeptidation and a transfer reaction analogous to 7-glutamyl transamidation are excluded from the mechanism of the synthesis of glutathione from its amino acids. No information has yet been reported on the precise role of ATP in the glutathione, as in the glutamine synthesis from its proximate constituents. IV. MECHANISM OF AMINO ACID INCORPORATION PROTEINS

INTO

1. E F F E C T OF INHIBITORS

We shall consider in this chapter only those findings on the incor­ poration of amino acids into proteins that bear on questions of mecha83

Bloch, K , and Anker, H. S., / . Biol. Chem. 169, 765 (1947). Bloch, K , «7. Biol. Chem. 179, 1245 (1949). 85 Johnston, R. B., and Bloch, K , J. Biol. Chem. 179, 493 (1949). 86 Johnston, R. B., and Bloch, K , J. Biol. Chem. 188, 221 (1951). 87 Snoke, J. E., and Rothman, F., Federation Proc. 10, 249 (1951). 88 Yanari, S., Snoke, J., and Bloch, K., Federation Proc. 11, 315 (1952). 89 Snoke, J. E., and Bloch, K., J. Biol. Chem. 199, 407 (1952). 90 Bloch, K., Snoke, J., and Yanari, S., Il e Congres International de Biochimie, Symposium sur la Biogenese des Proteines, Paris, p. 32 (1952). 84

204

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nism.90a It needs to be stated at the outset that nothing positive and specific is known. On the other hand, there is now a considerable body of periph­ eral information which defines the problem or problems. A summary of the major findings on the incorporation of labeled amino acids into proteins is: (1) every tissue that has been tested has been found to incorporate into its proteins, in vitro as well as in vivo, every common labeled amino acid (L-form) presented to it; (2) so far one amino acid which is not a normal protein constituent—ethionine—has been found to be incorporated, 91 two others—α-aminoadipic acid92 and α-aminobutyric acid93—were not incorporated; (3) the rate of incorpora­ tion varies with the amino acid and the tissue from approximately 0.1 to 10 micromoles per gram of protein per hour; (4) the incorporation of a single amino acid appears to be largely independent of the presence of others except for specific accelerating effects of a few amino acids; (5) labeled amino acids are incorporated by peptide linkage; (6) in most but not all cases inhibitors of respiration and phosphorylation inhibit amino acid incorporation and protein synthesis; (7) there is evidence that in some cases cofactors (unidentified) are involved. Two questions arise at the outset of a discussion of mechanism. One is: " I s the mechanism of incorporation in any one tissue the same for all amino acids?"; and the other: " I s the mechanism of incorporation the same in every tissue?" On the first question the evidence indicates that the mechanism of incorporation of most, if not all amino acids, in any one tissue is the same. Thus in rabbit bone marrow cells96 the incorporation of glycine, leucine, or lysine was inhibited completely by anaerobiosis, 0.001 M arsenite, and 0.001 M 2,4-dinitrophenol. Arsenate (0.001 M) inhibited the incorpora­ tion of glycine, leucine, and lysine, 96%, 77% and 80%, respectively, and azide (0.001 M) 84 %, 77 %, and 68 %, respectively. In rat diaphragm 97 anaerobiosis, arsenite (0.001 M), and 2,4-dinitrophenol (0.001 M) in­ hibited completely the incorporation of the same three amino acids, arsenate (0.001 M) inhibited their incorporation 67%, 58%, and 63%, 90a Thg reader will find the field as a whole reviewed in references 14, 40, 94, 95. Levine, M., and Tarver, H., J. Biol. Chem. 192, 835 (1951). 92 Borsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G., and Lowy, P. H., J. Biol. Chem. 187, 839 (1950). 93 Zamecnik, P. C., and Frantz, I. D., Jr., Personal communication. 94 Zamecnik, P. C , Cancer Research 10, 659 (1950). 95 Borsook, H., Fortschr. Chem. org. Naturstoffe, Springer Verlag, Vienna (1952), p. 292. 96 Borsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G., and Lowy, P . H., J. Biol. Chem. 186, 297 (1950). 97 Borsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G., and Lowy, P. H., J. Biol. Chem. 186, 309 (1950). 91

ENZYMATIC SYNTHESES OF PEPTIDE BONDS

205

respectively, and azide (0.001 M) 85%, 78%, and 85%, respectively. The similarity of the degree of incomplete inhibition of the three amino acids in each tissue is a stronger indication that the incorporation mecha­ nism of the three amino acids is the same than the similarity of the com­ plete inhibition. A more extensive test was carried out with rabbit reticulocytes. 41 There were four amino acids and more inhibitors. The following is a summary of the results, expressed as per cent inhibition of the incorpora­ tion of glycine, histidine, leucine, and lysine, respectively. Anaerobiosis: it was never complete and very variable, the average inhibitions of many experiments being 55, 23, 63, and 25. Arsenate (0.001 M): 29, 18, 20,'46. Arsenite (0.001 M): 90, 95, 96, 97. Azide (0.001 M): 30, 19, 13, 30. 2,4-Dinitrophenol (0.001 M): 93, 83, 81, 86. Diethyldithiocarbamate (0.001 M): 19, 19, 37, 53. α,α'-Dipyridyl (0.001 M): 64, 46, 62, 69. Fluoride (0.02 M): 99, 97, 99, 100. Fluoride (0.001 M): 0, 0, 1, 0. Hydroxylamine (0.02 M): 89, 89, 93, 95. Hydroxylamine (0.001 M): 0, 0, 0, 0. Iodoacetate (0.001 M ) : 88, 54, 51, 79. Ammonium molybdate (0.001 M): 42, 55, 61, 33. The degree of inhibition of each of the four amino acids with each inhibitor is sufficiently similar to conclude that the mechanism of the incorporation of each of the four amino acids is very similar, and also similar in the three tissues, rabbit bone marrow cells, rat diaphragm, and rabbit reticulocytes. Evidence of a different kind was found in the rabbit reticulocyte system 41 that the mechanism for the incorporation of at least four amino acids is the same. Glycine, histidine, leucine, and lysine are incorporated into the proteins several times faster in plasma than in Ringer's solution. A similar accelerating system can be extracted from dried liver powder. The accelerating system is a complex of at least three groups of factors. One consists of certain specific amino acids—histidine, leucine, phenylalanine, and valine. Another is generated by carbohydrate metabolism— it may be ATP. The third has been found in plasma, liver, spleen, and yeast; it is not a protein, and all the common amino acids, familiar cofactors, vitamins, metabolites, metals, and inorganic salts have been excluded. It is present in the plasma of every mammal so far investi­ gated, in their normal erythrocytes and in rabbit reticulocytes. The wide distribution of this factor suggests that its function may be more extensive than only that of accelerating amino acid incorporation into reticulocyte proteins. The incorporation of glycine, histidine, leucine, and lysine is accelerated to the same degree by each of the stimulating systems, whether these operate separately or together. This is strong evidence that in rabbit reticulocytes the mechanism of the incorporation of the four labeled amino acids is the same or very similar.

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This conclusion cannot be generalized. In guinea pig liver homogenate lysine is incorporated into the proteins by two different enzyme mecha­ nisms. 98 The enzyme in one is confined to the particle-free solution," its optimum pH is in the neighborhood of 6.5, and added Ca + + (optimum cone. 0.004 M) is obligatory; the other enzyme is largely in the mito­ chondria, its optimum pH is near to 7.5, and added Ca + + accelerates somewhat but is not obligatory. The following summary shows the striking differences in the effects of inhibitors; the numbers give the degree of inhibition, respectively, of the first (acid-calcium) and the second (alkaline) reactions. Anaerobiosis: 0, 24. Arsenate (0.001 M): 27, 2. Arsenite (0.001 M): 67, 0. Azide (0.001 M): 43, 8. 2,4-Dinitrophenol (0.001 M): 28, 0. Fluoride (0.02 M): 95, 14. The conclusions from the findings cited above may be summarized as follows. The mechanism of amino acid incorporation into proteins ap­ pears to be the same in many animal tissues and for many amino acids; but one special and different mechanism, that incorporating lysine in guinea pig liver homogenate, has already been found. Most of the work in this field has been done with animal tissues, cells, or cell extracts. From the little that has been done with microorganisms 1 it appears that some inhibitors may act differently on them from the way they act on animal tissues. For example, arsenite (0.001 M), which inhibited incorporation nearly completely in animal tissues, inhibited growth (colony formation) of E. coli 50 % and did not inhibit amino acid incorporation at all. Experiments on the effect of penicillin on E. coli showed that growth (i.e., ability to form colonies) is experimentally separable from amino acid incorporation. In the early stages of lysis by penicillin there is considerable increase in bacterial protein—it may be doubled or more—100-103 a n ( j ^ θ a m m o a c id incorporation observed at this stage may be associated with synthesis of protein. But in other experiments with other microorganisms bacterial cell fragments were obtained which could incorporate amino acids actively. 104 These frag­ ments may be analogous to the intracellular aggregates—mitochondria and microsomes—of animal tissues, which are able in vitro to incorporate amino acids into their proteins. 98

Borsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G., and Lowy, P. H., / . Biol. Chem. 179, 689 (1949). 99 Borsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G., and Lowy, P . H., / . Biol. Chem. 184, 529 (1950). 100 Gardner, A. D., Nature 146, 837 (1940). 101 Smith, L. D., and Hay, T., / . Franklin Inst. 233, 598 (1942). 102 Erikson, K. R., Ada pathol. Microbiol. Scand. 23, 221 (1946). 103 Park, J. T., and Johnson, M. J., J. Biol. Chem. 179, 585 (1949). 104 Lester, R. L., J. Am. Chem. Soc. 75, 5449 (1953).

207

ENZYMATIC SYNTHESES OF PEPTIDE BONDS 2. COMPARISON OF TRANSFER AND SYNTHETIC REACTIONS

When one compares the inhibitor picture of amino acid transfer by peptidases and of 7-glutamyl and glycine transpeptidases on the one hand, with that of amino acid incorporation on the other, one sees at a glance that they are quite different (Table V). Additional examples of TABLE V COMPARISON OF THE E F F E C T S OF INHIBITORS ON GLUTAMO-AMIDE

TRANSFER,

P E P T I D E AND P R O T E I N SYNTHESIS, AND AMINO ACID INCORPORATION

All reactions in vitro

Inhibitor Arsenate Cyanide 2,4-Dinitrophenol Fluoride Iodoacetate Malonate

Glutamo-amide transfer58·60 Activated Not inhibited Not inhibited Inhibition variable Not inhibited No data

Synthesis of hippuric Amino acid or of acid p-amino- Synthesis Synthesis incorhippuric of gluta- of serum poraton thione86 albumin105 14 · 40 · 41 acid2 No data No data Inhibited No data Inhibited No data Inhibited Inhibited Inhibited No data No data No data

Inhibited Inhibited Inhibited Inhibited Inhibited Inhibited

Inhibited Inhibited Inhibited Inhibited Inhibited Inhibited

contrast not shown in the table are HCN, which activates the transferase activity as it does the hydrolytic action of papain and inhibits amino acid incorporation; the amide transfer reactions are activated by arsenate, and the synthesis of serum albumin is inhibited. Both the amide transfer and synthetic reactions are activated by ATP, M g + + or Mn + + , and phosphate. Yet, despite this similarity, they are quite different in their effects in relation to inhibitors. Classified according to the effects of inhibitors amino acid incorpora­ tion belongs with the synthetic reactions (Table V). In varying degree arsenate, arsenite, azide, cyanide, dinitrophenol, and fluoride are in­ hibitory. The one exception is the incorporation of lysine by an enzyme in guinea pig liver homogenate (see above), which is not affected by the above inhibitors. Most of the syntheses and amino acid incorporations are inhibited by anaerobiosis. But it is an inconsistent inhibitor, which is not surprising, since respiration can participate only indirectly, by providing a reactive intermediate or by activating the enzyme through ATP or some other such means. The effects of anaerobiosis throw little or no light, therefore, on the immediate mechanism of either peptide synthesis, protein synthe-

208

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sis, or amino acid incorporation. The following is a summary of the in­ hibitory effects of anaerobiosis reported; the results are expressed as per cent inhibition. Synthesis of hippuric acid, 100 ;2 synthesis of serum albumin, 77 ;105 incorporation of glycine, leucine, and lysine into rabbit bone marrow cells, 100 ;96 and rat diaphragm, 100 ;97 incorporation of glycine, histidine, leucine, and lysine into rabbit reticulocytes, variable and incomplete; 41 incorporation of DL-alanine into rat liver slices, 90; 106107 glycine into rat liver mitochondria, 90 ;108 lysine by the acid-calcium reac­ tion in guinea pig liver homogenate, 0, and by the alkaline reaction, 24. 98 The following is a summary of the effects of oxidation and phosphorylation inhibitors in addition to those which have been cited above. In "resting" E. coli109 the incorporation of S35-methionine was inhibited by azide (0.005 M) 100%, 2,4-dinitrophenol (0.001 M)96%, and fluoride (0.02 M) 100%. Malonate, at concentrations of 0.02, 0.01 and 0.005 M, respectively, inhibited the incorporation of glycine in fetal rat liver homogenate 75%, 65%, and 40 %,110 and the incorporation of alanine, 74%, 60%, and 40 %.106 The latter observations indicate that the glycine and alanine are incorporated by the same mechanism. In the mitochondrial system of Peterson and Greenberg 111 the inhibi­ tions were by: arsenite (6 X 10~3 M) 95%; fluoride (1.2 X 10~2 M) 7 5 % ; malonate (4.5 X 10~2 M) 86%; phlorizin (1 X 10~3 M) 0%. And in the microsome system of Siekevitz:112 anaerobiosis 79%; azide (10~3 M) 59%; dinitrophenol (6 X 10~5 M) 88%; and fluoride (10~2 M) 40%. 3. AMINO ACID INCORPORATION AND PHOSPHORYLATION

The most consistent and most powerful inhibitor of amino acid incorporation and of peptide and protein synthesis is 2,4-dinitrophenol. This is the strongest evidence in favor of the participation of high-energy phosphate bonds, because 2,4-dinitrophenol uncouples many (but not all) oxidation and phosphorylation reactions. 113 ' 114 The coupling of phos105

Peters, T., Jr., and Anfinsen, C. B., J. Biol. Chem. 182, 171 (1950); ibid. 186, 805 (1950). 1C6 Frantz, I. D., Jr., Loftfield, R. B., and Miller, W. W., Science 106, 544 (1947). 107 Zamecnik, P. C , Frantz, I. D., Jr., Loftfield, R. B., and Stephenson, M. L., J. Biol Chem. 175, 299 (1948). 108 Winnick, T., Friedberg, F., and Greenberg, D. M., J. Biol. Chem. 175, 117 (1948). 109 Melchior, J. B., Mellody, M., and Klotz, I. M., / . Biol. Chem. 174, 81 (1948). 110 Winnick, T., Arch. Biochem. 28, 338 (1950). 111 Peterson, E. A., and Greenberg, D. M., J. Biol. Chem. 194, 359 (1952). 112 Siekevitz, P., J. Biol. Chem. 195, 549 (1952). 113 Cross, R. J., Taggart, J. V., Covo, G. A., and Green, D. E., / . Biol. Chem. Ill, 655 (1949). 114 Loomis, W. F., and Lipmann, F., J. Biol. Chem. 173, 807 (1948).

ENZYMATIC SYNTHESES OF PEPTIDE BONDS

209

phorylation in peptide synthesis was first suggested by Lipmann 115116 before the action of dinitrophenol was known. As phosphorylation is normally coupled with respiration, protein and peptide synthesis will ultimately depend, if phosphorylation is a necessary intermediate step, on respiration. ATP has been shown to be involved in the synthesis of quasi-peptides and small peptides and of hippuric, p-amino-, and ornithuric acids and glutathione (see above). Peterson and Greenberg 111 found that ATP accelerated incorporation in their system of mitochondrial plus supernatant fractions of rat liver homogenate. In addition to and in the presence of M g + + and ATP a mixture of amino acids—aspartic acid, glutamic acid, arginine, proline, alanine, and methionine—acceler­ ated the incorporation of a number of other amino acids, suggesting that the process is one of protein synthesis. Prior to these observations Winnick 110 · 117 had observed that amino acid incorporation in fetal homogenates, which had been inactivated by dialysis, could be reinstituted by M g + + and adenylic acid compounds, the latter being converted, presumably, to ATP by glycolysis and coupled oxidations. Siekevitz112 has reported that a soluble cofactor produced by mitochondria from α-ketoglutarate or succinate plus cofactors, stimulates the incorporation of alanine by rat liver microsomes. Although some stimulation by ATP and M g + + under certain conditions was observed, Siekevitz was of the opinion that ATP was only secondarily involved, i.e., that neither it nor any other pyrophosphate compound was a stimulating cofactor, but that ATP might be necessary for the formation of the cofactor. In rabbit reticulocytes 41 preincubated for 3 hours at 38°C. the sub­ sequent rate of incorporation is greatly reduced and the activity of the cells is restored to their original level by addition of a mixture of amino acids—histidine, leucine, phenylalanine, and valine—and glucose plus ATP. But added glucose and ATP do not accelerate incorporation in fresh cells, whereas certain (as yet unidentified) heat-stable, nonprotein factors in plasma, liver, and spleen accelerate incorporation greatly in both fresh and preincubated cells. In the synthesis of hippuric acid the role of ATP is clear: it partici­ pates in the formation of benzoyl-Co A. In two other cases where ATP has been shown to be involved in the primary process, e.g., the syntheses of glutamine and glutathione, the question is still an open one what is phosphorylated; no phosphorylated intermediates have been found. In these syntheses there is a stoichiometric correspondence between the degree of synthesis and liberation of inorganic phosphate from ATP, but 115

Lipmann, F., Advances in Enzymol. 1, 99 (1941). Lipmann, F., Federation Proc. 8, 597 (1949). 117 Winnick, T., Arch. Biochem. 27, 65 (1950).

116

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Co A is not involved. Stoichiometric correspondence between inorganic phosphate liberated and amino acid incorporation has not been shown. The published data indicate that several orders of magnitude more equivalents of ATP were broken down than amino acid incorporated. This may be because the enzyme systems contained much ATP-ase, or that other processes unrelated to amino acid incorporation were going on. It is clearly premature to implicate ATP in the primary process of amino acid incorporation. Spiegelman and Kamen 118 had suggested that nucleic acids may act as phosphate donors for protein synthesis; their later work casts doubt on this suggestion—a doubt which they them­ selves expressed.119 The findings on hippuric acid synthesis and amino acid incorporation cited above deprive this suggestion, even if it had been confirmed, of its cogency at the present time as far as the immediate mechanism is concerned. 4. Is

AMINO ACID INCORPORATION SYNTHESIS OF PROTEIN D E OR AN EXCHANGE?

Novo

In one of their first papers on amino acid turnover in proteins in vivo Schoenheimer et al.120 recognized that labeled amino acid incorporation may occur by two different mechanisms: There are two general reactions possible which might lead to amino acid replace­ m e n t : (1) complete breakdown of the proteins into its units followed by resynthesis or (2) only partial replacement of units. Metabolic studies with isotopes indicate only end-results b u t not intermediate steps of a reaction. We have no indication as to what had happened to the protein molecule in the animals. Both reactions are conceivable.

The question raised by Schoenheimer and co-workers may be stated as two extreme alternatives: (1) amino acid incorporation into proteins occurs by synthesis of protein de novo from amino acids; or (2) it occurs by an enzyme-mediated exchange of amino acids in the protein with the corresponding free amino acids in the medium, without hydrolysis of the moieties on either side of the two peptide bonds split in the exchange. Some alternatives between these two extremes are that the amino acid is first incorporated into a peptide by de novo synthesis or by replacement, and the peptide is then built into a protein either by de novo synthesis of the protein or by peptide replacement. A way to a direct experimental attack on this problem has not yet been found. The interpretation of what indirect evidence there is, is a 118 119

120

Spiegelman, S., and Kamen, M. D., Science 104, 581 (1946). Spiegelman, S., and Kamen, M. D., Cold Spring Harbor Symposia Quant. Biol. 12, 211 (1947). Schoenheimer, R., Ratner. S., and Rittenberg, D., / . Biol. Chem. 130, 703 (1939).

ENZYMATIC SYNTHESES OF PEPTIDE BONDS

211

matter merely of opinion. Thus Simpson et al.121 observed that ethionine inhibited, in vivo, the incorporation of both methionine (its naturally occurring analog containing one-CH 2 more in the chain) and also glycine, both to about the same extent. Administration of methionine relieved the ethionine inhibition. The authors interpreted these findings as favor­ ing, "superficially at least," incorporation of amino acids by de novo synthesis of protein from the free amino acids. But they are aware of other possible interpretations and do not stress the point. Arguing against de novo synthesis is the fact that amino acid incorporation is not depend­ ent on the administration of all the other amino acids, and that rapid incorporation occurs even in fasting animals. Under certain in vitro con­ ditions (see above) the evidence indicates that amino acids are incor­ porated independently of each other, both in fast- and slow-reacting tissues. If incorporation represented synthesis de novo from amino acids, one would expect the administration of other amino acids to have a great effect. But it maybe that the tissues, as used, contained sufficient amounts of all the other amino acids. In one case there is clear evidence of direct incorporation in the absence of other amino acids. Schweet122 has prepared and somewhat purified by precipitation a soluble enzyme from guinea pig liver homogenate that incorporates lysine. No other free amino acids or metabolites need to be added to the reaction mixture. But this incorporation of lysine appears to be a special case (see above). A better formulation of the question heading this section would be: " I s there more than one mode of protein synthesis?" All amino acids are incorporated, in vivo and vitro; they are incorporated in different loci of the protein. Amino acid incorporation represents, then, breakdown and resynthesis of many if not all peptide bonds in the protein molecule. De facto this is synthesis. But there may not, in this process, be a net increase in protein. And the energy for the reconstitution of the peptide bonds may reside in temporary enzyme-peptide bonds. Where there is a net increase in protein, as in growth or regeneration, the increase comes from amino acids, and no scheme of intermediates will obviate the neces­ sity of supplying the free energy gained in the over-all synthesis of peptides from amino acids. There is a tendency to consider only net increase in protein as protein synthesis de jure. Incorporation, as defined above, and net synthesis may be two different processes. This remains to be seen. In any event amino acid incorporation into proteins is a universal biological phenomenon. It is a relatively fast reaction. For these two reasons it is venturing little to assert that amino acid incorporation must 121 122

Simpson, M. V., Färber, E., and Tarver, H., «7. Biol. Chem. 182, 81 (1950). Schweet, R. S., and Borsook, H., Federation Proc. 12, 266 (1953).

212

II.

BOUSOOK

be of great biological importance. No other phenomenon we know illus­ trates as well the lability of the proteins in the tissues. Whether it is the same process as net synthesis of protein or not, it obviously figures largely in the economy of the dynamic steady state of the cell. 5. T H E POSSIBILITY OF PEPTIDES AS INTERMEDIATES IN PROTEIN SYNTHESIS

Where there is a net synthesis of protein, as in the production of serum albumin by chicken liver slices,105 the protein must have been synthesized either from free amino acids or peptides, or from both. Anfinsen and Steinberg 123-125 concluded that peptides are intermediates in the synthesis of ovalbumin by the hen's oviduct. They incubated minced oviduct with C 14 0 2 , crystallized the ovalbumin present after the incubation, and then digested it with a bacterial enzyme that hydrolyzes the protein to plakalbumin and three peptides: (1) a hexapeptide (3 alanine, 1 aspartic acid, 1 glycine, and 1 valine); (2) a tetra peptide(l alanine, 1 aspartic acid, 1 glycine, and 1 valine); and (3) a dipeptide (alanylalanine). (It is inferred that the tetra- and dipeptide were con­ stituents of the hexapeptide moiety.) The aspartic acid was isolated from the hexapeptide and from the plakalbumin and their specific activities compared. The specific activity of the aspartic acid in the hexapeptide was always greater than that from the plakalbumin, the ratio varying from 1.3 to 3.5. Labeled alanine similarly was incorporated unequally into different parts of the protein. In vivo and in vitro experiments gave similar results. In their latest paper Steinberg and Anfinsen125 give their argument and conclusions from these results. If the ovalbumin were synthesized directly from the amino acid pool by a template mechanism any one labeled amino acid would have the same specific activity throughout the protein molecule. As this was found not to be the case, a complete tem­ plate mechanism of synthesis is excluded. If there are intermediates through which the incorporated amino acids pass on their way to protein, then differences in specific activity of these intermediates would give different specific activities of the same amino acid at different loci in the protein according to the part of the protein molecule formed from a given intermediate. It is immaterial to the argument whether the intermediates are peptides, free or bound, or different amino acid conjugates. The differences in specific activities of the intermediates may arise in several ways. Steinberg and Anfinsen envisaged the possibilities as follows: 123 124 125

Anfinsen, C. B., and Steinberg, D., / . Biol. Chem. 189, 739 (1951). Anfinsen, C. B., and Steinberg, D., Federation Proc. 10, 156 (1951). Steinberg, D., and Anfinsen, C. B., J. Biol. Chem. 199, 95 (1952).

ENZYMATIC SYNTHESES OF PEPTIDE BONDS

213

." First, the labeled amino acid entering different sized pools of preformed, unlabeled peptides could undergo different degrees of dilution. The specific activity of t h a t amino acid would then differ from peptide pool to peptide pool. When these peptides com­ bined to form the protein, the different segments corresponding to these peptides could contain the labeled residue at different specific activities. " Second, if the possibility of true dynamic equilibrium between free amino acids and peptides is considered, differences in the rate constants would permit differences in the rate of isotope equilibration even a t the steady state. " Finally, if peptide fragments released by catabolic reactions from a preformed protein were utilized directly in the synthesis of another protein, these fragments would be expected to contain residues of considerably lower specific activity than those in the remainder of the molecule."

An additional fact needs to be included in the foregoing argument. It is that the specific activity of the labeled amino acid in the pool remains constant after the first few minutes throughout the experimental period of several hours. Steinberg and Anfinsen did not examine which of the three possi­ bilities they cited is the most probable. An estimate of their order of probability can be derived from the quantitative aspects of amino acid incorporation. The best data available at present for this purpose are from the rabbit reticulocyte system under study in the author's labora­ tory. Ninety per cent or more of the protein in rabbit reticulocytes is hemoglobin.126 In vitro the rate of incorporation of leucine commonly is 2.5 micromoles per gram of protein per hour, which is about the same rate as the synthesis of amylase in pigeon pancreas. 127 The reticulocytes containing 50 mg. protein are suspended in 4 ml. of 10~3 M labeled leucine. The amino acid pool contains, accordingly, 4 micromoles of leucine, of which 0.125 micromole are incorporated into the protein in 1 hour. This rate per kilogram of cells, on the basis of 320 g. of red cell protein per kilogram, is 0.8 micromole. Riggs, Christensen, and Palatine 128 found in rabbit reticulocytes 6 micromoles of glycine per kilo of water in rabbit reticulocytes. Glycine is one of the most abundant amino acids in rabbit reticulocytes. 128a Qualitative observations we have made indicate that there is far less free leucine—it could be as little as one-tenth that of glycine. If a peptide intermediate is the carrier of the labeled leucine into the cells its leucine must have a very much higher specific activity than the leucine found in the protein. We have searched for such a peptide inter126

Thoreil, B., Cold Spring Harbor Symposia Quant. Biol. 12, 247 (1947). Hokin, L. E., Biochem. J. (London) 50, 216 (1951-1952). 128 R i g g s > T. R., Christensen, H. N., and Palatine, I. M., J. Biol. Chem. 194, 53 (1925). i28a jf [i w e r e η ο ^ s o > assuming twenty different amino acids, the total amino acid con­ centration would be 0.12 M, which would alone account for nearly the total tonicity of the cells. As most of the tonicity is due to inorganic ions, the total amino acid concentration can hardly be more t h a n 0.05 M and is probably less.

127

214

H.

BORSOOK

mediate and found no evidence of any. If it exists at all it must be present in very low concentration, of the order of 1 % of that of the free amino acid. It must, therefore, have been completely used up by incorporation into the proteins in the first few minutes, and from then on the leucine in the peptide intermediate would have the same specific activity as the free leucine. At the end of 1 to 2 hours, if this be the mechanism, all the leucine in the reticulocyte protein would have the same specific activity. This was found not to be the case;129 differences were observed as large as those found by Steinberg and Anfinsen in the incorporated alanine, aspartic acid, and glutamic acid in ovalbumin. Different specific activi­ ties of the same amino acid in different loci of the protein would call for different peptide intermediates in equilibrium with the free amino acid— as many different such intermediates for one amino acid as there were different specific activities in the protein. Multiplying this number of intermediates by a fairly large factor for all the different amino acids (even if one intermediate is assumed to carry more than one amino acid into the protein) would probably give a concentration of intermediates as great or greater than that of total amino acids. Nor does the above hypothesis of intermediates account for another fact, that as the experi­ mental time is prolonged the inequality in specific activity grows less. The last alternative suggested by Steinberg and Anfinsen seems, then, the most probable of the three, that peptide fragments released by catabolic reactions from a preformed protein are utilized directly in the synthesis of parts of another protein in conjunction with free amino acids from the pool. Even this hypothesis will not, without modification, account for all the observed facts. It does not account for the convergence of specific activities in all loci with time. The last-mentioned hypothesis could be so modified as to account for the available observations, but it seems rather unwarranted to do so here at the present time. All the facts can be accounted for by another hypothesis, which Steinberg and Anfinsen mention, that labeled amino acids in proteins came there by a direct exchange of individual (labeled) amino acids from the pool with their unlabeled counterparts in the protein; in such a direct exchange there is a temporary rupture followed by reformation of two peptide bonds. Differences in specific activity would be the result of differences in rates of exchange at different loci. The loci of fastest exchange would come toward equilibrium with the pool soonest, the slowest loci later, and the effect of prolonging the experimental time would be to bring the specific activities at different loci closer. Steinberg and Anfinsen are of the opinion that "A priori such a mechanism (direct 129

Dubes, G., and Borsook, H., Unpublished observations.

ENZYMATIC SYNTHESES OF PEPTIDE BONDS

215

exchange) appears unlikely/' It may be that direct exchange occurs in peptides released by catabolic reactions from preformed proteins and that these peptides are then synthesized into protein. It is the writer's opinion that there is no a priori basis at present for favoring any one of the hypotheses cited, except in a negative sense that some appear to be excluded. One of these excluded mechanisms is a complete template mechanism of protein synthesis directly from free amino acids (if amino acid incorporation is protein synthesis); another excluded mechanism is that of preformed peptides in rapid equilibrium with the amino acid pool. No hypothesis yet proposed is in accord with all the observations which have been reported. To take the case of globin synthesis, Muir et al.u0 injected labeled glycine and valine into rats and determined the specific activities of the terminal and nonterminal valine in the circulat­ ing hemoglobin 12, 24, and 72 hours and 1 and 2 weeks after the injection. The valine in the two sets of loci had the same specific activity. Also the ratio of incorporated glycine to valine remained the same throughout the experimental period. The authors' conclusion was that The findings are compatible with the hypothesis that globin synthesis consists either of a simultaneous condensation of amino-acids or of a rapid successive formation of peptide bonds without any appreciable accumulation of intermediates.

The findings of Steinberg and Anfinsen with ovalbumin and those in the writer's laboratory with rabbit reticulocyte proteins, discussed above, favor the latter alternative hypothesis suggested by Muir and collabo­ rators. In microorganisms some observations have been reported and inter­ preted as favoring a complete template mode of protein synthesis, and others as supporting protein synthesis through intermediates. Halvorson and Spiegelman 131 compared the simultaneous effect of inhibitory amino acid analogs on growth and adaptive enzyme (maltase) formation in yeast. No analog was found which had a severe effect on growth which did not also inhibit adaptation; and both processes were inhibited in a parallel manner. In the inhibited cells the incorporation of free amino acids into protein was likewise inhibited. A search for peptide or other intermediates failed to find any. Labeled amino acids are readily incor­ porated into enzymes in tissue slices in vitro—ribonuclease (Anfinsen) and pancreatic amylase (Hokin). Halvorson and Spiegelman concluded that if there be a peptide intermediate it must be a very large molecule 130

Muir, H. M., Neuberger, A., and Perrone, J. C , Biochem. J. (London) 49, lv (1951). 131 Halvorson, H. D., and Spiegelman, S., J. Bacteriol. 64, 207 (1952).

216

H.

BOliSOOK

(peptide?) containing many amino acids. Mutant strains of E. coli1*2 form an adaptive enzyme (ß-galactosidase) only when supplied a par­ ticular free amino acid, even when they have previously synthesized polypeptides containing this amino acid; this suggests that, to synthe­ size the enzyme, they cannot use the polypeptide but must have the free amino acid. This evidence was taken as arguing against peptide intermediates. The following observations argue in favor of peptide intermediates. In enzyme adaptations of yeast cells carried out in the absence of an external supply of nitrogen there is a loss of one or more of the enzymes pre-exist­ ing in the cells.133 Yeast cells incubated in the presence of two metabolically unrelated substrates do not normally adapt simultaneously; either substrate alone does induce adaptation of the homologous enzyme. In E. coli there appears to be a relation between adaptively formed lactase and an enzymatically inactive but antigenically related protein which decreases during adaptation. 134 We have come here to one of the central problems. The possibility of unequal specific activities of the same amino acid in different loci of the protein is a condition any mechanism proposed must satisfy. Now, more and new kinds of data appear to be needed. 6. T H E MODE OF LINKAGE OF INCORPORATED AMINO ACIDS

A major assumption underlying nearly all the work on amino acid incorporation into proteins is that radioactivity in the protein after incubating a tissue with a radioactive amino acid, or finding N 15 in an amino acid after an experiment with an N15-labeled amino acid, means that the labeled amino acid had been incorporated into it by peptide bonds. The whole structure of interpretation rests on this assumption. It has been rigorously proved in only two cases; and the proof is almost complete in only one other. It may be said, at once, that since every amino acid that has been tested, indispensable or dispensable, in vivo or in vitro, has been found to be incorporated, it argues strongly for incorporation by peptide link­ age. There are simply not enough side chain loci that might conceivably combine with so many and so different amino acids. But to a limited extent nonpeptide combination can and does occur. Melchior and Tarver 135 found that after S35-methionine had been incubated with 132 133 134 135

Glass, B., Quoted in Phosphorus Metabolism 2, 286 (1952). Spiegelman, S., and Dunn, R., J. Gen. Physiol. 31, 153 (1947). Cohn, M. M., and Torriani, A. M., Compt. rend. 232, 115 (1951). Melchior, J. B., and Tarver, H., Arch. Biochem. 12, 301 (1947).

ENZYMATIC SYNTHESES OF PEPTIDE BONDS

217

liver slices, a large fraction of the radioactivity " b o u n d " to the protein was released by thioglycolic acids; i.e., S 35 -methionine (and cystine formed from it) were bound to the proteins by other than peptide linkage. Simi­ larly, when S35-ethionine was incubated with plasma some of the ethionine was liberated from the proteins by monothiolglycol. 91 When glycine was incubated with rat liver homogenate, 108 a large fraction of the counts in the protein were released after solution of the protein in dilute alkali followed by dialysis, and by heating at 90°C. with 5 % trichloroacetic acid. 111117 In these instances, except possibly the last, the linkage of the labeled amino acid appears to be through sulfur in the protein. Labeled amino acids may combine nonenzymatically with certain proteins in other ways than through sulfur linkage. This was shown in Brunish and Luck.136 Desoxypentose nucleohistone combined with alanine, glycine, and lysine. The alanine and glycine were C14-COOH labeled and after combination with the protein ninhydrin reagent, which liberates C 0 2 when the amino and carboxyl groups of an amino acid are free, did not liberate any counts from the protein. The combination with amino acids was dependent on time but independent of pH or the pres­ ence of ATP or oxygen. Potassium appears to increase the amount of uptake. The "incorporation" was increased by elevating the tempera­ ture to 100°C. The phenomenon described by Brunish and Luck is eliminated in all experiments where the degree of incorporation is zero or insignificant in the boiled controls, as it was in practically every instance reported other than theirs. In many experiments the C14-labeled amino acid was recovered as such after acid hydrolysis and in that form accounted for practically all the counts originally in the protein before hydrolysis. And the C14 was not liberated by ninhydrin treatment of the unhydrolyzed protein. Nevertheless, these two findings do not constitute proof that the labeled amino acid was incorporated by peptide bonds. It would be proof if a peptide could be isolated and identified among the partial hydrolysis products of a radioactive protein. This was done in the experiment cited above 125 where C14-labeled egg albumin was formed by minced hen's oviduct and radioactivity was found in the hexapeptide of the partial enzymatic hydrolysate. Proof, almost as rigorous, was furnished by Winnick et al.n7 in the case of carboxyl-C14-glycine incorporated into rat liver homogenate proteins. On treatment with ninhydrin the follow­ ing results were obtained: no C 1 4 0 2 was released from the unhydrolyzed protein; no C 14 was released after peptic or tryptic digestion; 7 5 % of the 136 137

Brunish, R., and Luck, J. M., J. Biol. Chem. 197, 869 (1952). Winnick, T., Peterson, E. A., and Greenberg, D. M., Arch. Biochem. 21, 235 (1949).

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C14 was released as C 1 4 0 2 following digestion with a mixture of trypsin, chymotrypsin, carboxypeptidase, and erepsin. Table VI contains an example of results obtained when the proteins of rabbit reticulocytes, after the incorporation of labeled amino acids, were submitted to a variety of treatments. All the results are in accord with the interpretation that the labeled amino acids were incorporated by T A B L E VI R E S U L T S OF T B E A T M E N T O F P R O T E I N S OBTAINED AFTER INCUBATION WITH C 1 4 - L A B E L E D G L Y C I N E , L - H I S T I D I N E , L - L E U C I N E , OR L - L Y S I N E 4 1

Amino acid incorporated Treatment

Glycine*

Histidine a

Leucine 6

Lysine a

None, c.p.m./mg. protein. Boiled with 5 % trichloroacetic acid. Specific activity as per cent of original protein. Dissolved in alkali and dialyzed. Specific activity as per cent of original protein. Radioactivity released b y ninhydrin. Per cent of original protein. Oxidized with performic acid. Specific activity as per cent of original protein. Hydrolyzed. Radioactivity in isolated amino acid corresponding to t h a t with which it was incubated as per cent in original protein. Radioactivity in amino acid corresponding to t h a t with which it was incubated and with N H 2 group free in the protein.

4.30

7.10

14.3

6.87

101

95

99

95

101

98

99

100

0

1

1

0

108

106 101

109 103

101 99

adenosine-5'-phosphate + adenosine diphosphate 2-Aminoadenosine + A T P —>· 2-aminoadenosine-5 / -phosphate + adenosine diphosphate

As with crude yeast extracts, transfer of phosphate is only to the 5' carbon of the nucleoside. 2. FLAVIN MONONUCLEOTIDE

Flavin mononucleotide (riboflavin-5-phosphate) is synthesized from riboflavin with a purified enzyme from yeast 84 according to the following equation: Riboflavin + A T P —> riboflavin-5'-phosphate + adenosine diphosphate 78

Kornberg, A., and Lindberg, O., J. Biol. Chem. 176, 665 (1948). Kornberg, A., and Pricer, W. E., / . Biol. Chem. 182, 763 (1950). 80 Ostern, P., Baranowski, T., and Terszakowec', J., Hoppe-Seyler's Z. physiol. Chem. 251, 258 (1938). 81 Ostern, P., Terszakowec', J., and Hubl, S., Hoppe-Seyler's Z. physiol. Chem. 255, 104 (1938). 82 Caputto, R., J. Biol. Chem. 189, 801 (1951). 8 3 Kornberg, A., and Pricer, W. E., J. Biol. Chem. 193, 481 (1951). 84 Kearney, E. B., and Englard, S., / . Biol. Chem. 193, 821 (1951).

79

NUCLEOTIDES AND NUCLEOSIDES

281

This system differs from the one which phosphorylates adenosine, because here adenosine diphosphate can also act as a phosphate donor. The enzyme is inert with isoriboflavin, and it does not catalyze the synthesis of flavinadenine dinucleotide. However, it does phosphorylate arabityl flavin and dichloroflavin, both of which are vitamin antagonists. 85 A preparation from the small intestine of rats carries out the same reaction, but is active only with ATP. 86 3. NICOTINAMIDE MONONUCLEOTIDE

Nicotinamide mononucleotide does not have any known coenzyme function but is an intermediate in the formation of DPN. It can be syn­ thesized by the direct phosphorylation of nicotinamide riboside. 87 Nicotinamide riboside + A T P —► nicotinamide-ribose-5'-phosphate + adenosine diphosphate

Other synthetic pathways, of course, are possible, and it has been specu­ lated that perhaps nicotinamide reacts with ribose-1, 5-diphosphate to give nicotinamide mononucleotide and inorganic orthophosphate. The formation of nicotinamide mononucleotide has been observed in intact red cells incubated with nicotinamide and glucose.88 4. DIPHOSPHOPYRIDINE NUCLEOTIDE AND TRIPHOSPHOPYRIDINE NUCLEOTIDE

a. Synthesis of DPN Synthesis of DPN has been observed in red blood cells incubated with nicotinamide 89 and in crude yeast fermentation mixtures. 90 Recently the synthesis has been observed with purified yeast and liver enzymes, 91 and found to proceed as follows: Nicotinamide mononucleotide + A T P «=± D P N + inorganic pyrophosphate

The reaction is reversible and the equilibrium constant is approxi­ mately 0.4. It is actually analogous to the phosphorolysis of nucleosides 85

Kearney, E. B., J. Biol. Chem. 194, 747 (1952). Englard, S., Federation Proc. 11, 208 (1952). 87 Kornberg, A., Phosphorus Metabolism 1, 392 (1951). 88 Leder, I. G., and Handler, P., J. Biol. Chem. 189, 889 (1951). 89 Handler, P., and Kohn, H. I., J. Biol. Chem. 150, 447 (1943). 90 Lennerstrand, A., Ark. Kemi. Mineral. Geol. 14A, No. 16 (1941). 91 Kornberg, A., / . Biol. Chem. 182, 779 (1950). 86

282

LEON A. HEPPEL

discussed earlier in this chapter. Thus DPN may be considered to under­ go pyrophosphorolysis: P-ribose-nicotinamide PP + P-ribose-adenine +± PPP-ribose-adenine + P-ribose-nicotinamide (inorganic (diphosphopyridine (adenosine (nicotinamide) pyrophosphate) nucleotide) triphosphate) mononucleotide)

This equation shows how inorganic pyrophosphate enters to break the internucleotide bond of DPN, just as orthophosphate breaks the iV-glycosidic linkage of a nucleoside. The correctness of this reaction mechanism was demonstrated 92 by incubating DPN, inorganic pyrophosphate labeled with P 32 , and enzyme, until equilibrium was reached. DPN and nicotinamide mononucleotide were isolated; they contained practically no radioactivity. The ATP fraction was isolated by ion-exchange chromatography and found to have the same specific activity as inorganic pyro­ phosphate. By enzymatic means it has been shown that P 32 was exclu­ sively in the two terminal phosphate groups of ATP. Thus, inorganic pyrophosphate reacted as a unit to become incorporated in ATP, as expected from the above equation. b. Synthesis of TPN TPN differs from DPN in having one more phosphate group, which has been assigned to carbon 2' of the adenylic acid moiety. 93 This is based on several lines of evidence. By splitting the pyrophosphate bond of TPN with potato nucleotide pyrophosphatase it was possible to separate a diphosphoadenosine fragment (compound XI) as the lead salt. Phosphate esterified to carbon 5' was hydrolyzed by an adenosine-5-phosphatase from potato, and the product was found to be identical with adenylic acid " a , " presumably adenosine-2'-phosphate. Confirmatory evidence has been provided by finding a deaminase active with adenosine-3'phosphate, adenosine-ö'-phosphate, ATP, and DPN but inert with adenosine^'-phosphate and TPN. 7 4 Synthesis of TPN from DPN was originally observed by incubating with ATP and yeast maceration juice. 94 Later, a similar reaction was 92 93 94

Kornberg, A., and Pricer, W. E., J. Biol. Chem. 191, 535 (1951). Kornberg, A,, and Pricer, W. E., / . Biol. Chem. 186, 557 (1950). von Euler, H., and Vestin, R., Ark. Kemi. Mineral. Geol. 12B, No. 44 (1938).

NUCLEOTIDES AND NUCLEOSIDES

283

observed with crude pigeon liver fractions. 95 More recently, an enzyme has been partially purified from autolysates of ale yeast 96 which carries out the following reaction: D P N + A T P -* T P N + adenosine diphosphate

The same system converts the reduced form of DPN to the reduced form of TPN. Manganese or magnesium ions are required for the reaction. c. Conversion of TPN to DPN TPN can be dephosphorylated to DPN by phosphatases present in crude yeast juice 97 and potato extract. 79 Recently the activity has been purified 450-fold from extracts of pig kidney cortex. 98 The properties of the purified enzyme resemble those of alkaline phosphatase, and it splits other nucleotides than TPN. Also, highly purified preparations of alkaline phosphatase are very active in converting TPN to DPN. The question remains open as to whether or not a specific enzyme exists which catalyzes this reaction. d. Pyridine Nucleotide Transhydrogenase Another mechanism for the interconversion of the pyridine nucleo­ tides involves the enzyme pyridine nucleotide transhydrogenase, which has been purified from extracts of Pseudomonas fluorescens.99'100 It cata­ lyzes a number of reactions, of which two are given below. (The reduced coenzymes are represented as T P N H and DPNH.) T P N H + D P N -> T P N + D P N H T P N H + desamino-DPN -> T P N + d e s a m i n o - D P N H

The reaction appears to involve an electron, or hydrogen, transfer rather than a transfer of phosphate; otherwise desamino-TPNH would be 95

Mehler, A. H., Kornberg, A., Grisolia, S., and Ochoa, S., J. Biol. Chem. 174, 961 (1948). 96 Kornberg, A., J. Biol. Chem. 182, 805 (1950). 97 von Euler, H., and Adler, E., Hoppe-Seyler's Z. physiol. Chem. 252, 41 (1938). 98 Sanadi, D . R., Arch. Biochem. and Biophys. 35, 268 (1952). 99 Colowick, S. P., Kaplan, N . O., Neufeld, E . F., Ciotti, M. M., / . Biol. Chem. 195, 95 (1952). 100 Kaplan, N . O., Colowick, S. P., and Neufeld, E. F., J. Biol. Chem. 195, 107 (1952).

284

LEON A. HEPPEL

formed from desamino-DPN. Some evidence for the reversibility of the first reaction has been obtained by coupling the system with TPN specific cytochrome c reductase. 101 The transhydrogenase reaction also occurs in rat heart. 5. FLAVIN ADENINE DINUCLEOTIDE

FAD is similar in structure to DPN. It differs in that riboflavin replaces the nicotinamide riboside moiety. It is formed when red blood cells are incubated with riboflavin.102 A purified enzyme system from yeast catalyzes the synthesis of this coenzyme from ATP and flavin mononucleotide. 103 A T P + flavin mononucleotide