[1st Edition] 9781483217055

Table of contents :
Content:
Contributors to Volume 6Page ii
Front MatterPage iii
Copyright pagePage iv
List of ContributorsPage ix
PrefacePage xiBERNARD L. HORECKER, EARL R. STADTMAN
Preface to Volume 6Page xiiBERNARD L. HORECKER, EARL R. STADTMAN
Contents of Previous VolumesPages xiii-xvii
Role of Proteases in SporulationPages 1-20ROY H. DOI
Regulatory Properties of Glucose-6-Phosphate DehydrogenasePages 21-62A. BONSIGNORE, A. DE FLORA
The Behavior of Intact Biochemical Control Systems*Pages 63-130MICHAEL A. SAVAGEAU
A Possible Role for Kinetic Reaction Mechanism Dependent Substrate and Product Effects in Enzyme RegulationPages 131-167DANIEL L. PURICH, HERBERT J. FROMM
Control of Biogenesis of Isoprenoid Compounds in AnimalsPages 169-207T. RAMASARMA
On Allosteric ModelsPages 209-226JEFFRIES WYMAN
Regulation of Uridylic Acid Biosynthesis in Eukaryotic CellsPages 227-265MARY ELLEN JONES
Flip-Flop Mechanisms and Half-Site EnzymesPages 267-310MICHEL LAZDUNSKI
AUTHOR INDEXPages 311-329
SUBJECT INDEXPages 330-334

Citation preview

Contributors to Volume 6 A. BONSIGNORE A . DE FLORA HERBERT J. FROMM ROY H. DOI MARY ELLEN JONES MICHEL

LAZDUNSKI

DANIEL L. PURICH T. R A M A S A R M A MICHAEL A . S A V A G E A U JEFFRIES W Y M A N

CURRENT TOPICS IN

Cellular Regulation edited

by

Bernard L Horecker

·

Albert Einstein College of Medicine Bronx, New York

E a r l R. S t a d t m a n National Institutes of Health Bethesda, Maryland

Volume 6 1972

ACADEMIC New

York

PRESS and

London

COPYRIGHT © 1972, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

A C A D E M I C PRESS, I N C . I l l Fifth Avenue, New York, New York 10003

United Kingdom Edition published by A C A D E M I C PRESS, I N C . ( L O N D O N ) L T D . 24/28 Oval Road, London N W l

LIBRARY OF CONORE3S CATALOG CARD NUMBER:

PRINTED IN THE UNITED STATES OF AMERICA

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List of Contributors Numbers in parentheses indicate the pages on which the authors' contributions begin.

Institute of Biological Chemistry, University of Genoa, Genoa, Italy A . D E FLORA ( 2 1 ) , Institute of Biological Chemistry, University of Genoa, Genoa, Italy HERBERT J. FROMM ( 1 3 1 ) , Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa ROY H . DOI ( 1 ) , Department of Biochemistry and Biophysics, University of California, Davis, California MARY ELLEN JONES ( 2 2 7 ) , Department of Biochemistry, School of Medicine, University of Southern California, Los Angeles, California MICHEL LAZDUNSKI ( 2 6 7 ) , Centre de Biologie Moleculaire du C.N.R.S., Marseille and Department of Biochemistry, Faculty of Sciences, Nice, France DANIEL L . PURICH ( 1 3 1 ) , Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa T . RAMASARMA ( 1 6 9 ) , Department of Biochemistry, Indian Institute of Science, Bangalore, India MICHAEL A . SAVAGEAU ( 6 3 ) , Department of Microbiology, The University of Michigan, Ann Arbor, Michigan JEFFRIES WYMAN ( 2 0 9 ) , C.N.R. Centre for Molecular Biology at the In­ stitute of Biochemistry, University of Rome, and Regina Elena In­ stitute for Cancer Research, Rome, Italy A. BONSIGNORE ( 2 1 ) ,

ix

Preface Recent years have witnessed rapid advances in our knowledge of the basic mechanisms involved in the regulation of diverse cellular activities such as intermediary metabolism, the transfer of genetic information, membrane permeability, and cellular differentiation and other organ functions. Information gained from the detailed analyses of a large num­ ber of isolated enzyme systems, together with results derived from physio­ logical investigations of metabolic processes in vivo, constitutes an everincreasing body of knowledge from which important generalized concepts and basic principles of cellular regulation are beginning to emerge. How­ ever, so rapid are the present advances in the general area of cellular regulation and so diverse are the disciplines involved, that it has become a formidable task for even the expert in a specialized area to keep abreast of the progress in his field. This series of volumes is concerned with such recent developments in various areas of cellular regulation. We do not in­ tend that it will consist of comprehensive annual reviews of the literature. We hope rather that it will constitute a medium which will, on the one hand, provide contributing authors with an opportunity to summarize progress in specialized areas of study that have undergone substantial de­ velopments and, on the other hand, serve as a forum for the enunciation of general principles and for the formulation of provocative theories and novel concepts. To this end editorial review of individual contributions will be concerned primarily with the clarity of presentation and con­ formity to publication policies. It is hoped in this manner to bring together current knowledge of various aspects of cellular regulation so as both to enlighten the uninformed and to provide a base of knowledge for those engaged in research in this subject. BERNARD L . HORECKER EARL R . STADTMAN

xi

Preface to Volume 6 Previous volumes in this series have focused attention on the now classical mechanisms for regulation at the enzyme level, including allosteric and feedback regulation, although several authors have dealt with broader concepts of regulation at the cellular level. The present volume contains several chapters which develop interesting models for regulatory systems at the cell as well as at the enzyme level, including an extension of the original Monod-Wyman-Changeux theory, and an attractive model for half-site enzymes. Other articles in this volume are concerned with regulatory mechanisms related to sporulation, to the pentose phosphate pathway, and to the biosynthesis of isoprene compounds and pyrimidines in animal cells. This volume thus represents a continuation of our efforts to bring to the reader reviews of rapidly de­ veloping areas of metabolic regulation as well as provocative and novel concepts which will promote further progress in this field. BERNARD L . HORECKER EARL R . STADTMAN

xii

Contents of Previous V o l u m e s

Volume 1 Conformational Aspects of Enzyme Regulation D. E. Koshland, Jr, Limitation of Metabolite Concentrations and the Conservation of Solvent Capacity in the Living Cell Daniel E. Atkinson The Role of Equilibria in the Regulation of Metabolism H. A. Krebs Regulation of the Biosynthesis of the Branched-Chain Amino Acids H. E. Umbarger On the Roles of Synthesis and Degradation in Regulation of Enzyme Levels in Mammalian Tissues Robert T. Schimke The Regulation of the Biosynthesis of a-l,4-Glucans in Bacteria and Plants Jack Preiss Allosteric L-Threonine Dehydrases of Microorganisms W. A. Wood The Aspartokinases and Homoserine Dehydrogenases of Escherichia coli Georges N, Cohen Pyruvate Dehydrogenase Complex Lester J . Reed xiii

Xiv

CONTENTS OF PREVIOUS VOLUMES

Pyruvate Carboxylase Merton F. Utter and Michael C. Scrutton Author Index—Subject Index

Volume 2

DPN-Linked Isocitrate Dehydrogenase of Animal Tissues Gerhard W. E. Plaut The Regulation of Biosynthesis of Aromatic Amino Acids and Vitamins J. Pittard and F. Gibson Regulation of Cholesterol Biosynthesis in Normal and Malignant Tissues Marvin D. Siperstein The Biogenesis of Yeast Mitochondria Anthony W, Linnane and J. M. Haslam Fructose 1,6-Diphosphatase from Rabbit Liver S. Pontremoli and B. L. Horecker The Role of Phosphoribosyltransferases in Purine Metabolism Kari 0. Raivio and J, Edwin Seegmiller Concentrations of Metabolites and Binding Sites. Implications in Meta­ bolic Regulation A. Sols and R. Marco A Discussion of the Regulatory Properties of Aspartate Transcarbamylase from Escherichia coli J. C. Gerhart Author Index—Subject Index

CONTENTS OF PREVIOUS VOLUMES

xv

Volume 3

The Regulation of Branched and Converging Pathways B. D. Sanwal, M. Kapoor, and H. Duckworth

The Role of Cyclic AMP in Bacteria Robert L. Perlman and Ira Pastan

Cell Surfaces in Neoplastic Transformation Max M. Burger

Glycogen Synthase and Its Control Joseph Larner and Carlos Villar-Palasi

The Regulation of Pyruvate Kinase Werner Seubert and Wilhelm Schoner

Author Index-Subject Index

Volume 4

The Regulation of Arginine Metabolism in Saccharomyces cerevisiae: Exclusion Mechanisms J. M. Wiame

The Lac Repressor Suzanne Bourgeois

L-Glutamate Dehydrogenases Barry R. Goldin and Carl Frieden

Regulation of Fatty Acid Biosynthesis

P. Roy Vagelos

XVi

CONTENTS OF PREVIOUS VOLUMES

Kinetic Analysis of Allosteric Enzymes Kasper Kirschner Phosphorylase and the Control of Glycogen Degradation Edmond H. Fischer, Ludwig Μ. G, Heilmeyer, Jr., and Richard H. Haschke Author Index—Subject Index

Volume 5 Phosphofructokinase Tag E. Mansour A Theoretical Background to the Use of Measured Concentrations of Intermediates in Study of the Control of Intermediary Metabolism F. S. Rolleston Memory Molecules Götz F. Domagk Protein Kinases Edwin G. Krebs Glutamine Phosphoribosylpyrophosphate Amidotransferase James B. Wyngaarden The Regulatory Influence of Allosteric Effectors on Deoxycytidylate Deaminases Frank Maley and Gladys F. Maley The Citrate Enzymes: Their Structures, Mechanisms, and Biological Functions Paul A. Srere

CONTENTS OF PREVIOUS VOLUMES

Regulation of Histidine Biosynthesis in Salmonella typhimurium Robert F. Goldberger and John S. Kovach Author Index—Subject Index

XVll

Role of Proteases in S p o r u l a t i o n ROY H . DOI Department of Biochemistry Biophysics University of California Davis, California I. Introduction A . Relationship between Protease Production and Sporulation . B. Occurrence of Proteolytic Activity during Sporulation . . II. Extracellular Proteases of Bacilli A. Bacillus suhtilis Proteases B. Bacillus cereus Protease C. Bacillus megaterium Protease D . Bacillus licheniformis Proteases E. Other Bacillus Proteases III. Regulation of Extracellular Protease Synthesis and Sporulation . IV. Protein Turnover during Sporulation A. Protein Synthesis during Sporulation B. Protein Degradation during Sporulation C. Intracellular Proteases V. The Modification of R N A Polymerase and Aldolase by Serine Protease VI. Conclusions References

and

1 3 4 5 5 6 7 7 7 8 10 11 12 12 14 16 18

1. Introduction Sporulation in bacteria has been studied increasingly as a model system for cellular differentiation. Several reviews have appeared on the bio­ chemical and cytological events which occur during the process of sporula­ tion (16, 23J 24, 29, 39, 57, 66). There are well defined stages of sporula­ tion which can be correlated with the appearance of several enzymes, chemical components, and ultrastructures associated with the developing spore (Table I). The isolation of mutants blocked at various stages of development has also made the study of sporulation amenable to genetic analysis (67, 72). One of the major areas of study has been the analysis of enzymes which appear during the initial stages of sporulation. These studies have been complicated by the fact that sporulation is initiated at the end of the log phase of growth of bacilli. At this same period many cataboliterepressed genes are also derepressed as the result of the depletion of glu1

BOY Η. DOI TABLE

I

STAGES IN BACTERIAL SPORULATION«

Stage

Time after end of log phase^ (hour)

0 I

0(«

II

2 («

I

ill)

III

3

ω

IV

4

ω

V

VI

5(ί5) 6

ω

Events that occur End of log phase of growth 1. Chromatin condensation into axial filament 2. Derepression of catabolite genes 3. Protease production 4. Modification of R N A polymerase 1. Appearance of forespore septum and formation of septum 2. Protein turnover 3. Synthesis of tricarboxylic acid enzymes 1. Engulfment of forespore 2. Segregation of nuclei 3. Production of glucose dehydrogenase 1. Cortex synthesis 2. Dipicolinic acid synthesis 3. Appearance of refractility 1. Coat deposition 2. Occurrence of heat resistance 3. Calcium incorporation 1. Maturation of spore

« See following references: (17, 23, 29, 67, 61, 80, 82), ^ These hourly designations will vary slightly with the organism and the medirnn.

cose from the medium. It is difl5cult then to decide whether the appear­ ance of an enzyme was the result of derepression of a catabolite-repressed gene or the expression of a sporulation-specific gene. As will become more obvious in the review, it is likely that the expression of the initial sporulation genes and the catabolite-repressed genes are both under identical or similar catabolite control mechanisms. In this review I will summarize the characteristics of the proteases, which are prominently expressed during sporulation of bacilli and which have been linked with the sporulation process. A recent finding indicates that a serine protease modifies the DNA-dependent RNA polymerase of Bacillus subtilis during sporulation resulting in a change in its template specificity. These results indicate that proteases are intimately involved in cellular regulatory processes and play a significant role during cellular differentiation. It is unlikely that all the proteases of the sporulating cell have been identified; this is particularly true of the intracellular proteases, which appear to have a high degree of specificity and are in-

ROLE OF PROTEASiS IN SPORULATION

3

volved in enzyme modification and protein turnover. Although much more information is required for the final evaluation of proteases as regu­ latory enzymes during developmental processes, this concept may have application for the analysis of differentiating tissues of higher forms. A . Relationship between Protease Production and Sporulation During the growth cycle of bacilli a relationship was observed ini­ tially between the appearance of extracellular proteolytic activity and the initial stages of sporulation (4, 12, 34, 44) \ the efficiency of spore formation was correlated to the normal production of extracellular pro­ tease activity. The earlier studies did not distinguish the different pro­ teases which were being produced, since the usual assay methods con­ sisted of agar plate tests or tests for total protease activity in the supernatant fraction of the culture medium. These initial observations were followed by genetic analysis which provided a more direct link between the expression of protease activity and regulation of spore formation. The analyses of asporogenic mutants of bacilli revealed that many of these mutants did not produce extracellular proteases and that reversion to sporogeny was accompanied by the ability to produce proteases {65, 72). The absence of protease production was noted in those mutants blocked early in sporulation, and those mutants blocked in the later stages of sporulation could produce proteases suggesting that any putative role of protease in sporulation was connected to some early event in the sporulation process, i.e., up to stage III (66). However, it was considered a possibility that a mutation that blocked protease production also blocked some early step in sporulation by a pleiotropic effect and that these two phenomena were only fortuitously linked, e.g., a mutation in some regulatory gene could effect derepression of a series of cataboliterepressed genes which could include the protease gene and some gene involved Γη the initiation of sporulation. Also the absence of protease activity in an asporogenous strain could be the result, for instance, of a mutation in the membrane which prevented excretion of the protease, of lack of activation of proteases, or of proper assembly of the protease molecule. The mere absence of protease activity therefore could not be directly linked to asporogeny since factors which affected protease pro­ duction could obviously affect the more complicated sporulation process. The great difficulty lay in finding asporogenous strains which were directly affected in the structural genes of the proteases produced during sporulation.

4

ROY Η . DOI

Β. Occurrence of Proteolytic Activity during Sporulation Proteolytic activity is observed both intracellularly and extracellularly in bacilli during spore formation. The intracellular activity that has been analyzed most extensively consists of studies on protein turnover during spore information. Although the enzymology of protein turnover has not been analyzed thoroughly, the in vivo studies have revealed the extensive nature of this process. On the other hand, the extracellular proteolytic activity of bacilli has been known for some time and has been used for practical purposes such as the classification of this genus and for the production of proteases for commercial purposes. The extra­ cellular enzymes have been studied much more thoroughly; however, it is difficult to correlate some of this information, since the optimum condi­ tions for obtaining high yields of protease were not necessarily ideal for sporulation nor for obtaining native enzymes. The presence of several different extracellular proteases also complicated the studies, and many of the earlier physiological and biochemical studies on total proteolytic activity are difficult to interpret. As will become more evident in this review, the question of whether the extracellular proteases have an in­ tracellular role prior to being released from the cell may be very impor­ tant. Therefore the terms "extracellular" and "intracellular" will be used with regard to the cellular location of the enzyme at the time of experi­ mentation and with the knowledge that a small, but significant, portion of the extracellular enzyme may be intracellular at any given time. These studies raised the following questions concerning the functions and roles of the extracellular proteases: Did these enzymes have any intracellular functions prior to excretion into the medium, or were they strictly scavenger-type enzymes which provided amino acids for the cell that was differentiating in a deficient medium? Did they have any role in protein turnover, which occurs at a high rate in most bacilli under­ going sporulation? Did they have any specific role in the initiation and continuation of spore formation? If an intracellular function occurred prior to excretion of the protease, the extracellular function could be of secondary importance. In any case, the early physiological and genetic studies established a relationship between sporulation and extracellular protease production and have led to the studies to be discussed in this review. Although the exact roles for all the proteases have not been definitely established, there are enough data to suggest that proteases play a key role in the differen­ tiation process, and they should be considered as regulatory enzymes at the posttranslational level.

BOLE OF PROTEASES I N SPORULATION

5

IL Extracellular Proteoses of Bacilli Extracellular protease production occurs from the late log phase of growth (stage O-I) and continues into the latter stages of sporulation (Table I). Two types of extracellular proteases produced during this period have been characterized particularly well from B. subtilis and re­ lated strains. They include the subtilisins, which are serine proteases and whose properties have been summarized by Ottesen and Svendsen (5S), and the B, subtilis neutral protease, which has been described by Yasunobu and McConn (81). The serine protease is inhibited by diisopropylphosphofluoridate (DFP)* and phenylmethanesulfonyl fluoride (PMSF), but not by ethylenediaminetetraacetate (EDTA) or o-phenanthroline. It has an optimum pH in the neutral to alkaline region (pH 7.5-9.0), and does not hydrolyze 3-(2-furylacryloyl)glycyl-L-leucine amide (FAGLA). The complete amino acid sequence of three of the subtilisins and a great deal of the other chemical and physical properties are known (70). Their structural relationship to other proteases and their evolutionary significance have been discussed by Smith et al (71). The neutral protease requires Zn^* for activity and Ca^+ for stability and is referred to as a neutral metal protease. It is inhibited by EDTA and o-phenanthroline and has an optimum pH around neutrality. In many cases, these enzymes were obtained from commercial sources, and the lack of information concerning the exact stage of growth at which these enzymes were obtained makes it difficult to assess their relationship with the proteases which are produced during sporulation. However, from the properties reported for the serine and metal proteases produced dur­ ing sporulation of B. subtilis (7,18, «0, S2, 27, 28, 32, 48, 56), it is likely that they are similar or identical with the subtilisins and neutral pro­ teases, respectively. A . Bacillus wbtilis Proteases During sporulation J5. subtilis produces two proteases and an esterase (47, 4^). The esterase and one of the proteases have a serine in the active site. These two are inhibited by DFP and PMSF, which react with an essential serine residue (41), The serine protease shows optimum activity • T h e following abbreviations are employed: B T E E , benzoyl-L-tyrosine ethyl ester; DFP, diisopropylphosphofluoridate; EDTA, ethylenediaminetetraacetate; FAGLA, 3-(2-furylacryloyl)-glycyl-L-leucine amide; PEP, phosphoenolpyruvate; PMSF, phenylmethanesulfonyl fluoride; TAME, p-tosyl-L-arginine methyl ester.

6

ROY Η . DOI

in the alkaline range and has esterolytic activity on p-tosyl-L-arginine methyl ester (TAME) and on benzoyl-L-tyrosine ethyl ester (BTEE). The other protease has properties much like the neutral protease (43), since it is inhibited by EDTA and o-phenanthroline, its optimum activity is at neutral pH, and it lacks esterolytic activity. The proteases are produced actively from stage 0 to stage IV or V. In B, subtilis Marburg (48) and B. subtilü WB746 (33), 80% of the proteolytic activity is due to the serine protease and about 20% to the metal protease. Of the total esterolytic activity, 85% is due to the esterase and only 15% to the serine protease (48). In an analysis of 5 different classes of early blocked sporulation mutants of B. subtilis Marburg, it was found that the production of the serine protease and the esterase were reduced in all the classes (45). In contrast the metal protease activity remained constant in all the strains. These results suggested that the serine protease and the esterase were related to sporulation, but that the metal protease was not. In support of this contention one strain was found which sporulated normally (45) but produced an undetectable level of the metal protease. Further support for the serine protease's role in sporulation has been obtained by Leighton et al (32, 33), who have isolated a temperaturesensitive serine protease mutant which does not sporulate at the nonpermissive temperature (47°C) and exhibits little or no protease activity by plate assays at 47°C. That the mutation is in the structural gene for the protease has been demonstrated by 8 criteria, including differences from the wild-type enzyme in thermal stability, mobility in acrylamide gels, specific activity, temperature dependence, in vitro stability, and rates of inactivation by PMSF. Morphological analyses by electron microscopy have revealed that the mutant is blocked at nonpermissive temperatures at stage 0 or stage I, since no forespore membrane formation was observed (64). The pattern of RNA, alkaline phosphatase, and aconitase synthesis was altered in the organism at 47°C indicating that inactivation of the protease has far-reaching effects. A more specific role of this protease will be discussed in Section V. B. Bacillus cereus Protease An extracellular neutral protease has been reported for B. cereus, but the analysis of this enzyme suggests that it has no relationship either to sporulation or to protein turnover. Hypoprotease mutants still sporu­ lated normally and the turnover rate of one of these mutants was identical to that of the wild type (1, 34). A small amount of serine protease is also produced in B. cereus, since a protease activity was reported which was inhibited by DFP and stable to dialysis against distilled water (62).

ROLE OF PROTEASES I N SPORULATION

7

This enzyme has the ability to convert vegetative cell aldolase to spore aldolase and will be discussed in Section V. C. Bacillus megaterium

Protease

Bacillus megatenum produces only one extracellular protease during sporulation (46, 50). This enzyme is inhibited by o-phenanthroline and EDTA, but not by DFP, has maximum activity at pH 7.2, and is stabil­ ized by Ca^+. It is therefore classified as a neutral metal protease. It is produced during the growth phase when grown in minimal medium, and during sporulation when grown on a complex medium. A protease-minus mutant of B, megaterium was isolated which sporulated normally and turned over protein at the same rate as does wild type. It is believed therefore that this protease is not involved in either sporulation or pro­ tein turnover {52). D. Bacillus licheniformis Proteases

Two proteases are produced by B, licheniformis (28). One is sensitive to DFP and has optimum activity at pH 8.5 to 9.0; it also has esterolytic activity. The other is insensitive to DFP, but is inhibited by EDTA and stimulated by Ca^+. It shows optimum activity at pH 7.2-7.6 and is stable at 65°C whereas the DFP-sensitive protease is inactivated at 65°C. These two enzymes are related in these particular properties to the proteases produced by ß . subtilis, E. Other Bacillus Proteases Several alkaline proteases produced by various Bacillus strains have been compared by Keay and Moser (28). They fell generally into two groups, which could be distinguished by the ratio of esterase to protease activities, the amino acid composition, and serological cross reactions. One group contained subtilisin Carlsberg, Β, licheniformis^ and Β, pumüus; the other group contained subtilisin Novo, Nagarse, B. subtilis NRRL Β 3411; and B, subtilis var. amylosacchariticus, A comparison of neutral protease from B, subtilis NRRL Β 3411 and B. subtilis var. amylosacchariticus showed that they were very similar; however, these two enzymes differed from thermolysin (from B. thermoproteolyticus) when compared by immunochemical, heat stability, and amino acid composition studies (27), The extracellular proteases of the various Bacillus species have been summarized in Table II. Essentially all the bacilli produce one or two extracellular proteases of either the serine or neutral type. B. brevis sporulated without produc­ tion of any extracellular protease (69), and, as discussed previously, mutants of B. cereus and B, megaterium have been obtained which sporu-

ROY Η . DOI

ο TABLE

II

EXTRACELLULAR PROTEASES PRODUCED DURING SPORULATION BY BaciUus SPECIES Organism B, suhtüis

Β, megaterium Β, cerem Β. licheniformis

Types and properties

References

1. Alkaline serine protease; inhibited by D F P and PMSF 2. Neutral metal protease; inhibited by EDTA and o-phenanthroline 3. Serine esterase; inhibited by D F P 4. Basic serine protease; inhibited by D F P and PMSF 5. Acidic serine protease; inhibited by D F P and PMSF 1. Neutral metal protease; inhibited by E D T A 1. Neutral metal protease; inhibited by E D T A 1. Alkaline serine protease; inhibited by D F P 2. Neutral metal protease; inhibited by E D T A

(33, 48, 60) (33, 48, 60) (48, 60) (7, m (7, m) (46, 62) a, 34) (22) (22)

late normally but do not produce extracellular neutral protease. Thus the production of an extracellular protease is not an absolute requirement for sporulation. This does not mean, however, that the intracellular function of these enzymes are not essential for sporulation, since low levels of these enzymes are found inside the cell. III. Regulation of Extracellular Protease Synthesis and Sporulation The synthesis of extracellular protease and the initiation of sporulation are regulated by catabolite control systems {66), Both of these phe­ nomena do not occur when cells are growing in a medium with high levels of glucose; protease synthesis and sporulation occur only after the depletion or reduction of glucose in the medium. In minimal medium neutral protease is produced during growth and sporulation of B, megatenum; however, in complex medium protease is produced only during sporulation (52). The effect of glucose was demonstrated with B, licheniformis which produces protease when grown in a minimal medium with glutamate as the carbon source; when this growth medium was suddenly enriched with glucose, protease production stopped immediately {31). As with the other organisms B, subtilis sporulation and protease pro­ duction are repressed by the presence of glucose (68), In fact sporulation can be prevented by the addition of glucose as the end of the log phase is approached. However, if the cells enter into the stationary phase, there is a point at which the cells become "committed" to sporulation and

ROLE OF PROTEASES IN SPORULATION

9

glucose addition does not repress sporulation.. An explanation has been found for this commitment with B. subtilis. With B. subtilis there is a decrease in the uptake of glucose which is accompanied by a decrease in the glucose-PEP transferase activity when glucose becomes limiting in the medium (19). This decline in glucose-PEP transferase activity commits the cell to sporulation by preventing the entrance of glucose into the cell, and thus allowing derepression of the catabolite-repressed sporulation genes. The decline in glucose-PEP transferase activity can be explained in terms of enzyme turnover or by an alteration in the membrane that may affect the activity of the enzyme. The constant ratio of the serine and neutral proteases during sporulation of B, subtilis suggests that synthesis of both these enzymes may be controlled by a similar mechanism (48). Protease production is also strongly repressed by the presence of amino acids or peptides (8, 9, 34, 4^, S2, 68), All the amino acids do not repress with equal efiSciency, and the same amino acids do not cause repression in the various Bacillus species. In most cases a mixture of amino acids or peptides is required to obtain maximum repression. There is no definite pattern of repression by the individual amino acids, since very poor repression was obtained with glutamate or aspart^,te in B, megaterium, while these two amino acids repressed very strongly in B, subtilis, Isoleucine and threonine, however, did repress rather strongly (about 60%) with both organisms. Protease was synthesized during growth of B, megaterium in a glucosecontaining minimal medium, but the addition of threonine and isoleucine inhibited 80-90% of the synthesis (10), Interestingly, the amino acids repressed the synthesis of extracellular protease, but did not reduce the synthesis of intracellular protease. This indicates that different mecha­ nisms may be involved in regulating their respective synthesis (8), The synthesis of extracellular and intracellular proteases was also not coordinately controlled in B. licheniformis (6), However, it is possible in both these cases that the excretion process was involved in regulating the amount of enzyme in the medium. Furthermore, since the total intra­ cellular enzyme activity was determined in these studies, it is still possible that one or more of the intracellular proteases was regulated in a co­ ordinate fashion with the extracellular enzyme. In one case a stimulation of extracellular protease synthesis by amino acids was observed. This stimulation occurred, however, only in the absence of glucose from the medium (15), A relationship between the regulation of protease synthesis and of sporulation was also observed in B. cereus. High levels of amino acids inhibited both phenomena. Mutants were isolated which sporulated in

10

ROY Η. DOI

the high amino acid medium and which produced high levels of protease in normal and inhibitory medium. These results were interpreted as the absence in these mutants of a common catabolite involved in the control of several functions (including protease production and sporulation) {34), It was shown with B. cerem that the protease excreted during sporulation is made during this period, since labeling of the enzyme occurred only if the labeled amino acid was present at the end of the log phase of growth. Prelabeling of log phase cells did not create a pool of precursor molecules which would have been converted to active protease molecules during sporulation {34) · These results lead to the conclusion, first proposed by Schaeffer et al. {68), that the initiation of sporulation and many of the enzymatic activities associated with the sporulation stage, including the extra­ cellular proteases, are regulated by catabolite type repression. IV. Protein Turnover during Sporulation In addition to extracellular proteolytic activity, protein turnover has been observed in various Badllm species during sporulation and must play a key role in furnishing amino acids to a system which is under­ going differentiation in a medium devoid of the usual carbohydrates and amino acids {5, 29). This rate has been reported to be between 1% to 18% per hour (Table III). In growing cells the rate is less than 0.5% per hour {78). Turnover of proteins occurs at the early stages of sporula­ tion slightly prior to forespore membrane formation and continues throughout sporulation. When the general pattern of protein turnover was analyzed, it was observed that proteins which were made during the log phase of growth and those made during sporulation were turned over at the same rate {14). It will be shown later that all protein species are not turning over at the same time or rate. In species with a sufficiently high turnover rate radioisotope studies indicate the possibility of com­ plete turnover of all the proteins of the vegetative cell by the end of the sporulation cycle {14)TABLE

III

TURNOVER RATE OP PROTEIN DURING SPORULATION OF BACILLI Organism B, B. B. B, B. B, B,

hrevis cereus cerem licheniformis subtilis subtilis thuringiensis

Percent per hour

Reference

1.6 7 6 20 8-10 18 10

(69) (78) (1) (6)

(40) (74) (66)

ROLE OF PROTEASES IN SPORULATION

11

Analysis by immunological techniques has revealed that spore antigens appeared during the midstages of sporulation (stages II and IV) and that a relative decrease in vegetative cell antigens occurred. Thus there was a synthesis of spore specific antigens and a concomitant turnover of some of the vegetative cell antigens {79). When individual enzymes were studied during sporulation, different patterns of turnover were observed; these studies were carried out by harvesting cells at different periods of sporulation and determining their total and specific activities. Generally three patterns were observed {H). Several enzymes such as DNA polymerase and adenylate kinase were found in cells before sporulation commenced and their levels stayed fairly constant throughout the sporulation cycle and fell only during the later stages of sporulation. The second group of enzymes which included IMP dehydrogenase was produced at a rapid rate at the initial stages of sporulation, attained a high level, but at about the midpoint of sporulation their activities disappeared due to rapid degradation; the third group of enzymes, including malic dehydrogenase, arginase, and ornithine transcarbamylase, appeared well after the initial stages of sporulation and remained at constant levels until the later stages of sporulation. These analyses of individual enzymes indicate the complexity of the problem. There is obviously a regulation of the synthesis, and the degradation of various enzymes and a distinct pattern of turnover may occur for each enzyme. A . Protein Synthesis during Sporulation As stated earlier the appearance of enzymes appears to be regulated by at least two types of mechanisms. In one case there is reasonable evidence that catabolite repression controls the synthesis of many en­ zymes that occur during the initial stages of sporulation [see reviews of Schaeffer {66) and Hanson et al, {24)], These enzymes include the enzymes of the tricarboxylic acid cycle {25), of amino acid catabolism {30, 31), and of extracellular nature such as proteases {1, 52), The synthesis of other enzymes and proteins may be dependent on the positive control of transcription, since they occur later in the sporulation cycle and appear to be independent of catabolite repression. These en­ zymes include dipicolinic acid synthase {3, 83), diaminopimelic acidadding enzyme {76), and spore coat protein {73), The other aspect of this type of control is the observation by Losick and Sonenshein {36, 37) that the RNA polymerase of the vegetative cell is modified during the initial stages of sporulation. This modification consists of a change in the template specificity and in the structure of vegetative cell RNA polymerase. The structural change consists of reduction of the molecular weight of the beta subunit of RNA polymerase from 155,0(X) to 110,000.

12

ROY Η . DOI

The modified RNA polymerase is also unable to transcribe RNA from phage φe DNA efficiently. This modification is a most significant observa­ tion, since it can explain how the cell switches from transcription of vegetative cell genes to sporulation genes. The altered RNA polymerase must recognize different promoter sites (26). The modification of the RNA polymerase could occur in two ways. One possibility is the transcription and translation of a completely new beta subunit gene resulting in the beta subunit with the reduced molecular weight. The other possibility is the specific cleavage of the beta subunit of the vegetative cell RNA polymerase by a protease. This second pos­ sibility appears to be the case, since in vivo and in vitro experiments support this idea. This modification and the role of serine protease will be discussed thoroughly in Section V. For the synthesis of enzymes under catabolite repression, a system analogous to the lac system of Escherichia coli may be necessary (13); in addition to the modification of RNA polymerase structure, the syn­ thesis of later sporulation-specific enzymes may be under control of the same factors which control the specificity of RNA polymerase as demon­ strated fori;, coll (13) andT4 (77). B. Protein Degradation during Sporulation In B, subtilis there is a correlation between the degradation of intra­ cellular proteins and the increases of intracellular protease activity (60, 80). Protein breakdown is also dependent on the de novo synthesis of proteins, since it is prevented by the addition of chloramphenicol before the initiation of turnover. Once turnover commences, protein degradation continues even in the absence of protein synthesis (Jf^)- Available data do not indicate whether this is the synthesis of "turnover" proteases or the synthesis of another protein required for the activation of the pro­ teases involved in turnover. C. Intracellular Proteases The presence of intracellular proteolytic enzymes has been reported in various J5. subtilis strains (6, 21, 33, 53, 54, 60, 75). Their relationship to protein turnover has not been established. At one time there appeared to be some relationship between the neutral extracellular protease activity and intracellular protein turnover (40). This relationship was probably the result of a pleiotropic effect on a regulatory system which affected the production of both the intracellular turnover protease system and the extracellular protease, since subsequent studies have indicated that the absence of extracellular neutral protease did not affect either protein turnover or sporulation (45). Similarly in B. cereus (1) and B. mega-

ROLE OF PROTEASES IN SPORULATION

13

terium {51, 52), it has been shown that the extracellular metal protease is not involved in intracellular turnover and is not essential for the sporulation process. This was demonstrated by isolating mutants which contained a normal level of the enzyme, but which did not sporulate and had low turnover rates, and other mutants which sporulated and had high turnover rates, but no detectable level of the protease. With B, licheniformis {6) an intracellular proteolytic activity was found to be much higher against denatured protein than native protein {35), ^nd the amount of protease activity was not reflected in the turnover rate. It was proposed that the intracellular proteases acted as scavengers; however, it is possible to consider these proteolytic activities as having other roles; for instance the broad specificity of the enzyme would allow it to attack diJBferent peptide bonds in regions of the proteins which had been locally "denatured" by some modification. These enzymes may not have a role in general protein turnover, but may have a much more specific role such as modifying enzymes by specific cleavage. Therefore from the available data it is fair to say that no definitive role is ascribable to the intra­ cellular proteases. One may ask whether a large amount of protein turnover is necessary for sporulation. As Table III indicated, most Bacillus species display a rather large turnover rate during sporulation. One exception to this rule has been found with B, brevis. In B, brevis there is very little protein turnover during sporulation, no production of extracellular proteases, and little or no intracellular protease activity {69). Only a small amount of turnover amounting to 1-1.6% per hour was detected at stage t 2 . These results indicate that a large amount of protein turnover is not absolutely necessary for the sporulation process; however, the small turnover activity detected at stage tg may play a significant role. The study of intracellular proteases appears to hold great potential for understanding protein turnover and the enzymatic modifications that occur during sporulation. Many of the earlier studies on intracellular proteases have been limited by the fact that the activity of protease mixtures or the total protease activity of an extract was examined. Re­ cently an intracellular protease of B. megaterium was purified 100-fold and characterized {49, 53). It was inhibited by both DFP and EDTA, but not by o-phenanthroline, and it had a narrower specificity than the extracellular metal protease of B. megaterium or the extracellular serine protease of B. subtilis. It had esterolytic activity and was classed with the extracellular serine proteases of B. subtilis; however, it differed from them in that it displayed a much narrower specificity on carboxymethylated insulin Β chain and had much lower activity on casein. No function has been assigned to this enzyme as yet.

14

ROY Η . DOI

Two intracellular proteases have been purified from B. subtilis 168 (21). One has properties similar to the extracellular neutral protease and is inhibited by EDTA. The other is a new DFP-inhibitable serine protease which is different from the two proteases reported previously for this particular organism (7). A mutant which did not have a detect­ able level of the intracellular neutral protease, but which did have twice the usual level of the new serine protease initiated sporulation sooner than the parental strain and sporulated to the same degree as the parental strain. Another mutant which had the normal amount of neutral protease, but which lacked the new serine protease was incapable of producing spores. It appears that this new serine protease plays some role in the sporulation process, although the exact stage at which it was required was not defined. The question of whether extracellular enzymes appear intracellularly has been tested by the use of PMSF-treated cells of B. subtilis WB746. The cells were pretreated with PMSF before breakage, since PMSF in­ activates all extracellular and periplasmic serine protease but does not penetrate the cytoplasmic membrane (Leighton, unpublished results). The ratio of the intracellular serine protease and neutral protease was 4:1, a ratio found extracellularly. This result is suggestive that the extra­ cellular enzymes occur intracellularly. Besides the role of proteases in protein turnover during sporulation, two cases of proteolytic modification of enzymes have been reported. It is this role of proteases which may be crucial to regulation in the sporu­ lating cell. V. The Modification of R N A Polymerase and Aldolase by Serine Protease The proteolytic modification of two enzymes from their vegetative cell forms to that found in sporulating cells or spores has been reported (32, 33, 62). Leighton et al {33) isolated a temperature-sensitive serine protease mutant of ß. subtilis which sporulated and produced extra­ cellular serine protease normally at 30°C, but did not produce an active protease nor sporulate at the nonpermissive temperature (47°C). The cells did not develop beyond stage 0 or 1 at 47^C, Furthermore when the RNA polymerase from cells grown to sporulation stage II at the nonpermissive temperature was examined, it did not save a modified beta subunit as reported by Losick et al (36) (Table IV); the modification did occur during sporulation at 30°C when the protease was active or when cells grown at 47°C to stage I were shifted to 30°C (33). These results imply that the serine protease was necessary for sporulation and that the conversion of the vegetative RNA polymerase to the sporulation

ROLE OF PROTEASES I N SPORULATION

15

TABLE

IV

MOLECULAR WEIGHTS OF THE SUBUNITS OP R N A POLYMERASE FROM Bacillm suhtilis Subunits« Growth phase

Alpha

Beta

Beta'

Beta*

References

Vegetative ceUs Sporulating cells Spores In vitro conversion by serine protease: 0 min 60 min

45,000 45,000 43,000

155,000 — —

155,000 155,000 146,000

— 110,000 129,000

(2,33,36) {33,36) (38)

45,000 45,000

155,000 —

155,000 155,000

— 110,000

(33) (33)

» The terms alpha, beta, beta', and beta* are operational labels for R N A polymerase subunits which band at specific locations after gel electrophoresis (33, 36).

form was either directly or indirectly dependent on serine protease activity. In in vitro experiments the purified vegetative cell RNA polymerase was mixed with purified extracellular serine protease, and the beta modification was obtained as in the in vivo situation (33) (Table IV). Furthermore the template specificity change noted between the RNA polymerase from vegetative cells and sporulating cells (37) was also noted with the RNA polymerase which had been modified in vitro. These results indicate that the serine protease is involved directly in the modi­ fication while it is intracellular and before it is excreted into the medium. In several cases a low but significant amount of proteolytic activity similar to the extracellular serine protease has been found within cells (33,34,60). Another well-documented case of protease modification involves the proteolytic modification of the fructose 1,6-diphosphate aldolase which has a molecular weight of 79,000 in vegetative cells and 44,000 in spores (62, 63). These two forms of the aldolase enzyme have similar enzymatic and immunochemical properties and heat resistance in the absence of Ca^+. They differ in molecular weight. Stokes radii, mobility in gels, and heat resistance in the presence of Ca^*. This initial report of aldolase modification in B. cereus was the first indication that proteases may play a more specific role than extracellular scavengers. The role of protease modification may be extensive, since a temperature shift-up of the temperature sensitive serine protease mutant of JB. subtilis at any stage of sporulation reduces the total spore yields (33). Therefore although the modification of RNA polymerase occurs early in the sporu­ lation cycle at stage 0 or I, the protease appears to be required for later

16

ROY Η . DOI

functions. One interpretation is that vegetative cell polymerase is con­ tinually made and modified during sporulation and this modification is essential for transcribing sporulation genes. Another possibility is that protease modification of other enzymes is required at all stages of sporulation. The modification of RNA polymerase by the serine protease is the most direct evidence for the role of proteases in sporulation. Since its activity appears to be essential for normal sporulation and since it affects the activity of a very crucial enzyme, the regulation of its synthesis and its intracellular function (s) will probably turn out to be the key steps in the initiation of sporulation. V I . Conclusions Several points can be made concerning protease activity and its rela­ tionship to the sporulation process. The extracellular neutral metal proteases are not directly related to the sporulation, since mutants which have little or no metal protease activity can sporulate normally. Further­ more, the rate of protein turnover during sporulation of these mutants is normal. Therefore no function during sporulation is known for this enzyme. Its synthesis, however, is regulated in a fashion similar to that of the serine proteases and they appear to be produced coordinately during the initial stages of sporulation. The role of the serine protease has been defined at least partially. Both in vivo and in vitro experiments indicate that the serine protease is involved in the modification of RNA polymerase during sporulation. Although its level is low intracellularly relative to that found extra­ cellularly, its function as a modifying enzyme may play a crucial role in initiating and continuing the sporulation process. A current model for the initiation of sporulation is as follows (16): 1. As glucose becomes limiting in the growth medium, serine protease is released from catabolite repression. 2. The serine protease cleaves the beta subunit of the vegetative cell RNA polymerase, changes its template specificity, and converts it to the sporulating cell RNA polymerase. 3. The sporulation RNA polymerase begins to transcribe sporulation genes. 4. The serine protease continues to modify the RNA polymerase and other enzymes necessary for sporulation. The serine protease does not appear to be involved in protein turnover, since turnover in the temperature-sensitive serine protease mutant con­ tinues at the.usual rate at the nonpermissive temperature whereas RNA

ROLE OF PROTEASES IN SPORULATION

17

polymerase modification is totally inhibited. Also an asporogenous mutant lacking serine protease activity was able to turn over protein at the normal rate. A review of the current status of our knowledge of proteases in sporu­ lating bacilli indicates a need for an extensive investigation of the intra­ cellular proteases. One would like to know the number of intracellular proteases in a cell, the factors that regulate their synthesis, their functions and substrates, and the factors that may regulate their activities. Since the substrate specificity may be very high, it is likely that some proteases will remain undetected by the use of unnatural substrates, e.g., azocasein is "apparently" hydrolyzed very slowly as compared to RNA polymerase when its hydrolysis is measured by loss of enzymatic activity. Will there be many specific proteases, or will there be few proteases that can recognize different proteins which have a common substrate site? Are there factors that convert a protein into a substrate for a specific protease by "local denaturation"? This raises the question of whether a substrate-modifying factor may be part of the regulatory sequence. Also are proteases of sporulating cells in the active state at all times, or are their protease activating factors? These and many other questions require answers before the role of proteases in sporulation can be fully evaluated. The modification of existing proteins and the rapid turnover of pro­ teins may be somewhat imique for sporulating cell, since sporulation is occurring under harsh nutritional conditions. The degradation of pre­ existing proteins would furnish amino acids for the synthesis of sporulation-specific proteins; the process of partial cleavage of preexisting enzymes for the modification of their functions would allow the cell to survive with a minimum of genes and would not require the synthesis of a series of new enzymes. In this way the usual polypeptide chains would be made, and the modification of these chains would depend on the absence or the presence of the modifying protease. If this is indeed the case, then the regulation of the expression of the protease genes would be of greatest importance. Although there is a relationship between catabolite repression and protease formation, the data do not reveal the exact mechanism. This is another area that requires considerably more study in sporulating cells (11), How general is the protease modification of RNA polymerase in differ­ entiating systems? In differentiated cells specialized functions are being performed by a restricted number of gene products. There are several possible mechanisms to restrict transcription of a few genes. One such model could involve proteases that would regulate differential transcrip­ tion by modifying a basic RNA polymerase structure at the posttransla-

18

BOY Η. DOI

tional level. In order to maintain the differentiated state, the continuous presence of protease function would be required. It will be of interest to see how widespread this phenomenon is. ACKNOWLEDGMENTS I wish to express my appreciation to Dr. Terrance J. Leighton, Mr. Phillip Freese, and Mrs. Leatrice Santo^ who contributed their ideas and skills to the work reported from my laboratory; these studies were supported by grants from the National Science Foundation (GB-26409X) and the U. S. Atomic Energy Com­ mission (AT (04-3)34). REFERENCES 1. Aronson, A. I., Angelo, N., and Holt, S. C , / . Bacteriol. 106, 1016 (1971). 2. Avila, J,, Hermoso, J. M., Vinuela, E., and Salas, M., Eur, J. Biochem. 21, 526 (1971). 3. Bach, M. L., and Gilvarg, C , / . Biol. Chem. 241, 4563 (1966). 4. Bemlohr, R. W., / . Biol. Chem. 239, 538 (1964). 5. Bemlohr, R. W., in "Spores III" (L. L. Campbell and H. 0 . Halvorson, eds.), pp. 75-87. Amer. Soc. Microbiol., Ann Arbor, Michigan, 1965. 6. Bemlohr, R. W., and Clark, V., / . Bactenol. 105, 276 (1971). 7. Boyer, H. W., and Carlton, B. C , Arch. Biochem. Biophys. 128, 442 (1968). 8. Chaloupka, J., and Kreckova, P., Biochem. Biophys. Res. Commun. 8, 120 (1962). 9. Chaloupka, J., Kreckova, P., and Rihova, L,, Biochem. Biophys. Res. Commun. 12,380 (1963). 10. Chaloupka, J., and Kreckova, P., Folia Microbiol. 11, 82 (1966). 11. Clark, V., and Bemlohr, R. W., in "Spores V" (H. O. Halvorson, R. S. Hanson, and L. L. Campbell, eds.), pp. 167-173. Amer. Soc. Microbiol., Bethesda, Mary­ land, 1972. 12. Coleman, G., J. Gen. Microbiol. 49, 421 (1967). 13. De Crombrugghe, B., Chen, B., Anderson, W., Nissley, P., Gottesman, M., Pastan, I., and Perlman, R., Nature (London) 231, 139 (1971). U. Deutscher, Μ. P., and Kornberg, Α., Biol. Chem. 243, 4653 (1968). 16. Din, F. U., and Chaloupka, J., Biochem. Biophys. Res. Commun. 37, 233 (1969). 16. Doi, R. H., and Leighton, T. J., in "Spores V" (H. 0 . Halvorson, R. S. Hanson, and L. L. Campbell, eds.), pp. 225-232. Amer. Soc. Microbiol., Bethesda, Mary­ land, 1972. 17. Ellar, D . J., and Lundgren, D . G., J. Bactenol. 92, 1748 (1966). 18. Feder, J., Biochemistry 6, 2088 (1967). 19. Freese, E., Klofat, W., and Galliers, E., Biochim. Biophys. Acta 222, 265 (1970). 20. Hageman, J. H., and Carlton, B. C , Arch. Biochem. Biophys. 139, 67 (1970). 21. Hageman, J. H., and Carlton, B. C , in "Spores V" (H. 0 . Halvorson, R. S. Hanson, and L. L. Campbell, eds.). Amer. Soc. Microbiol., Bethesda, Maryland, 1972. 22. Hall, F. F., Kunkel, Η. Ο., and Prescott, J. Μ., Arch. Biochem. Biophys. 114, 145 (1966). 23. Halvorson, H. 0., Symp. Soc. Gen. Microbiol. 15, 343 (1965). 24. Hanson, R. S., Peterson, J. Α., and Yousten, A. Α., Annu. Rev. Microbiol. 24, 53 (1970).

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26, Hanson, R. S., Srinivasan, V. R,, and Halvörson, Η. Ο., Bacteriol. 86, 45 (1963). 26. Hussey, C , Losick, R., and Sonenshein, A. L., J. Mol, Biol. 57, 59 (1971). 27. Keay, L., Biochem. Biophys. Res. Commun. 36, 257 (1969). 28. Keay, L., and Moser, P. W., Biochem. Biophys. Res. Commun. 34, 600 (1969). 29. Komberg, Α., Spudich, J. Α., Nelson, D . L., and Deutscher, Μ. P., Annu. Rev, Biochem. 37, 51 (1968). SO. Laishley, E. J., and Bemlohr, R. W., Biochem. Biophys. Res. Commun. 24, 85 (1966). 31. Laishley, E. J., and Bemlohr, R. W., J. Bacteriol. 96, 322 (1968). 32. Leighton, T. J., Freese, P. K., and Doi, R. H., Fed. Proc, Fed. Amer. Soc. Exp. Biol. 30, 1069 (1971). 33. Leighton, T. J., Freese, P. K., Doi, R. H., Warren, R. A. J., and Kelln, R. Α., in "Spores V" (H. 0 . Halvorson, R. S. Hanson, and L. L. Campbell, eds.), pp. 238-246. Amer. Soc. Microbiol., Bethesda, Maryland, 1972. 34. Levisohn, S., and Aronson, A. I., J. Bacteriol. 93,1023 (1967). 36. Linderstr0m-Lang, K., Hotchkiss, R. D., and Johansen, G., Nature (London) 142, 142 (1938). 36. Losick, R., Shorenstein, R. G., and Sonenshein, A. L., Nature (London) 227, 910 (1971). 37. Losick, R., and Sonenshein, A. L., Nature (London) 224, 35 (1969). 38. Maia, J. C. C , Kerjan, P., and Szulmajster, J., FEBS Lett. 13, 269 (1971). 39. Mandelstam, J., in "Microbial Growth" (P. Meadow and S. J. Pirt, eds.), pp. 377-401. Cambridge Univ. Press, London and New York, 1970. 40. Mandelstam, J., and Waites, W. M., Biochem. J. 109, 793 (1968). 41. Matsubara, H., J. Biochem. 46,107 (1959). 42. May, B. K., and Elliott, W. H., Biochim. Biophys. Acta 157, 607 (1968). 43. McConn, J. W., Tsuru, D., and Yasunobu, K. T., J. Biol. Chem. 239, 3706 (1964). 44· Michel, J., Ann. Inst. Pasteur 111, 14 (1966). 46. Michel, J., and Mulct, J., J. Appl Bacteriol. 33, 220 (1970). 46. Millet, J., Bull. Soc. Chim. Biol. 51, 61 (1969). 47. Mület, J., Bull. Soc. Chim. Biol. 51, 457 (1969). 48. Mület, J., J. Appl. Bacteriol. 33, 207 (1970). 49. Millet, J., C. R. Acad. Sei. Paris 272, 1806 (1971). 60. Mület, J., and Acher, R., Eur. J. Biochem. 9, 456 (1969). 61. Mület, J., Acher, R., and Aubert, J.-P., Biotech. Bioeng. 11, 1233 (1969). 62. Millet, J., and Aubert, J.-P., Ann. Inst. Pasteur 117, 461 (1969). 63. MiUet, J., and Aubert, J.-P., in "Spores V" (H. 0 . Halvorson, R. S. Hanson, and L. L. Campbell, eds.). Amer. Soc. Microbiol., Bethesda, Maryland, 1972. 64. Minamura, N., Yamamoto, T., and Fukumoto, J., Agr. Biol. Chem. 30, 186 (1966). 66. Monro, R. E., Biochem. J. 81, 225 (1961). 66. Morihara, K., Tsuzuki, H., and Oka, T., Biochem. Biophys. Res. Commun. 42, 1000 (1971). 67. Murrell, W. G., Advan. Microbiol Physiol. 1, 133 (1967). 68. Neumark, R., and Citri, Ν., Biochim. Biophys. Acta 59, 749 (1962). 69. Ottesen, M., and Svendsen, L, in "Methods in Enzymology," Vol. X I X (G. E. Perlmann and L. Lorand, eds.), pp. 199-215. Academic Press, New York, 1970. 60. Prestidge, L., Gage, V., and Spizizen, J., / . Bacteriol. 107, 815 (1971).

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61. Ryter, Α., Ann. Inst. Pasteur 108, 40 (1965). 62. Sadoff, H. L., Celikkol, E., and Engelbrecht, H. L., Proc. Nat. Acad. Sei. U. S. 66, 844 (1970). 63. Sadoff, H. L., Hitchens, A. D., and Celikkol, E., J. Bactenol. 98, 1208 (1969). 64. Santo, L. M., Leighton, T. J., and Doi, R. H., Bacteriol. Proc. p. 135 (1971). 65. Schaeffer, P., Folia Microbiol. 12, 291 (1967). 66. Schaeffer, P., Bacteriol. Rev. 33, 48 (1969). 67. Schaeffer, P., lonesco, Η., Ryter, Α., and Balassa, G., Colloq. Int. Centre Nat. Reck. Sei. (Paris) 124, 553 (1963). 68. Schaeffer, P., Millet, J., and Aubert, J.-P., Proc. Nat. Acad. Sei. U. S. 54, 704 (1965). 69. Slapikoff, S., Spitzer, J. L., and Vaccaro, D., J. Bacteriol. 106, 739 (1971). 70. Smith, E. L., DeLange, R. J., Evans, H., Landon, M., and Markland, F. S., J. Biol. Chem. 243, 2184 (1968). 71. Smith, E. L., Markland, F. S., and Glazer, A. N., in "Structure-Function Rela­ tionships of Proteolytic Enzymes" (P. Desnuelle, H. Neurath, and M. Ottesen, eds.), pp. 160-172. Munksgaard, Copenhagen, 1970. 72. Spizizen, J., in "Spores ΠΙ" (L. L. Campbell and H. 0 . Halvorson, eds.), pp. 125-137. American Society for Microbiology, Ann Arbor, Michigan, 1965. 73. Spudich, J. Α., and Kornberg, Α., J. Biol. Chem. 243, 4588 (1968). 74. Spudich, J. Α., and Kornberg, Α., Biol. Chem. 243, 4600 (1968). 76. Sussman, A. J., and Gilvarg, C , Annu. Rev. Biochem. 40, 397 (1971). 76. Tipper, D . J., and Pratt, I., J. Bactenol. 103, 305 (1970). 77. Travers, A. Α., Nature (London) 225, 1009 (1970). 78. Urba, R. C , Biochem. J. 71, 513 (1959). 79. Waites, W. M., Biochem. J. 109, 803 (1968). 80. Warren, S. C , Biochem. J. 109, 811 (1968). 81. Yasunobu, K. T., and McConn, J., in "Methods in Enzymology," Vol. X I X (G. E. Perlmann and L. Lorand, eds.), pp. 569-575. Academic Press, New York, 1970. 82. Young, I, E., and Fitz^ames, P. C , / . Biophys. Biochem. Cytol. 6, 467 (1959). 83. Yugari, Y., and Gilvarg, C , / . Biol. Chem. 240, 4710 (1965).

Regulatory Properties of G l u c o s e - 6 Phosphate D e h y d r o g e n a s e A . BONSIGNORE A . D E FLORA Institute of Biological University of Genoa Genoa, Italy I. Introduction II. General Properties A. Substrate Specificity B. Kinetic Properties III. G6PD from Microorganisms A. Escherichia coli Β. Leuconostoc mesenteroides C. Candida utilis D. Brewer's Yeast (Saccharomyces E. Penidllium dupontii IV. G6PD from Mammalian Cells A. Ovary B. Adrenal Cortex . C. Mammary Gland D . Liver E. Human Erythrocytes V. Concluding Remarks References

carlshergensis)

.

.

.

.

Chemistry

21 23 23 23 24 24 24 25 26 31 31 31 32 34 36 41 56 56

I. Introduction The widespread occurrence of the pentose phosphate pathway in living cells testifies to the importance of this metabolic route, whose primary functions are (a) the production of pentose phosphate for mono-, di-, and polynucleotide synthesis, (b) the formation of N A D P H to be used by the cell in a variety of biosynthetic pathways and reductive processes {72,187). It is now also well established that in most living organisms the syn­ thesis of pentose phosphate is not restricted to the oxidative decarboxyla­ tion of gluconate 6-P, but may be the result of the combined activity of transketolase and transaldolase on different precursors.* The limiting •Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; 6-PgD, gluconate 6-phosphate dehydrogenase; GSH, reduced glutathione; GSSG, oxidized glutathione; G-6-P, glucose 6-phosphate; EDTA, ethylenediaminotetracetate; ACTH, adreno­ corticotropic hormone; Tris, tris(hydroxymethyl)aminomethane. 21

22

Α. BONSIGNORE AND Α. DE FLORA

function in the oxidative shunt is thus the formation of NADPH through the action of G6PD* and G-PgD. Control of G-6-P oxidation appears therefore to be of major importance for modulation of the NADPH-utilizing processes, including synthesis of fatty acids and of other lipids, several hydroxylation reactions involved in the synthesis of steroids, amino acids, and heterocycles, formation of glutamate and of GSH. On the other hand, the NADPH-consuming path­ ways may in turn regulate the flow of G-6-P through the pentose phos­ phate cycle. In various cells the rate-limiting step of NADPH formation, as judged by maximal catalytic capacities of the two dehydrogenases involved, is oxidation of G-6-P to 6-phosphogluconolactone; moreover, the G6PD reaction has been shown to be markedly displaced from equilibrium values in Krebs ascites cells (70) as well as in rat liver cells (69). These findings, together with the key position of G6PD at a branch point in glucose metabolism, suggest that the reaction catalyzed by G6PD may be an important site for control of the pentose phosphate pathway. Con­ sistent with this view is the fact that G6PD from several sources dis­ plays a number of structural and functional properties which are shared with other regulatory enzymes, while some features, although unusual, may represent interesting models for enzymatic regulation. Examples of the former properties are the fluctuations of G6PD activity under differ­ ent hormonal and nutritional states, its quaternary structure, the occur­ rence of afiSnity interactions following the binding of a number of ligands, the inhibition by metabolites (the three last parameters are typically encountered in allosteric proteins). On the other hand, the susceptibility of erythrocyte G6PD to dissociation by several factors (25, 26, 179, 18) provides the molecular basis for specific control of turnover of a protein through a mechanism which is relatively unknown in enzyme regulation (152,153). The purpose of this review is to illustrate the regulatory properties of G6PD from several sources, with particular reference to those cells in which these properties may operate in vivo in the control of carbohydrate metabolism. In addition we will discuss some molecular characteristics of G6PD which may provide a structural basis for the understanding of the properties of the enzyme which are related to important cell functions. For historical background and a full description of the general proper* Among such precursors it has been previously demonstrated that two molecules of fructose 6-P are converted to sedoheptulose 7-P and to xylulose 5-P, respec­ tively, by means of a coupled reaction catalyzed by transketolase and transaldolase [see A. Bonsignore, S. Pontremoli, G. Mangiarotti, A. De Flora, and M. A. Mangiarotti, / . Biol, Chem. 237, 3597 (1962)].

REGULATORY PROPERTIES OF

G6PD

23

ties of G6PD from a variety of sources, the reader is referred to previous detailed reviews {127,137). II. General Properties A . Substrate Specificity G6PD catalyzes the oxidation of D-glucopyranose 6-P to 6-phosphogluconolactone {^2) according to the following equation: /3-D-glucopyranose 6-P -|- NADP+

D-gluconolactone 6-P + N A D P H -h H+

Although the enzyme exhibits strict anomeric specificity, G6PD from a number of sources acts also on analogs of G-6-P like 2-deoxyglucose 6-P, 3-deoxyglucose 6-P, and galactose 6-P {H8, 81, 181) even though at consistently reduced rates and with low aflSnities in comparison with the natural substrate. The coenzyme specificity is rather broad: NAD can in fact replace NADP in the reaction catalyzed by G6PD from different organisms. Olive et al {132) have stressed the occurrence of three different types of G6PD based on their dinucleotide specificity. A first group, such as G6PD from yeast {176), Candida utilis {50), and Escherichia coli {150), is specific for NADP; a second group, including the enzymes from Leuconostoc mesenteroides {132, 47), Pseudomonas aeruginosa {90), Hydrogenomonas Η 16 {9), and Thiohadllus jerroxidans {165), reacts with both NAD and NADP at comparable rates. Finally, a third group of enzymes from most animal sources display maximal activity with NADP and lower and variable activity with NAD and some analogs of both NAD {91) and NADP. B. Kinetic Properties Studies on the kinetic mechanism of G6PD have been seriously limited by the diflSculty of evaluating the reverse reaction (reoxidation of NADPH), due to the marked instability of the δ-gluconolactone 6-P, the half-life of which is 24 minutes at pH 6.4 {65) and 1.5 minutes at pH7.4 {71), Therefore presently available information was derived by studying, under steady-state conditions, the reciprocal effect of either substrate on binding of the other, as well as by exploring the type of inhibition by NADPH, first reported to be competitive toward NADP with yeast G6PD {65). Use of such kinetic approaches suggested that in most cases the reaction catalyzed by G6PD obeys a sequential mechanism with NADP binding first and NADPH being released last {161, 132),

24

Α. BONSIGNORE AND Α. DB FLORA

III. G6PD from Microorganisms Α. Escherichia coli

Some lines of evidence indicate the occurrence of metabolic control at a still unidentified site(s) of the pentose phosphate pathway in E, coli {150). Since this regulation does not involve any significant change in steady-state concentration of any of the crucial enzymes in this pathway, namely G6PD, 6-PgD, transketolase, and transaldolase, two possible mechanisms have been suggested to explain the variable contribution of oxidative and nonoxidative segments to pentose synthesis: (a) a fluctuat­ ing demand for NADPH and (b) modulation by some effectors of one or more enzymes involved. The results obtained by Sanwal {150) on the kinetic properties of G6PD purified from this microorganism could be relevant to the latter mechanism. G6PD extracted from E. coli, besides being activated by Mg^^, sper­ midine, and a number of cations, undergoes a marked inhibition by NADH {150). The patterns of the inhibition are linearly noncompetitive toward G-6-P, while a sigmoidal behavior was found when the reaction velocity was plotted versus increasing NADP at high NADH levels (at low concentrations of NADH the inhibition was linearly noncompetitive as well). Moreover increasing NADH at fixed concentrations of both substrates elicited a sigmoid-shaped response of the inhibition curve. These kinetic data, which are clearly consistent with a multisite binding of the inhibitor on the protein, have been proposed to act as a physio­ logical control mechanism in E. coli {150). Β. Leuconosfoc mesenteroides 1. MOLECULAR PROPERTIES

The enzyme from Leuconostoc mesenteroides has been obtained in homogeneous form by Olive and Levy {130, 131), and some of its molecular parameters were carefully investigated {131). The molecular weight was found to be 103,700, which is very similar to the values found for dimeric G6PD from other sources (see below). The Leuconostoc enzyme has a partial specific volume of 0.717 and an isoelectric point of 4.6. 2. COENZYME SPECIFICITY

G6PD from L. mesenteroides exhibits a dual dinucleotide specificity {132, 47, 130), leading to production of both NADH and NADPH. Analysis of the purified enzyme showed a for NAD of 1.5 X 10"* Μ

REGULATORY PROPERTIES OF G 6 P D

25

and a for NADP being 7.4 X 10"^ Μ, while the ratio between the two catalytic activities, as measured at saturating concentrations of the dinucleotides, was FNAD/^^NADP = 1 . 7 7 - 1 . 8 3 (130), This fact may be re­ lated to the finding that NAD concentrations largely exceed those of NADP in L, mesenteroides (94), therefore possibly compensating for the 20-fold lower affinity displayed by G 6 P D for NAD as compared with that for NADP. It has been shown (79) that the NAD-linked and NADP-linked reactions fulfill two distinctive functional requirements in L. mesenteroides, the former producing NADH which is used for ethanol and lactate production, the latter generating NADPH which is specifically involved in lipid synthesis. 3. KINETIC FEATURES

The kinetic mechanism of the purified enzyme has been the object of a detailed investigation by Olive et al, (132), Their results showed that as far as the NADP-dependent reaction is concerned, a sequential mechanism in which NADP is bound first and NADPH released last, is most likely, although a Theorell-Chance mechanism could not be ex­ cluded. The NAD-dependent reaction shows a partially different behavior as the data obtained are still consistent with sequential mechanism where binding of NAD precedes combination with G - 6 - P but isomerization of the free enzyme appears to be involved. The evidence for an interconversion between two isomeric forms of the enzyme, one binding NAD and the other combining with NADP, as well as with NADPH and NADH, bears an obvious relationship with the dual physiological function of G 6 P D in L, mesenteroides. Thus, the interconversion between the two forms should be a crucial mechanism in switching on and off, alternatively, important cellular processes like reductive biosynthesis, on one hand, and energy-yielding fermentations on the other. No clear-cut evidence is, however, available so far for factors affecting the isomerization step, since the only known conditions eliciting a distinctive response in the NAD-dependent and the NADPdependent reactions, namely the pH optimum and susceptibility to high concentrations of phosphate (130), do not necessarily involve a displace­ ment of the equilibrium between the two above isomeric species. C. Candida utilis 1. GENERAL CONSIDERATIONS

In C. utilis the rather peculiar situation has been observed that four enzymes involved in the pentose phosphate pathway exist as two distinct isoenzymes. This finding was obtained with G 6 P D (59, 51, 54, 56), 6-PgD

26

Α. BONSIGNORE AND Α. DB FLORA

ms)j transketolase (80), and transaldolase [a third isoenzyme of which was shown to be a hybrid of the two primary forms {173, 174)], No clear correlation could be shown so far between the above enzyme hetero­ geneity and control of the pentose phosphate pathway in this organism. 2. CHEMICAL PROPERTIES

As far as G6PD is concerned, a number of structural and functional parameters of the two isoenzymes have been collected by Domagk and co-workers, and this information may reasonably anticipate a detailed understanding of the specific regulatory properties of such isoenzymes. The two enzyme forms are easily separated in the course of the purifica­ tion procedure (59), by fractionation with ammonium sulfate (AS 75 and AS 95, respectively). The two forms have been shown to differ from each other on the basis of a distinctive inactivation by homologous anti­ bodies, fingerprint analysis of tryptic peptides, their behavior in both disc-gel electrophoresis and electrofocusing (51), slightly different in­ activation patterns following photoxidation by rose Bengal (54), sig­ nificant differences in α-helical content (66), and, to a partial extent, amino acid composition {69, 50), Conversely, other properties, such as the apparent molecular weight ( 1 0 4 , 0 0 0 ) , the afiSnity constants for both substrates, and the pH optimum, were found to be quite similar in both isoenzymes. Further work by the same investigators on relationship be­ tween structure and activity of G6PD, was mostly performed with the AS 95 isoenzyme. Preliminary data favor a subunit structure of this isoenzyme {53). Furthermore, on the basis of chemical modifications, the involvement of histidine {54), tyrosine (57), arginine {52), and lysine {55) residues in the catalytic activity has been proposed. 3 . REGULATORY PROPERTIES

A possible regulatory property of G6PD from C. utilis may arise from the susceptibility of both isoenzymes to the inhibition by various nucleo­ tides {59); the inhibition by 5'-AMP appears to be of particular interest since at high concentrations of this modifier the saturation curve relating catalytic activity of the AS 95. species to varying G-6-P concentrations deviates from the hyperbolic shape and becomes sigmoidal {50). D. Brewer's Yeast (Saccharomyces

carlshergensis)

1. PHYSICAL PROPERTIES

Since its preliminary purification by Glaser and Brown {65) and description of some thermodynamic and kinetic properties, G6PD from

REGULATORY PROPERTIES OF

G6PD

27

brewer's yeast was further purified and obtained in a homogeneous form {126, 88, 190). This allowed a careful analysis of the physical properties of this enzyme {190, 191), which were found to be markedly affected by presence or absence of apoenzyme-bound NADP (Table I). The NADP-free G6PD (apoenzyme) shows a close molecular similarity to the dimeric form of human erythrocyte enzyme in that this catalytically active form has a molecular weight of 101,650 and consists of two appar­ ently identical subunits (each with 51,000 MW) {191); however, presence of bound coenzyme in the erythrocyte dimer and complete absence of it in the corresponding yeast species provide a completely different basis for control of structure and catalytic function (see below). The effect of NADP on the yeast apoenzyme has been investigated in detail and found to involve an induced association process yielding a tetramer of MW 205,000-212,000. On the basis of the physical param­ eters reported in Table I, Yue et al. {191) elaborated a model according to which the tetrameric species, represented by the four-subunit NADPenzyme species, should have the shape of a prolate ellipsoid in which the major axis of each individual subunit is parallel to the major axis of the tetramer and perpendicular to the major axis of each half-molecule (i.e., dimer). EDTA has been found to prevent the NADP-induced tetramerization and this effect could be accounted for, in the opinion of Yue et al. {191), by a modification of solvent structure leading to a perturbed interaction between the solvent itself and the protein, rather than by the chelating action of EDTA. The molecular basis for the associating action of NADP remains unclear. It is possible, however, that binding of NADP to the apoprotein modifies the interactions be­ tween hydration water and protein and accordingly leads to a new orientation in the structure of the bound solvent in the resulting tetramer TABLE I PHYSICAL PROPERTIES OF YEAST G6PD« Parameter 820°,w

M..D

f/fo ^20°,app

Apoenzjnne (dimer)

NADP-enzyme (tetramer)

6.14 X 10-13sec (pH6.9) 5.77 X lO-^cmVsec (pH6.9) 101,600 gm/mole 101,700 gm/mole 1.17 (a/b = 3.6) 0.744 (c = 1.90%)

9.6 X 10-» (pH6.8) 4 . 5 X 10-7cmVsec 205,000 gm/mole 212,000 gm/mole 1.19 (a/b = 4.1)

« From R. H. Yue, E. A. Noltmann, and S. A. Kuby, J. Biol. Chem. 244, 1353 (1969), by permission of the American Society of Biological Chemistry.

28

Α. BONSIGNORE AND Α. DE FLORA

(191), Preliminary calculations indicated a maximum binding of 1 mole of NADP per mole of monomer (180), As a result of the apoenzymecoenzyme interactions, the protein may display a marked variability in kinetic parameters which could account for the discrepancies reported by various investigators (see below). 2. THERMODYNAMIC PROPERTIES

Glaser and Brown (65) calculated that the equilibrium constant of the reaction catalyzed by yeast G6PD: ^ , _ (NADPH) (gluconolactone 6-P)(H-^) (NADP+)(G.6-P)

is 1.22 at 28°C, as measured at pH 6.4. From these data, the AF"" was found to be +8600 cal/mole. Moreover, the value of Eo for the reaction glucose 6-phosphate —^ δ-gluconolactone 6-phosphate was determined to be -0.26 V, while ΕΌ = - 0 . 2 8 V at pH 7.0, which is comparable with a calculated value of ΕΌ = -0.32 V for the reduction of NADP at pH 7.0 (33,144)^ 3. REGULATION BY IONS AND METABOLITES

a. Effects of NADPH, Glucosamine β-Ρ, and Various Ions. A number of ions and metabolites have been found to affect the catalytic function of yeast G6PD. Glaser and Brown (6δ) first showed that this enzyme is competitively inhibited by NADPH (toward NADP) and by glucosa­ mine 6-P (toward G-6-P); moreover, they confirmed the earlier finding by Theorell (171) of an inhibitory mechanism exerted by phosphate, which they reported to act in a competitive manner with respect to NADP. Different conclusions were reached by Rutter (147), Passonneau et al. (133), and Anderson et al. (6), based on kinetic results consistent with a competitive mechanism between phosphate and G-6-P. The ex­ planation for such discrepancies and for further conflicting observations concerning the effect of sulfate (111, 133, 5) may be related not only to the interactions of the enzyme with NADP, but also to differences in the assay conditions (i.e., presence or absence of interfering electrolytes, see 111) as well as in the degree of purification of the protein. Yeast G6PD is inhibited by ammonia by a competitive mechanism toward both substrates (111). It has been postulated that this effect may bear some relevance to control of nitrogen metabolism: thus, inhibition of G6PD by ammonia could depress the operation of the pentose phos­ phate pathway, thereby lowering the NADPH-dependent synthesis of glutamate via glutamate dehydrogenase (111). Other ions affecting the activity of the yeast enzyme are and Na+,

REGULATORY PROPERTIES OF

G6PD

29

both reducing the y^ax and decreasing the affinity for G-6-P and NADP (112); 01- leading to activation (112); Mg^^, formerly known as an activator of G6PD (65, 86) and displaying a dual effect, namely en­ hancement of Fraax and a decreased aflSnity for both G-6-P and NADP (113), b. Regulatory effects of Adenine Nucleotides, The effect of adenine nucleotides on yeast G6PD was found independently by Bonsignore et al, (16, 17), by Passonneau et al. (133), and by Avigad (8). The inhibition by ATP appears to be of the fully competitive type (49) with respect to G-6-P (16, 8), and a very good agreement exists between the reported values of K^, namely 5.0X10"* Μ (16) and 5.5 X 10"* Μ (8), both calculated at pH 7.4. ADP and AMP were also found, although to a lower extent, to be slightly inhibitory to the reaction, still by a mecha­ nism fully competitive toward G-6-P (16). The effect of ATP, evaluated at varying concentrations of NADP and at saturating concentrations of G-6-P, fits kinetically the model of the partially competitive inhibition according to Dixon and Webb (49) and is consistent with formation of a ternary substrate-enzyme-inhibitor complex. Further investigations (17) showed, however, that the latter inhibitory effect involves occurrence of homotropic cooperative inter­ actions between more than one ATP-binding site and of heterotropic (36) antagonistic interactions between ATP-binding regions and catalytic site(s) for NADP (Fig. 1). The marked pH-dependence of the ATP inhibition toward NADP is shown in Fig. 2. Preliminary experiments, worked out by sucrose gradient centrifugation, showed that the inhibiting

FIG. 1. Inhibition of yeast glucose-6-phosphate dehydrogenase by ATP. Ο Ο, 4.1X10-''M glucose-6-phosphate (G-6-P), 5 . 8 x 1 0 " * Μ N A D P ; φ 4.1 Χ 10-*Μ G-6-P, 2.9 Χ 1 0 » Μ N A D P ; © C 1.9 Χ 10"»Μ G-6-P, 2.9Χ10Γ''Μ NADP. From Bonsignore et al. {17) by permission of the Italian Biochemical Society.

30

Α. BONSIGNORE AND Α. DE FLORA

FIG. 2. Effect of pH on the inhibition of yeast glucose 6-phosphate dehydrogenase by ATP. From Bonsignore et al. (17) by permission of the ItaUan Biochemical Society.

effect of ATP does not involve any appreciable variation of the S2oo,w of G6PD (A. De Flora, M. A. Mangiarotti, and I. Lorenzoni, unpublished data). So far as the physiological significance of ATP inhibition is concerned, it is of interest that such inhibition was confirmed with G6PD from other sources (5, 193, 30) and that the reported intracellular concentrations of ATP (and also of ADP and AMP) in yeast are of the same order of magnitude as the calculated Ki values [136). In addition, the inhibition by ATP is not prevented or reversed by proteins, and the two effects which are displayed toward G-6-P (competitive) and NADP (allosteric) are independent of each other, therefore enhancing the sensitivity of the mechanism. If the in vivo relevance of the ATP inhibition is assumed, the rather interesting situation ensues that ATP controls both pathways of glucose degradation, namely the glycolytic route at the phosphofructokinase level {139) and the hexose monophosphate shunt by its effect on G6PD. Evidence has recently been presented {31) that ATP may indeed be considered as the most important metabolite regulating glucose metabolism. c. G6PD and Glucose Dehydrogenase Activities. The previous finding that yeast G6PD also displays weak catalytic activity with )S-D-glucose as substrate {148, 88, 41) stimulated Nordlie and co-workers to a de­ tailed comparative investigation of the two activities of this enzyme, namely toward G-6-P and glucose, respectively {73, 6, 5). Their results provided further evidence for the identity of G6PD and glucose de­ hydrogenase in yeast, since some divalent cations (Mg^^, Ca^+, and Ba^^) activated the two activities, whereas a number of modifiers including long-chain fatty acyl-CoA compounds, Co^^, ATP, and other nucleoside

REGULATORY PROPERTIES OF

G6PD

31

5'-triphosphates and diphosphates inhibited the activity with both sub­ strates to the same extent and through similar kinetic mechanisms. Despite the foregoing results, providing unequivocal evidence for one protein being involved in both activities with G-6i-P and glucose, certain anions (phosphate, sulfate, and bicarbonate) were found to display dis­ tinctive effects on these activities: thus, the glucose dehydrogenase activity was significantly stimulated, while that on G-6-P was inhibited by the three above anions, under comparable assay conditions (5), through a competitive mechanism toward G-6-P and noncompetitive effects toward NADP (6), Nordlie and co-workers have elaborated a model, based on the induced-fit concept (87), according to which a single binding site common to G-6-P, anions, and inhibiting nucleotides might account for the aforementioned effects on the catalytic activities with G-6-P and with glucose (73,6). An understanding of the possible regulatory significance of the dis­ tinctive effects of phosphate, sulfate, and bicarbonate on the two cata­ lytic activities of yeast G6PD, still awaits further experiments on the enzyme from additional sources, which have been initiated by Nordlie and co-workers (74) on rat liver hexose phosphate dehydrogenase (see below). E. Penidllium

dupontii

G6PD from the thermophilic fungus Penidllium dupontii displays some interesting kinetic features. The purified enzyme shows sigmoidal saturation curves for both substrates G-6-P and NADP (32). Hill plots yielded η values of 1.61 when the variable substrate was G-6-P and of 1.08 when NADP was varied at saturating G-6-P concentrations. Occur­ rence of positive interactions between sets of binding sites for both sub­ strates may result in the amplification of the catalytic function of G6PD according to fluctuations of intracellular levels of G-6-P and NADP, this representing a potentially important and rather unusual regulatory mechanism. IV. G6PD from Mammalian Cells A . Ovary Limited information is at present available on the physical properties of G6PD from the ovary. The enzyme partially purified from bovine corpus luteum has been investigated particularly with respect to its susceptibility to be inhibited by a number of steroid hormones, a property that the ovarian enzyme shares with G6PD from other mammalian cells

32

Α. BONSIGNORE AND Α. DE FLORA

(116, 124). The inhibition experiments showed that out of a variety of compounds tested, estradiol-17-α, estradiol-17-j5, and pregnenolone are uncompetitive inhibitors of this enzyme with respect to NADP (124). These effects appear, however, to lack physiological significance on the basis of several considerations quoted by Nielson and Warren (124), the most important of which is a consistently lower concentration of the steroids in the ovary than that producing inhibition in vitro. More interesting, as concerns its in vivo regulation, could be the report that ovarian G6PD shows cyclic variations of activity during the estrous cycle (with lowest values at diestrus and highest at estrus (104)). More­ over, ovarian G6PD has been found to be stimulated by some gonado­ tropins both in vivo and in vitro, and these results suggested a tentative model for the action of FSH on the inmiature ovary (104). It has been reported that G6PD partially purified from corpus luteum loses most of its catalytic activity when it is washed with ammonium sulfate according to the procedure developed by Kirkman (81) to remove the apoenzyme-bound NADP from the human erythrocyte enzyme (124) · This fact supports the view, highly important from a standpoint of regu­ lation (see section on erythrocyte G6PD), that the ovarian enzyme con­ tains some "structural" coenzyme playing an essential role in maintain­ ing its normal structure and catalytic function. B. Adrenal Cortex The activity of G6PD in the adrenal cortex seems to fulfill an impor­ tant requirement for the physiological functions of this gland, such as the rate of steroidogenesis (102) and also the growth and cell replication of the adrenals (105). This role and additional findings (see below) stimulated a number of investigations on both the molecular and regula­ tory properties of adrenal G6PD. 1. PURIFICATION AND PROPERTIES

Two methods have been described for the crystallization of G6PD from bovine adrenal cortex (43, 162), and final preparations obtained by both procedures fulfill current criteria of homogeneity. Some minor discrepancies have been found for the physical properties of the crystal­ line enzyme, since Squire and Sykes found an S2o«,w of 9.47 S and a MW of 190,000 (162) slightly different from the values of 9.8 S and 235,000238,000, respectively, reported by Criss and McKerns (43). The purified enzyme is exceedingly susceptible to inactivation by ascorbate even at concentration levels several orders of magnitude lower than those in the adrenals (151). A protection against the ascorbateinduced inactivation is afforded by albumin in the presence of NADP,

REGULATORY PROPERTIES OF

G6PD

33

as well as by other still unidentified substances found in the adrenal extract, most probably proteins. Additional experiments concerning in­ activation of adrenal G6PD following an extensive dialysis and reactiva­ tion by NADP and jS-mercaptoethanol, point to the similarity with the erythrocyte enzyme and are consistent with presence of bound NADP involved in stabilizing the enzyme in an active conformation (Ιδί), Adrenal G6PD contains 32 cysteine residues per enzyme molecule (106). Moreover, two cysteine residues, most probably involved in the binding of NADP, are essential to catalytic activitv. 2. REGULATION

McKerns (105) has developed a model, tentatively accounting for the action of ACTH at the molecular level, which is based essentially on the previous finding that this hormone is an activator of adrenal G6PD (103). The McKerns model implies that binding of ACTH to the enzyme occurs through hydrogen bonding between charged amino groups at the amino end and c-amino groups of the lysine residues of the hormone to specific groups on G6PD. Experiments on the homogeneous enzyme showed that binding of ACTH results in a rapid exposure of two essential cysteine residues. Since these sulfhydryl groups appear to be required for NADP binding, it has been proposed that the ACTH-G6PD interaction results in a con­ formational change of the structure of the enzyme and ultimately in a facilitated binding of NADP at its own catalytic site (106). This view provided a partial explanation for the previous report of a significant decrease of values toward NADP and G-6-P following the interaction of ACTH with the purified enzyme (44) · Although the hexose phosphate shunt has a remarkably high potential activity within the adrenals (105, 101), its catalytic operation is limited in vivo by the intracellular levels of NADP and G-6-P (103). Therefore the higher affinity of G6PD to­ ward both substrates as a specific consequence of the hormone-enzyme interaction could be responsible for an accelerated flow of G-6-P through the hexose phosphate shunt. The enhanced rate of NADPH production would stimulate those enzyme systems specifically requiring this com­ pound and particularly those concerned with steroid synthesis and fatty acid synthesis in the adrenal gland itself. The involvement of the foregoing effects in determining the specific response of the adrenal cortex to the specific ACTH stimulus is, however, far from being demonstrated with certainty. One of the most important observations militating against the McKerns model is the finding ob­ tained with adrenal cells in culture, according to which ACTH exerts its stimulatory effect on steroid production without penetrating the target

34

Α. BONSIGNORE AND Α. DB FLORA

cell (164)' Further work is therefore necessary to assess the in vivo role of the activation of G6PD by A C T H and to shed light on those mecha­ nisms mediating the action of this hypophysial hormone on the adrenal cortex. C. Mammary Gland G6PD from the mammary gland displays typical regulatory properties, as revealed by its changes of activity during lactation and by occurrence of an active interconversion among multiple molecular forms. Whether a correlation exists between the two facts is, however, still unknown. 1. FLUCTUATIONS OF ACTIVITY DURING THE LACTATION CYCLE

Like G6PD from ovary and liver, the enzyme from mammary gland shows wide variations of activity which are clearly related in the latter organ to the lactation cycle. Thus, Glock and McLean (66) found that levels of both G6PD and 6-PgD activities increase rapidly in rat mam­ mary gland from the end of pregnancy to the end of lactation, this in­ crease being approximately 60-fold and reaching the highest levels observed in mammalian cells for G6PD; finally involution of the pa­ renchymal tissue is accompanied by a sharp decrease to levels still lower than those found in pregnancy. This interesting finding stimulated further research, the results of which indicated a significant parallelism between operation of the hexose monophosphate shunt and the above changes of G6PD and 6-PgD activities (107, 108). It became evident that a close correlation occurs in lactating mammary gland between the activity of the pentose phosphate pathway and synthesis of fatty acids (1, 34), a mutual regulation existing between the two processes (109). Such a marked variability of G6PD patterns suggests the involvement of some specific regulatory mechanisms, possibly hormone dependent, affecting the turnover of this enzyme protein as well as of 6-PgD. Barry and co-workers (89, 68) have approached the problem by investigating the effect of some hormones and inhibitors of DNA, RNA, and protein synthesis on cultures of mammary tissue from mice. Cultured cells from mammary tissue of pregnant mice were shown to reproduce the increases of G6PD activity observed in the living animal at parturition. This rise of activity was attributed to the enhanced uptake of glucose, occurring both in the mammary gland and in cultures of mammary tissue, which follows the disappearance of placental lactogen, namely of a factor dis­ playing an anti-insulin action (89). The greater uptake of glucose by the mammary tissue would in turn result in an increased concentration of a still unidentified product of glucose metabolism, stimulating the synthesis of specific mRNA for both G6PD and 6-PgD and therefore leading to an increased rate of synthesis of these enzymes. The data

REGULATORY PROPERTIES OP

G6PD

35

obtained point to an efifective control of G6PD synthesis, which could be either induced or derepressed according to the model of Green et al {68), rather than of degradation of this enzyme. Consistent with this view is the absence of apoenzyme-bound N A D P , which is thought to be an essential requirement for the initiation of degradation processes of G6PD within other mammalian cells, such as the erythrocytes (see below). 2. MOLECULAR, KINETIC, AND REGULATORY PROPERTIES

G6PD was first crystallized from bovine mammary gland by Julian et al (77), who obtained an overall 10,000-fold purification. The enzyme has also been purified from the mammary gland of lactating rats by Levy (92), whose work elucidated another mechanism of G6PD regula­ tion within this tissue. The mammary enzyme has been shown to exhibit a dual coenzyme specificity, since N A D can replace N A D P in the G6PD-catalyzed reac­ tion. The high Km values for N A D in comparison with those calculated for N A D P (1.5 X 10"^ Μ and 8.9 X 10"« M, respectively) are, however, consistent with the view that the NAD-dependent dehydrogenation of G-6-P is of only minor importance in the mammary gland (92). Similarly to the G-6-P and glucose dehydrogenase activities of yeast G6PD (5, 6) the NAD-linked and NADP-linked reactions catalyzed by the mammary enzyme are distinctively affected by various reagents (92). The reactions with the two dinucleotides showed further significant differences in the pH optimum, a strong pH dependence being observed in presence of N A D while the NADP-linked activity is relatively in­ sensitive to pH changes. The mammary enzyme contains no bound N A D P (92), a rather unusual property for G6PD in contrast to other mammalian sources such as human erythrocytes, adrenal cortex, ovary, and, most probably, liver. A theoretically important analogy with the yeast enzyme, which does not require any bound coenzyme for catalytic activity (191), is the association of two dimeric units (with a molecular weight approximating 130,000) to yield a tetramer. This aggregation is specifically determined by N A D P , provided that the protein concentration is sufficiently high (122). On the other hand, the reversible dissociation of the active, N A D P free dimer to inactive monomers ( M W 63,000)* was reported to follow • A t variance with Levy and associates (122, 93, 123), the term "monomer" is here referred to the 63,300 MW species, rather than to the form of 130,000 gm/mole, just to adopt a common nomenclature for G6PD from various sources (yeast, erythrocytes). N o evidence has in fact been presented so far for identity or nonidentity of the 63,000 MW subunits; the same reason has prompted us to modify the terminology used by Yue et al. (191) with yeast G6PD.

36

Α. BONSIGNORE AND Α. DB FLORA

incubation overnight with 0.04 Μ Tris, pH 9.1, containing 7 mAf ^-mercaptoethanol (122), Further experiments suggested the following model (93): Monomer ;=± dimer X (NAD)

dimer Y ^ tetramer (NADP)

where dimer X and dimer Y are assumed to be the catalytically active forms of G6PD (no information is available, however, on the activity of the tetramer). The NADP-dependent activity is higher for Y than for X, an opposite situation being observed with the NAD-linked activity, which is greater for X than for Y. This model unifies in a single con­ cept the above-mentioned differences in activities with NADP and NAD, respectively, as well as the factors modifying the equilibrium between the two activities. Moreover it provides a reasonable explanation for the selective inhibition of the NADP-dependent activity by dehydroepiandrosterone (91) in terms of an exclusive binding of this steroid to the Y species. A detailed investigation on the effects of o-phenanthroline and of two analogs of it, phenanthridine and m-phenanthroline, on mammary G6PD, provided evidence for a conformational change of the whole protein being involved in the reversible transition of dimer Y to dimer X (123), Nevaldine and Levy (123) postulated on a kinetic basis the presence of different and partially interacting sites for the binding of the two dinucleotides on dimer X and Y. The availability of these sites to inter­ action with NAD and NADP has been suggested to provide the struc­ tural counterpart of the equilibrium between the two dimeric forms (123), which is affected by a number of reagents and conditions. In this connection, it is important to recall (see Section III, B) that quite re­ cently Levy and co-workers provided kinetic evidence for isomerization between two different forms of free enzyme in the course of the NADlinked reaction catalyzed by G6PD from Leuconostoc mesenteroides (132). D . Liver 1. GENERAL CONSIDERATIONS

In mammalian liver the oxidation of G-6-P is catalyzed by at least two proteins, which were shown to differ with respect to the following parameters: (a) subcellular localization, the first being located in the soluble fraction while the second is present in the microsomal fraction (12); (b) genetic inheritance, the soluble enzyme being controlled by an X-linked gene whereas the microsomal protein is autosomal (156,157, 129); (c) specificity toward dinucleotides and substrates other than

REGULATORY PROPERTIES OF

G6PD

37

G-6-P, which is consistently broader for the microsomal, autosomal en­ zyme, hence designated as "hexose phosphate dehydrogenase" (12, 110), in contrast to the soluble G6PD. The microsomal, autosomally inherited enzyme is most likely to be identical with the glucose dehydrogenase previously purified and characterized by Metzger et al (118), The presence of a third enzyme catalyzing dehydrogenation of G-6-P cannot be excluded with certainty, although the possibility exists that the liver particulate G6PD described by Zaheer et al (194) is identical with hexose phosphate dehydrogenase (12), 2. HEXOSE PHOSPHATE DEHYDROGENASE

Hexose phosphate dehydrogenase has received particular attention by Beutler and co-workers in an effort to define its physiological role and its properties (12, 110, 163, 164)- These investigations have shown that the enzyme is present in liver as well as in other organs (163), The liver enzyme oxidizes G-6-P, galactose 6-P, 2-deoxyglucose 6-P, and glucose, using either NAD and NADP as coenzymes (12), In contrast to G6PD (Ιδδ), hexose phosphate dehydrogenase is not inhibited by any of the several steroids tested (110), Furthermore its levels of activity were found to be unaffected by both starvation and refeeding with various carbohydrate diets (110), a finding which suggests that changes of soluble G6PD account for the well known variations of glucose 6-P dehydro­ genase activity which follow several nutritional conditions. A critical appraisal of both the substrate and dinucleotide specificity of the hexose phosphate dehydrogenase as well as of its relative aflSnity constants led Mandula et al (110) to postulate that the microsomal enzyme physiologically operates as a G6PD, perhaps fulfilling the role of generating NADPH. This view contrasts with the conclusion reached by Home and Nordlie (74), who found a considerable activation of the glucose dehydrogenase activity by physiological concentrations of vari­ ous anions and therefore postulated, in agreement with previous sugges­ tions by Metzger et al (118, 119), an involvement of this enzyme in the metabolism of free glucose. It is clear, however, that a definitive assess­ ment of the in vivo role of the autosomal enzyme still awaits a detailed evaluation of its molecular properties and their comparison to those of the soluble, sex-linked G6PD. 3. G6PD a. Induction, Several observations have indicated that liver glucose 6-P dehydrogenase activity is under long-term regulation and that various dietary states result in changes of this activity. The previously men­ tioned invariability of hexose phosphate dehydrogenase levels following

38

Α. BONSIGNORE AND Α. DB FLORA

starvation and refeeding with a number of diets (110) points to G6PD, which is the sex-linked enzyme, as the control site of these nutritional parameters. Glock and McLean (67) first reported a marked reduction of both dehydrogenases of the hexose monophosphate shunt in livers of starved rats. This finding was confirmed and extended by other investigators, who observed that refeeding of starved animals led to a characteristic and peculiar "overshoot" of G6PD activity (169, 170, 61, 62, 125, 2, 76, 128). A 3-fold increase, as compared with untreated animals, was ob­ served when a balanced diet was employed; the increase of G6PD ac­ tivity was still more pronounced (from 10- to 12-fold) if rats were fed with a high carbohydrate diet, a maximum rise being observed after refeeding with a fructose-containing diet {61, 62). The administration of a high fat diet following starvation does not induce recovery of G6PD activity {125) and, similarly, no increase of this enzymatic activity could be observed on refeeding with a diet consisting solely of carbohydrates {125, 138, 135). A number of questions arose from the foregoing observations, concern­ ing especially the nature of the molecular mechanisms which elicit such responses to the starvation and to dietary stimuli. A first problem, con­ cerning the nature of the increase in G6PD activity after refeeding, received partial explanation from experiments in which the effects of various inhibitors of protein synthesis on this phenomenon were examined. The results of these studies were consistent with the view that a de novo synthesis of G6PD occurs on refeeding with high carbohydrate diets {170, 2,138, 78), whereas activation of a preexisting precursor seems to account for the observed increase of activity following refeeding of starved ani­ mals with a balanced diet {2). Strictly related to this problem is the question whether the de novo synthesized enzyme shows any structural or functional difference in comparison with native G6PD: this question is so far unanswered, although no substantial differences with respect to some parameters emerged from the investigations of Kagawa et al. {78) on the properties of the enzymes from unfed and refed rats. The evidence for a synthesis of G6PD following high carbohydrate diets stimulated Johnson and Sassoon {76) to investigate which dietary component is specifically triggering the increase of G6PD, and although they where unable to find carbohydrate playing the role of "actual effector of induction," their results indicated that the site at which in­ duction of G6PD occurs is probably the transcription level. Since glucose is known to result in the release of insulin from the pancreas {11), Rudack et al. {1^6) examined whether this specific mechanism could be responsible for the increased synthesis of G6PD, but

REGULATORY PROPERTIES OP

G6PD

39

their investigation indicated that this is not the case, although insulin remains a likely candidate as a primary inducer for other enzymes (177). Furthermore, the mechanisms whereby increase of liver G6PD occurs, were analyzed in terms of rates of synthesis and degradation of this pro­ tein in vivo, and this procedure showed that high carbohydrate diets result in the acceleration of both phases of G6PD turnover. In particular, a 73-fold increase was recorded for the rate of G6PD synthesis following high carbohydrate refeeding of fasted rats, in comparison with values found in animals fed a balanced diet; conversely the corresponding in­ crease of the first-order rate constant of degradation was found to be 4.6-fold (146), A further question concerns the physiological meaning of the specific "overshoot" of G6PD activity in refed animals, namely, its possible cor­ relations to other metabolic responses elicited in liver by refeeding itself. It soon became apparent that a marked enhancement of lipid synthesis parallels the diet-induced increases of G6PD activity (169, 170) and that a correlation exists between the two events is confirmed by the recent finding that refeeding is followed by a sharp decrease of the NADPH/NADP quotient (69), Following these observations, two opposite views were expressed to account for such a correlation between the enhanced activity of the shunt enzymes and the increased rate of lipogenesis: these alternative hypotheses [reviewed in Tepperman and Tepperman (170)] were de­ signed as the "push" hypothesis (according to which the increased rate of fatty acid synthesis is a consequence of the greater availability of NADPH following the increase of the shunt enzymes) and the "pull" hypothesis (relating the rise of G6PD and, perhaps of 6PgD activities to the enhanced rate of lipogenesis, therefore considered as the primary event.) Recently, evidence has been provided in support of the "pull" hypothesis by Taketa et al, (167), who showed that.the induced levels of G6PD are not necessarily required to bring out the increased produc­ tion of NADPH under conditions of adaptive hyperlipogenesis. According to their findings, the increased synthesis of fatty acids may rather depend on an increased activity of additional enzymes catalyzing the carbonchain supplying reactions in lipogenesis, such as malic enzyme, citrate cleavage enzyme, and glucokinase (167). Therefore the induction of G6PD by the high carbohydrate diet appears to be secondary to the enhanced lipogenesis and the physiological significance of this induction in the economy of liver metabolism remains obscure. h, Inactivation of G6PD, Although the changes of G6PD activity fol­ lowing a number of nutritional states seem to depend primarily on a modified rate of G6PD synthesis, it has also been demonstrated that

40

Α. BONSIGNORE AND Α. DB FLORA

variations in both extent and rate of specific degradation of this enzyme protein contribute to the phenomenon (146). Schimke (1δ2, 153) has emphasized the general lack of knowledge on those mechanisms con­ trolling degradation of specific enzyme proteins. Despite this general fact, some lines of evidence suggest that specific degradation of G6PD in a number of mammalian cells is initiated by factors acting at the level of apoenzyme-bound NADP which in several cases is known to play a struc­ ture-stabilizing effect. Although direct spectrofluorometric evidences, such as those reported for the erythrocyte enzyme (81), are still lack­ ing for liver G6PD, presence of bound coenzyme and its involvement in maintenance of this protein in (a) functionally active structure (s) are suggested by (a) the antagonistic effect of NADP against the fatty acidinduced disaggregation of crystalline liver G6PD (192), (b) by the sharp inactivation of this enzyme by NADP-glycohydrolase (60, 18) and NADP-pyrophosphatase (18) and by the specific protecting effect displayed by NADP on this inactivation. Three different G6PD-inactivating proteins were shown in rat liver cells (19): (a) NADP-glycohydrolase, which is present in the microsomal fraction, (b) NADP-pyrophosphatase, located in the soluble cytoplasm, (c) a third soluble inactivating protein, not identifiable with a protease, which is devoid of activity on free NADP, although its inactivating effect on G6PD is prevented and reversed by NADP itself. The inactivating action of the foregoing factors was investigated, using erythrocyte G6PD as substrate, in order to explore more carefully the kinetic properties of the individual proteins. The combined action of these proteins on the liver enzyme is, however, apparent from the marked loss of G6PD activity occurring in dialyzed liver extracts (18, 19). The in vivo relevance of the three inactivating proteins to G6PD catabolism is still undefined: it is, however, possible that the primary attack by the three above-named proteins at the level of enzyme-bound NADP results in a destabilized structure of G6PD (probably, but not necessarily, subunits) being more accessible than the native enzyme to subsequent proteolytic digestion. c. Structural and Regulatory Properties. Rat liver G6PD was crystal­ lized by Matsuda and Yugari (117), who investigated some of the molecular properties of the homogeneous enzyme (192). Active G6PD is a tetramer of 110,000 MW as evaluated by ultracentrifugal analysis. On gel filtration, the tetramer is dissociated to two active dimeric units of 57,000-60,000 MW. Further disaggregation to inactive monomers (28,000 MW) occurs on incubating the active enzyme in presence of free fatty acids. NADP specifically antagonizes the inactivating effect exerted by fatty acids, and this relationship appears to be of a competitive type

REGULATORY PROPERTIES OF

G6PD

41

(192). ATP and other nucleotide triphosphates were found to facilitate the inactivation by fatty acids, although showing no inhibition per se (192): the latter finding contrasts with earlier results by Avigad (8), who described inhibition of rat liver G6PD by ATP. This discrepancy is likely to be due to the different extent of purification of the enzyme. At variance with the inhibitory effect of long-chain acyl-CoA esters on rat liver G6PD (58, 166), the physiological significance of which was excluded on the basis of a complete lack of specificity of the inhibi­ tion patterns (166), the inactivation of this enzyme by free fatty acids could play a role in vivo in view of the known correlation between the hexose monophosphate shunt (generating NADPH) and fatty acid syn­ thesis (consuming NADPH). In this connection, free fatty acids may be considered as negative feedback inhibitors for lipogenesis (192). E. Human Erythrocytes 1. ROLE OF THE HEXOSE PHOSPHATE SHUNT IN ERYTHROCYTE METABOLISM

The mature erythrocyte depends almost exclusively on glucose as its main source of energy. G-6-P formed by the hexokinase reaction is mostly metabolized through the Embden-Meyerhof pathway leading to reduction of NAD and to synthesis of ATP. Under normal conditions, from 5 % to 10% of G-6-P is channeled through the hexose phosphate shunt, thereby providing NADPH which is specifically involved in reduc­ ing GSSG via glutathione reductase and hence in maintaining a number of structural and functional proteins in an active state. GSH is also effec­ tive, by means of glutathione peroxidase, as a detoxifying agent, and this function is achieved through reduction of the hydrogen peroxide that may be formed within the erythrocyte especially in presence of some hemolyzing drugs (13), even under physiological conditions. Therefore the NADPH-producing enzymes, G6PD and 6-PgD, are ultimately con­ cerned, by way of the glutathione reductase system, with the protection of the erythrocyte against those structural damages produced by specific drugs, or by some metabolites thereof or, more generally, by any oxidative agent possibly being responsible for the hemolysis (see below). 2.

GENETIC POLYMORPHISM OF

G6PD

Due to the peculiar metabolism of the mature red cell, control of the hexose phosphate shunt and particularly of G6PD activity appears to be of primary importance in determining the life-span of the erythrocyte, despite the fact that this pathway represents only a minor route of G-6-P metabolism. Additional reasons, however, exist which account for

42

Α. BONSIGNORE AND Α. DE FLORA

the increasing interest of investigators from various fields in studying the properties of erythrocyte G6PD. Among these, the wide genetic poly­ morphism of this enzyme deserves consideration, as more than 50 variants have been so far detected (13, 75, 121) on the basis of several criteria which have been standardized since 1967 by the World Health Organization (178). This number, which by no means appears to be final, places erythrocyte G6PD as the most genetically heterogeneous mammalian enzyme (188), while among other mammalian proteins, only hemoglobin is known to exhibit a wider polymorphism. Like the soluble, more specific liver enzyme, also erythrocyte G6PD is controlled by the X chromosome (83): available evidence suggests that a single locus on the X chromosome is involved in G6PD synthesis, as the subunits of the enzyme appear to be identical on the basis of several criteria (181, 187, 14, 182, 25). The structural gene for G6PD is probably located close to the genes for color blindness of protan and deutan types (3, 158) and for hemophilia A and Β (159). From the X-linked mode of inheritance of G6PD, the situation ensues that males are always hemizygous, whereas females can be homozygous or heterozygous with respect to the G6PD trait. Inactivation of one of the two X-chromosomes of each female cell according to the "Lyon hypothesis (100)" is responsible for the identity of G6PD levels between normal hemizygous males and homozygous females [see Teplitz and Beutler (168) for a detailed review]. In all cases of G6PD polymorphism, the genetic change is assumed to be a structural mutation leading to an abnormal enzyme protein, since no clear evidence is available for nonstructural mutations affecting either the rate of synthesis or the rate of destruction of G6PD. On the other hand, most G6PD variants can be shown to differ from the normal wildtype enzyme (designated as "B") on the basis of parameters other than simply a modified catalytic activity (75, 178) which provides less valuable information on both the nature and mechanism of the genetic change. Besides the screening of new G6PD variants, which represents a par­ ticularly diflScult task due to the requirement of perfectly standardized criteria (178), the most important problem is concerned with the cor­ relation between structural mutation and possible alterations of the in vivo function of the enzyme (i.e., catalytic activity, susceptibility to effectors, etc.). With regard to this, it has been recently shown, for instance, that the A" variant (found in Negro subjects and known as a "deficient" variant on the basis of in vitro measurement of activity) exhibits a distinctive pattern of interaction with NADP and NADPH with respect to the catalytically "normal" variants A and Β (4). Such

REGULATORY PROPERTIES OP

G6PD

43

peculiar kinetic behavior was found to affect in vivo the metabolism of G-6-P by erythrocytes in such a way and to such extent as to com­ pensate for the defect of activity detected in vitro and to account for the normal metabolism and life span of the A" cells (99). An additional question concerns the nature of the structural mutation resulting in an altered enzyme protein. This problem was approached by Yoshida with purified homogeneous forms of the single variants, by means of standard analytical microscale procedures and particularly by fingerprint analysis of the tryptic and chymotryptic digests of the proteins. Two G6PD variants, A (associated with normal catalytic activity) and Hektoen (characterized by a 4-fold higher activity with respect to the Β type), were analyzed by Yoshida. He found in both cases that a single amino acid substitution—from asparagine in the Β type to aspartic acid in the A variant (188, 186) and from histidine in the Β type to tyrosine in the G6PD Hektoen [188) represented the only structural difference between the normal and variant enzymes, as the result of a single-step base transition in the G6PD structural gene. Most probably, also the structural mutation leading to the synthesis of the A" variant is a single amino acid substitution, as indicated by the analysis of the tryptic peptides from the Β type and the A" variant which revealed the presence of a single different peptide in the two electrophoretic maps (R. Cancedda and L. Luzzatto, personal communication). The genetic polymorphism of G6PD, leading to a "natural" change of its protein structure and to corresponding variations of functional parameters, may provide a profitable tool for a better understanding of both mechanism of action and regulation of this enzyme. The previ­ ously reported indications of some G6PD variants in which the mutation affects specifically the aflSnity for NADP and NADPH and of a much more effective utilization of NADP by the A" variant with respect to other G6PD types (4), prove the validity of this kind of approach. 3. G6PD

DEFICIENCY

a. General Considerations, Some of the known G6PD variants are associated with decreased catalytic activity. The above-mentioned view that in each female cell only one of the two loci responsible for G6PD deficiency is genetically active accounts for the finding in heterozygous females of levels of G6PD activity ranging between those of normal and of affected hemizygous males (obviously in the latter case the enzymatic activity is severely reduced). The degree of expression of the G6PD deficiency trait in heterozygous females is directly related to the mosaic of erythrocytes, some of which have normal catalytic activity and others have conversely a markedly impaired activity. Evidence for such a pheno-

44

Α. BONSIGNORE AND Α. DE FLORA

typic heterogeneity was provided by means of appropriate histochemical techniques {149, 172), b. Molecular Basis for G6PD Deficiency, On theoretical grounds the genetically determined G6PD deficiency could be accounted for by each of the following facts: (a) a lower rate of G6PD synthesis, (b) a more rapid degradation, (c) synthesis of a protein having an intrinsically low catalytic activity, (d) combination of these situations. A quite opposite situation, namely the previously mentioned G6PD Hektoen, associated with some 4-fold increases of activity, has been found to be due to an increased rate of synthesis of this structural variant (48), As concerns the A" variant, the structural mutation (95, 186, 184) does not appear to result in an intrinsically "deficient" enzyme protein, as shown by the specific activity of homogeneous preparations of this variant which approximates that of the normally active types, Β and A (R. Cancedda, 0. Babalola, and L. Luzzatto, personal communication). Therefore, the in vitro finding of G6PD deficiency associated to the A" variant, could be ascribed to an accelerated degradation of the mutant enzyme rather than to a decreased rate of production {184, 186), Confirmatory evidence for this view was provided by experiments of Piomelli et al, {134), who found a half-life of 13 days for G6PD from A" erythrocytes as com­ pared with 62 days for G6PD from normal cells. Only circumstantial evidence is available for mechanisms leading to G6PD deficiency with other variants, due to incomplete extent of puri­ fication. For instance, it has been reported that the B" (or Mediterranean) variant oxidizes G-6-P and 2 deoxyglucose 6-P at about the same rate, by contrast with the Β type which transforms this analog at a rate of about one-tenth as compared with the activity on G-6-P {84). The abovemodified specificity might reflect a structural modification responsible for the decreased activity with G-6-P as substrate, yet additional variations in the rate of synthesis and more probably of degradation {84) may also be involved. Indeed, the decay of G6PD activity in B" erythrocytes was found to be even more rapid than in the case of A~ cells {134), therefore pointing to an extreme in vivo instability of the mutant enzyme as the main determinant of the defect. For detailed reviews on the heterogeneity of G6PD deficiency the reader is referred to Marks {116), to Kirkman et al, {85), to Beutler {13), and to Fornaini and Bossü {64). c. Hematological Effects, As concerns the clinical effects of the G6PD defect, a marked variability has been observed. The life-span of the erythrocytes in subjects affected by G6PD deficiency may be normal or slightly reduced and accordingly no important clinical effects are ob­ served, due to eventual compensation from the bone marrow. However, a number of factors [see {13) and {178)], including a variety of drugs

REGULATORY PROPERTIES OF

G6PD

45

and exposure to fava beans, may determine in the G6PD-deficient sub­ jects several clinical manifestations, the most critical of which is the acute hemolytic anemia. Mechanisms correlating the effect of the above agents to the hemolysis remain to be elucidated. A most general hypothesis tends to explain those effects with an "oxidative stress" somehow induced by these agents and challenging the erythrocyte to a NADPH production which can presumably be fulfilled by normal cells but not by the deficient ones. This view received support by experiments in which normal (15, 145, 29, 27, 99), as well as deficient (28, 29, 99), red cells were incubated with methylene blue. Under these particular conditions, operation of the hexose phosphate shunt is increased to more than 10-fold levels in normal erythrocytes (15, 145, 27, 29, 99), and a much more limited stimulation was observed with G6PD-deficient cells, amounting to nearly 3-fold in the case of A" erythrocytes (99) and to 1.6-fold with B" erythrocytes (28, 29). These findings are obviously related to the differences between normal and deficient cells as to their content of active G6PD; further­ more, they account for a distinctive NADPH production under condi­ tions of "oxidative stress," which appears to correlate through still un­ known mechanisms to the different resistance of the erythrocyte structure to hemolyzing agents. Recently, evidence has been presented for an increased resistance against malarial infection being conferred by G6PD deficiency to hetero­ zygous females. This situation, accounting for the geographical correla­ tion between high frequency of the G6PD deficiency trait and high malarial endemicity (120,178),wsiS conclusively rationalized by Luzzatto et al (98), who showed a significantly higher presence of the parasite (Plasmodium falciparum) in normally active erythrocytes than in G6PD deficient cells. 4. G6PD

AND AGING OF THE ERYTHROCYTES

An additional reason for current interest in the structural and regula­ tory properties of G6PD is the striking correlation between decrease of erythrocyte G6PD activity and aging of the cell. This phenomenon, first suggested by Marks et al (114) j was subsequently confirmed and studied by a number of investigators in the hope of shedding light on mech­ anisms of physiological and pathological hemolysis. We refer to Marks (116) and to Fornaini (63) for detailed reviews of this topic. 5. MOLECULAR WEIGHT AND SUBUNIT STRUCTURE

Two discrete oligomeric species of human erythrocyte G6PD, type B, have been demonstrated, corresponding to tetramers (205,000-210,000 MW) and dimers (101,000-105,000 MW), respectively (38, 39, I40, 24,

46

Α. BONSIGNORE AND Α. DE FLORA

25, 179). Both species are catalytically active while the isolated subunits, consisting of a single polypeptide chain of nearly 50,000 MW, show no intrinsic activity, although they can be reactivated to various extents and rates under a number of experimental conditions. As previ­ ously mentioned, a number of lines of evidence point to the identity or to a close similarity between the subunits, although the final proof of this must necessarily await the complete sequence.* The demonstration of two oligomeric G6PD species, both displaying catalytic activity, provided an explanation for the previous observation of two bands staining for G6PD activity after electrophoresis of the par­ tially purified enzyme on Polyacrylamide gel [Jß): furthermore, the multiple-banded electrophoretic patterns obtained with less purified preparations could be ascribed to formation of mixed disulfides between SH groups of both tetramers and dimers and of some contaminating proteins (23). As far as the geometry of G6PD is concerned, no definitive conclusions are possible regarding the type of the contacts between two individual monomers within the dimeric forms. Results obtained by Cohen and Rosemeyer (39) would suggest, however, in good agreement with an earlier hypothesis (37), that presence of apoenzyme-bound (also desig­ nated "structural") NADP stabilizes hydrophobic interactions between subunits. Association of two dimeric units to form the tetramer occurs through interactions which are mainly affected by pH and ionic strength (38, 39, 24) y as well as by divalent cations (24, 179, 26), and which appear there­ fore to be quite dissimilar from those involved in the monomer-monomer contacts. These data suggest that the tetramer has two different planes of symmetry, at least from a chemical standpoint (39): NADP is thought to stabilize the contacts, perhaps hydrophobic, across a first plane, while a second one, determining the reversible dissociation to dimers, is probably maintained by ionic bonds. All attempts to dissociate the tetramer to dimers across the first plane of symmetry were so far unsuc­ cessful (A. De Flora, I. Lorenzoni, and R. Cancedda, unpublished data), and this suggests a possible cooperative mechanism by means of which dissociation of the first plane (i.e., that where structural NADP is invoh''ed) results in the consequent dissociation across the second one, therefore leading to a direct disaggregation to the single subunits. This view appears to be supported by the dissociation to monomers following treatment with mercuribenzoate (A. De Flora, unpublished data). • T h i s is the reason why the subunits of erythrocyte G6PD are here referred to as "monomers" rather than "protomers," the latter designation implying the unequivocal assessment of identity.

REGULATORY PROPERTIES OP

G6PD

47

TABLE

II

PHYSICAL PROPERTIES OP HUMAN ERYTHROCYTE G6PD« Molecular species 8200 .w ( 1 0 - " sec)

Stokes radius (lO^^ cm) DMO.W ( 1 0 - 7 cmVsec)

Molecular weight^.o Molecular weight SDS-enzyme

///o

Buoyant density (gm/ml)

Monomer 4.04 30.7

Dimer 5.75 40.7

6.95

5.26

52,900

101,400

Tetramer 9.18 51.5 4.15 204,800

49,700

—

1.27

1.33

1.328

1.344

—

—

—

1· The strong inhibition of homoserine dehydrogenase (55) and the weak or nonexistant inhibition of homoserine kinase (54) by threonine in this organism is consistent with the observation of reduced methionine levels. Recently the same physiological phenomenon has been reported in Bacillus subtilis by Vapnek and Greer (86).

BEHAVIOR OF INTACT BIOCHEMICAL CONTROL SYSTEMS

101

IV. O n the Significance of Feedforward Inhibition in α Biosynthetic Pathway The dominant themes in the regulation of biosynthetic pathways are feedback control by end-product inhibition (83, 95) and repression ( ^ ) . Since the discovery of these mechanisms in the 1950's, their presence has been well documented in practically every biosynthetic pathway that has since been examined (47, la, 84). Some of the functions of these mechanisms, such as their ability to maintain a relatively constant supply of the end product, have been intuitively obvious from the time of their first discovery. This is partly because of their analogy to, and our previous experience with, technological control systems (83), The more rigorous mathematical analysis of biosynthetic pathways con­ trolled by alternative patterns of feedback inhibition that was given in Sections II and III has confirmed this intuitive understanding as well as revealed additional, less obvious, features of such systems that are beneficial for the organism. In this section I will examine a novel control mechanism that has recently been discovered in the arginine biosynthetic pathway of several microorganisms (52, 78, 96), This mechanism consists of a ieedforward inhibition of the arginyl-tRNA synthetase by argininosuccinate, the im­ mediate precursor of arginine. Although the existence of this phenomenon has been clearly established, its significance has yet to be elucidated. When applied to this question the techniques outlined in Section II pro­ vide provocative results. These results, presented in this section, suggest that feedforward inhibition serves to amplify the changes in concentra­ tion of critical metabolites that act as signals to effect modifications in the system. In this way the overall control of the pathway is improved. The order of presentation will be the following. First, the effects of feedforward inhibition per se will be examined. Second, its operation in concert with the well-known feedbacfc inhibition mechanism will be analyzed. Finally, predictions of the consequences of this mechanism for the physiology of the organism are discussed in relation to the ex­ perimental observations that have been reported in the literature. Por­ tions of the material in this section have been presented elsewhere (71), Feedforward Inhibition per se For this analysis consider only feedforward inhibition in the biosyn­ thetic sequence from the intermediate citrulline to the end-product arginyl-tRNA as shown in Fig. 6. It is evident intuitively that the response of this system to changes in the steady-state value of the A.

102

MICHAEL Α. SAVAGEAU

Citrulllne —• Argininosuccinate —•Arginine —^Arginyl -t-RNA-

•X2

^ X 3 -

F i G . 6, A model for the arginine biosynthetic pathway of Chlamydomonas reinhardi from citrulline to arginyl-tRNA. Argininosuccinate inhibits the arginyltRNA synthetase and Xa represents the exogenous concentration of arginine.

exogenous concentration of arginine, Xs, is unaltered by the presence of the feedforward inhibition. In other words, the response to such a change in this system and in an internally equivalent system without feedforward inhibition is identical. The same conclusion cannot be reached if we consider the responses to a change in X i , the concentra­ tion of citrulline. In general, an increase in the steady-state concentration of citrulline, X i , will lead to an increase in argininosuccinate, X2. This in turn increases the level of arginine X3, which tends to increase arginine utilization, but also leads to an increased inhibition of arginine utiliza­ tion. It is not immediately obvious which of these two effects will pre­ dominate or to what degree the concentration of arginyl-tRNA, X4, will be affected. What is obvious is that the behavior may be quite different from what we would expect in the equivalent system without feed­ forward inhibition. To go beyond these intuitive ideas I will analyze the system in a more rigorous manner. By utilizing the mathematical approach illus­ trated in Section II, one can write the following set of steady-state equations which describe the system in Fig. 6: 62 - ^212/1 = -A222/2 63 64

gsbVB

=

(öf82 -

= A322/2 +

^32)2/2 -

hszVz

hzzyz -

Ä442/4

(68)

As previously described in detail, the 6 parameters are related to the ratio of apparent rate constants for the reaction producing a given metabolite and the reaction utilizing the same metabolite; the g's and A's represent the apparent kinetic order of the synthetic and degradative reactions with respect to the various modifiers and reactants involved with each reaction; the y^s represent the logarithms of the corresponding concentration variables. Solving for the dependent variables y2, 1/3, and y4 in terms of the independent variables yi and 2/5, we find

BKHAVIOR OF INTACT BIOCHEMICAL CONTROL SYSTEMS

_ gayi -

103

hi

hn ^'

hi _

g2iyi

*

hu

— &2 gz2 ^

A22

^^^^

g^y^ — fcs — &4

Λ44

A44

First we ask what the percent change in Z 2 , -X3, and Z4 will be in this system corresponding to a 1% change in the exogenous concentration of arginine, X^, This is obtained from Eqs. (69) by taking the partial derivatives of y^, 2/3, and 2/4 with respect to y^: ^ 2 5 = θΐ/2/θ!/δ = 0 ί/35 = dyz/dy^ = L45 = dyjdy^

> 0

(70)

= öf35/A44 > 0

These equations indicate that there is no change in X2] Xz always in­ creases, as does X^ for an increase in Ζ5· These results follow because all of the parameters in these equations are positive. /ΐ32 represents the apparent kinetic order of the arginyl-tRNA synthetase reaction, the reaction utilizing X3, with respect to changes in argininosuccinate, X2. It is the only apparent kinetic order in this system that is normally nega­ tive, i.e., Ä32 < 0. The internally equivalent system without feedforward inhibition is described by the same equations except that Ä32 must be set to zero. When this is done we find that 1/25

= L^,

L35 =

and

Li6,

L45 =

(71)

where the primed symbols denote the case without feedforward inhibition. Therefore we conclude that the resulting changes in X2, Xz, and XA are identical for the system with feedforward inhibition and the internally equivalent system without this mechanism. This result confirms the intuitive explanation given earlier in this section. The other perturbation that was mentioned earlier in this section, and for which the response was not obvious, can be analyzed by the same approach. In this case the responses to a change in the steady state level of citrulline, X i , are described by the following partial derivatives:

£.,-^'-pai^>0 öyi J

Λ22

Λ33

_ ^2/4 flf21 g32

(72)

104

MICHAEL Α. SAVAGEAU

Thus, all the metabolite concentrations increase to a new steady state value in response to an increase in the steady-state level of citrulline, Jfi.* The same expressions for the case without feedforward inhibition are obtained by simply setting Ä32 = 0. Taking the ratio of corresponding expressions in the two cases yields -ί'2ΐ/-ί'2ΐ ~

1

L3i/L^i = 1 -

(hM

> 1

(73)

= 1 where again the prime denotes the case without feedforward inhibition. From the results in Eqs. (73) it is evident that only arginine, X g , behaves differently in the case with feedforward inhibition compared to the case without it. In general the changes in X3 are magnified by the presence of this mechanism. From the analysis to this point we can conclude that a function of feedforward inhibition is to amplify the changes in the concentration of the metabolite immediately preceding the inhibited step. Other than this fact, no discernible advantage seems to accrue to an organism having suih a mechanism. For this mechanism to be of any use to the organism it must function, and derive its advantage, in intimate relation with other cellular control mechanisms, since feedforward inhibition per se appears to be of little value. I should emphasize that this situation is in contrast to ieedback inhibition, for which we can ascribe an advan­ tage to the mechanism per se (Section II). B. Feedforward Inhibition in Concert with Other Control Mechanisms Consider feedforward inhibition, together with the well known feed­ back inhibition by arginine of the first physiologically important step in this pathway (87, 89, 26). Again we ask how the presence of feedforward inhibition affects the performance of the system in comparison to an internally equivalent system without this mechanism. The system is represented in Fig. 7 together with a more symbolic version. The qualitative effect of an increase in the exogenous arginine con­ centration, X5, can be easily discerned in the system of Fig. 7 and in one lacking the feedforward mechanism. Such a change causes the internal concentration of arginine, Z3, to increase and this leads to an increased production of arginyl-tRNA, X^. The elevation of X3 also inhibits an •Although the details will not be presented, one can show that the system in Fig. 6 is completely stable in the dynamic sense. If the system is slightly per­ turbed from its normal steady-state operating conditions, it will return to these conditions without oscillations in any of the metabolite concentrations.

BEHAVIOR OF INTACT BIOCHEMICAL CONTROL SYSTEMS

105

F _J

^X^

FIG. 7. A model for the arginine biosynthetic pathway of Chlamydomonas reinhardi. Argininosuccinate inhibits the arginyl-tRNA synthetase, and arginine inhibits the utilization of iV-acetylglutamate. Xs and Xe represent the exogenous concentrations of arginine and citrulline, respectively. The first five reactions in this pathway have been lumped together for simplicity. This procedure in no way affects the validity of the analysis or the results described in the text.

early step, leading to a depletion of argininosuccinate, X g , and thus to the de-inhibition of arginyl-tRNA synthetase. This de-inhibition of the synthetase activity together with the elevated levels of arginine results in the augmented production of arginyl-tRNA. In the internally equivalent system without feedforward inhibition there would be no de-inhibition of the synthetase activity and the stimulation of arginyl-tRNA production would be correspondingly less. The effects due to a modification in the other independent concen­ tration variables are not so easily discernible. The comparison of these effects in systems with and without feedforward inhibition is even more difficult because quantitative considerations are involved. To uncover these effects we must proceed with a systematic analysis as was done in Section IV, A. The steady state equations describing the system in Fig. 7 are written in the logarithmic notation as follows: bi -

gioyo -

bi bs -

giaye

= =

gz5yb

=

-hnyi hnyi

+ —

(gz2 -

gim

Ä222/2 ^32)2/2 -

. hszyz

= A322/2 + hzzyz - A442/4 The symbols in these equations have the same meaning that was pre­ sented in the preceding section. All of the g and h parameters are nor­ mally positive with the exception of Λ32 and gis] these represent the apparent kinetic order of reactions with respect to the concentrations of inhibitors and are therefore negative. These equations may be solved bi

106

MICHAEL Α. SAVAGEAU

for the dependent concentration variables 2/1, 2/2, t/3, and 2/4 as functions of the independent concentration variables 2/0, t/5, and ye. The analysis is so similar to that in the previous section that I will not write out the solutions in detail; instead, I will go directly to the study of the varia­ tions in the system's concentrations. The responses in the dependent concentration variables for a change in the steady-state level of exogenous arginine, Z 5 , are given by the following expressions: Lis = dyi/dys

= gizgz5h22hii/A




0

where Δ = ÄHA22Ä33A44 + AiiA44öf 13(^32 - 0^32) > 0. As expected, Z i and X2 decrease in response to an increase in X 5 , whereas ^ 3 and X4, increase.* The internally equivalent system without feedforward inhibition is described by the same set of equations except with A32 set to zero. Recall that A32 is the apparent kinetic order of the synthetase reaction with respect to argininosuccinate, and this is zero without an inhibition by argininosuccinate. Taking the ratio of the changes in the system of Fig. 7 to the changes in the system without the feedforward inhibition yields: UJL[,

< 1

Lzb/V^ < 1 U,/L[, > 1

^^^^

The primed symbols represent the case without feedforward inhibition. The magnitudes of the changes produced are always less in the system with feedforward inhibition, except in the case of the end-product arginyl-tRNA, which always experiences a greater magnitude of change in this system. These results confirm the intuitive explanation given earlier: the presence of feedforward inhibition tends to cause a more efficient funneling of the exogenous arginine into the arginyl-tRNA pool. In other words, this mechanism allows the organism to make more efiicient use of the environmentally supplied, preformed metabolite. Consider now the more diflScult cases involving changes in the other independent concentration variables Xo and Xq. The responses in the •There are conditions on the molecular parameter values sufficient to ensure the stability of these systems, and these conditions will be assumed in the re­ mainder of this section.

BEHAVIOR OF INTACT BIOCHEMICAL CONTROL SYSTEMS

107

dependent concentration variables for a change in the exogenous citrulline concentration, Ze, are given by the following expressions Lu

= dyi/dyt

i/26

=

dy^/dyt

= giJk^JiisfiAA/^ > 0

= öfieAuAssWA > 0

Lee = dyz/dy^ = SfieÄii/i44(g82 -

Α32)/Δ > Ο

Lifi = dyi/dyfi = öfieö'32ÄnWA > Ο

The results from these equations imply that all the dependent concen­ tration variables from citrulline on increase in response to an increase in the exogenous level of citrulline, X e . The comparison with the in­ ternally equivalent system lacking feedforward inhibition is achieved by setting Λ32 = 0 and taking ratios of the corresponding expressions as in the previous cases. The results of these operations are Li,/LU

< 1

L^&IV^ < 1 L M > 1 L M < 1

. ^^^^

These results demonstrate that the magnitudes of the changes in all the intermediates from citrulline on are always greater in the system without feedforward inhibition, except in the instance of arginine, X3, where the magnitude of change is less. Very similar results can be demonstrated for the response to an increase in the initial substrate concentration, X o , All the intermediate concen­ trations increase, and all of them increase to a greater extent in the system without feedforward inhibition with the exception of arginine, which increases to a lesser extent. Thus, the concentrations in the sys­ tem possessing feedforward inhibition are less responsive to changes in the level of the initial substrate which might in turn result from per­ turbations elsewhere in the cellular metabolic network. This comparative analysis of the system in Fig. 7, and its internal equivalent without feedforward inhibition, gives a clue to the functional significance of the magnified arginine response that was noted in the preceding section. The magnified response in the arginine level, X3, causes this metabolite to act as a more effective feedback signal in the overall control system. Thus, the end-product arginyl-tRNA is less responsive to changes in substrate or precursor concentrations as a result of the feedforward mechanism. Another way of stating this is to say the end product is "buffered" to a greater extent with respect to these variations by the presence of this mechanism. These results follow from this analysis, and they are independent of the particular parameter

108

MICHAEL Α. SAVAGEAU

values that might obtain in particular systems of the form shown in Fig. 7. C. Physiological Implications The growth rate of Chlamydomonas reinhardi is reduced by the addi­ tion of excess ornithine or citrulline, and the discovery that argininosuc­ cinate inhibits the arginyl-tRNA synthetase activity in this organism has lead Sussenbach and Strijkert (78) to propose a model as an ex­ planation for this growth-retarding effect. Thus, data obtained at the molecular level have led to the formulation of a mechanism that intui­ tively appears to provide an explanation for the observed phenomena in the intact cell. Although their model includes repression of the single enzyme argininosuccinase with arginyl-tRNA as corepressor, one can show that this mechanism in their model has no effect on the qualitative behavior of arginyl-tRNA in response to the addition of citrulline. Their model is therefore essentially equivalent to that in Fig. 2 for the purposes of this discussion. However, the detailed analysis of this model has shown that the concentration of arginyl-tRNA must increase in response to an addition of ornithine or citrulline. This result is inconsistent with the experimental growth behavior, and we must conclude that such a model is inadequate. This case illustrates one of the important uses of biochemical systems analysis. Given a molecular model believed to account for a behavior pattern in the intact system, it is possible to perform a detailed analysis and determine whether or not it is in fact capable of producing the ob­ served system behavior. This is a powerful technique for eliminating unsuitable models from consideration. If the model in Fig. 7 is inappropriate, what type of model might provide an adequate explanation of the observed growth behavior? One important feature of arginine metabolism lacking in the model of Fig. 7 is an alternative metabolic fate for arginine, e.g., the alternative fate represented by the arginase reaction. I will briefly consider the analysis of the model in Fig. 8, which is similar to the model in Fig. 7 except for the inclusion of an alternative reaction for arginine. Omitting the detailed steps in the analysis and going directly to the most important result, one finds the change in arginyl-tRNA concentration, X4, resulting from the addition of exogenous citrulline, -Ye, is given by ^

dya

Ä44(A22A33

+fifl8Ä32-

Öfl30f32)

The denominator of this expression is always positive since all the parameter values are positive, with the exception of 0Ί3, ^42, and Λ32.

BEHAVIOR OP INTACT BIOCHEMICAL CONTROL SYSTEMS

109

X,

Τ FIG. 8. A model for the arginine biosynthetic pathway of Chlamydomonas reinhardi. This model is exactly the same as the one in Fig. 7 except for the in­ clusion of an alternative fate for arginine represented by the reaction catalyzed by the enzyme arginase.

The numerator, on the other hand, can be either positive or negative depending on the particular values of the parameters involved. A con­ straint on the allowable values of these parameters is necessary if a single type of qualitative behavior is to be obtained from this model. If, for instance, gz2 >

hi

—

{hzz/g^)g42

(80)

then 1/46 = dVi/dya

>

0

That is, if the apparent kinetic order for the rate of arginine production with respect to argininosuccinate is sufficiently large, then the concen­ tration of arginyl-tRNA will always increase in response to an increase in exogenous citrulline. However, if gz2
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174

τ . RAMASARMA

3 . LOCALIZATION OP THE PRIMARY SITE OP CONTROL

There appears to be general agreement that the primary site of control lies between acetyl-CoA and mevalonate. Several workers were able to show greater changes—inhibition or stimulation—in the incorporation of acetate-"C into cholesterol as compared with that of mevalonate-2-i*C. In such experiments, in many cases, it could be shown that incorporation of acetate-^*C into ketone bodies and fatty acids remained unchanged or increased to a small extent. These facts were interpreted as indicative of the site of action being before mevalonate and after HMG-CoA. These changes were shown to occur when tested in vivo or in vitro with slices or homogenates as well as by assaying the activity of the enzyme, HMG-CoA reductase, under a number of experimental condi­ tions known to alter the biogenesis of cholesterol. 4. VALIDITY OP EXPERIMENTS

In most of the work using incorporation of acetate-^^C and mevalonate2-^*C into cholesterol, the incorporation rates were equated to the syn­ thetic rates. It has remained a vexing problem whether this would reveal the true picture. White and Rudney {1δ7) raised objection to the use of incorporation rates for the study of alterations in synthesis. They pointed out that the "changes in incorporation into products will reflect altered synthesis only if specific activity of precursors remain unchanged" (157). This will be true when the pool size of the precursors remains unaltered. Although some investigators have checked the simultaneous incorporation of acetate-^*C into fatty acids and into ketone bodies, other products derived from the intermediates before mevalonate, and found these to be unaltered, even this was not regarded as conclusive because "increased entry of unlabeled substrate at pre-mevalonic site" would give just the same picture while the synthetic rate may not have altered at all. The alternative would be to study activity of the rate-limiting en­ zymes. The activity of HMG-CoA reductase showed corresponding changes, but the assay of this enzyme has not been standardized, and different authors report different specific activities. Furthermore, it is not clear whether the full activity of the enzyme as assayed in vitro is ex­ pressed in the cell. Under some conditions another enzyme, e.g., HMGCoA synthetase, may indeed play the role of rate-limiting step (158). Therefore, conclusions as to the regulatory role of an enzyme based on its properties in vitro must be corroborated by studies in vivo before its physiological significance can be accepted. Additional evidence was obtained that the cholesterol biogenesis was indeed altered by the study of incorporation of Ή from water into

CONTROL OF BIOGENESIS OF ISOPRENOID COMPOUNDS

175

cholesterol which showed changes comparable to the incorporation of acetate (9). After an extensive series of experiments, White and Rudney (158) also came to the conclusion that in conditions of fasting, choles­ terol feeding, and Triton administration, "alterations in incorporation were paralleled by alterations in the same direction in synthesis" and not due to alterations in pools of the intermediates—a conclusion that previous workers had assumed implicitly. In the study of alterations in biogenesis of bile acids and steroid hor­ mones no such careful study has been attempted so far. The diflSculties encountered in the use of lipid substrates such as cholesterol with iso­ lated enzyme systems further complicates the interpretations. B. Conditions of Altered Cholesterol Biogenesis Three types of alterations of biogenesis of cholesterol in the liver are known: (I) large decrease, with about 5% residual activity; (II) small decrease, not usually exceeding 50% of the initial activity; (III) severalfold increase in activity. Some of the typical cases are listed in Table I. In all cases the effect appears to be at the level of HMG-CoA-mevalonate. Thus, each of the three types appears to produce a different re­ sponse on the enzyme, HMG-CoA reductase, only one form of which is known to exist. This enzyme is located in microsomal fraction and ac­ counts for most of the synthesis of mevalonate although an insignificant fraction may be formed by the soluble, malonyl-CoA-dependent system described by Brodie et al (24); this will not be considered further. Cholesterol synthesized by the acetate-mevalonate pathway in the liver is used for the structure requirements in the liver cells, the produc­ tion of bile acids and for export as part of the low density lipoprotein to other tissues via the blood. It will be of interest to understand the changes obtained in the concentration of cholesterol and cholesterol prod­ ucts as a consequence of the control of biogenesis and the enzymes in the pathways. In view of the implication of cholesterol in the etiology of atherosclerosis, control of its endogenous synthesis has assumed great importance. Some of the conditions which involve the natural regulatory mechanisms are discussed below. 1. CHOLESTEROL FEEDING

The effects of cholesterol feeding on the endogenous hepatic synthesis of cholesterol were extensively studied and the results were analyzed in the excellent review by Siperstein (134)- Therefore only the salient fea­ tures are discussed here.

III

II

I

Type

Midnight vs noon Actinomycin D

Triton-WR 1339

X-irradiation

Decreased to 50% (96) Decreased to

Ubiquinone (1.5 mg/day) Estrogens

(33)

Increased severalfold

(10, 37, 1S7, 144)

Increased about 3-fold

(25, 46)

Increased severalfold

(5S)

Increased severalfold

50% (110)

Decreased to 50% (9) Decreased (125)

5% (S5)

Decreased to less than 5% (13) Decreased to less than 5% (153) Decreased to less than

Increased about 5-fold

-

(10,1S8)

Increased severalfold

-

No change (144)

-

-

No change (96)

-

Increased (33)

No change (144)

Increased (4-9)

Decreased (1, 20)

Decreased (96)

Decreased

-

No change (144)

-

Increased over 2-fold (94-) Increased over 2-fold (76) Increased over 2-fold (96)

Decreased (14-9)

No change

I

No change (1#), Decreased about decreases 50% (77) after days

No change (93)

No change (95)

Ubiquinone in the liver

Decreased (77)

No change (76)

Decreased (110)

(158)

Increased due to absorption

Increased due to absorption No change (93)

-

Serum

Liver

Cholesterol

Effect on concentration of cholesterol and ubiquinone

-

Decreased (156)

Decreased (lSl)

Decreased (S5)

Decreased (4-3)

Decreased (138)

Decreased to less than

5% (S5)

HMG-CoA reductase

Acetate-14C ~ cholesterol, % initial activity

CPIB 0.5%, diet Thyrotoxicosis

Starvation

Cholesterol, 1% diet Cholic acid, 0.5% diet ~ 4-Cholestenone

Condition, supplement

Effect on biogenesis and HMG-CoA reductase

TABLE I CONDITIONS OF ALTERED CHOLESTEROL BIOGENESIS

~

~ ~ > r.Jl ~ ~ >

~

~

CONTROL OF BIOGENESIS OF ISOPRENOID COMPOUNDS

177

a. Decrease in Cholesterol Biogenesis by Dietary Cholesterol It was recognized that cholesterol feeding for short periods depressed markedly the incorporation acetate-^*C into hepatic cholesterol (51). This was con­ firmed by a number of investigators (25, 47, 55, 95, 188,147, 152). It was also found that such changes were observed in intact animals when the tracers were injected {54, 95) or with tissue slices and homogenates in vitro {25, 54, 95, 138). It was conjectured that this represented a feed­ back mechanism, in view of the simultaneous increase in hepatic choles­ terol concentration, with the inhibited step conforming to the accepted pattern of the unique, irreversible and rate-limiting reaction. b. Addition in Vitro. When a cholesterol suspension or solution in pro­ pylene glycol was added to a system synthesizing cholesterol from acetate-^^C in vitro, either with liver slices or liver homogenates, no inhibi­ tion was observed {133). Attempts to show the inhibitory effect with artificial emulsions of cholesterol have also failed {133). It was consid­ ered possible that a metabolic product obtained from cholesterol, or a lipoprotein-cholesterol complex, and not cholesterol itself, may be acting as the feedback inhibitor {136). c. Intracellular Localization of the Site of Inhibition. It was found that biogenesis of cholesterol from acetate-^^C required both micro­ somal fraction and soluble supernatant. The effect was located primarily in the microsomal fraction {25) and the soluble supernatant provided the enzyme system to generate HMG-CoA. Starting from HMG-CoA as the substrate, it could be shown that its reduction to mevalonate, localized in the microsomal fraction, was markedly affected {103). d. Concentration and Time of Feeding of Dietary Cholesterol Diets mixed with cholesterol at a level of 0.5-5% have been used. The concen­ tration of liver cholesterol increased following ingestion and the inhibi­ tion of biogenesis of cholesterol can be correlated inversely with the hepatic concentration {47). However, Siperstein and Guest {139) re­ ported that in some of their experiments inhibition was noticed prior to the appearance of detectable elevation in the concentration of hepatic cholesterol, although the local concentration at the appropriate site may have increased. The effects were obtained more rapidly with higher concentrations of dietary cholesterol; while it required several days at the 1% level, ef­ fects could be seen in hours with the 5% level, with respect to concen­ tration, biogenesis from acetate of cholesterol and HMG-CoA reductase activity {128). With cholesterol-containing chylomicrons the effect was obtained 2.5 hours after an intravenous injection {136). e. Occurrence in Different Species. It is now found that regulation of biogenesis of cholesterol by dietary cholesterol occurs in several species

178

τ . RAMASARMA

of animals, birds, rodents, dogs, and primates; this regulatory mechanism appears to be a basic component of the capacity for cholesterol synthesis in the liver (134). The existence of feedback control of cholesterol biosynthesis in the man was questioned by Taylor et al (14^). However, using biopsy samples of human liver, Bhattathiry and Siperstein (16) could demonstrate that dietary cholesterol depressed acetate-^*C incorporation into cholesterol. Ahrens and co-workers (57, 116) developed methods for sterol balance by which changes in synthesis, absorption, and excretion of cholesterol in the man were studied. It was found that feeding cholesterol to normocholesterolemic subjects expanded the body pool without any detectable change in the body synthesis of cholesterol. However, feedback control was demonstrable as compensatory increase in biogenesis of cholesterol under conditions wherein absorption of endogenous and exogenous choles­ terol was prevented by feeding plant sterols. /. Specificity of the Liver Tissue. The effect was obtained primarily in the liver. Because the liver was considered to be the sole source of cir­ culating cholesterol (72) and the absorbed exogenous cholesterol accumu­ lated in the liver, this supported the hypothesis of feedback regulation. Other tissues tested were found to be unaffected by cholesterol feeding. Dietschy and Siperstein (35) obtained evidence that intestinal biogenesis of cholesterol was inhibited by bile, but not by cholesterol. g. Absence in Hepatoma. The elegant studies of Siperstein and co­ workers (134y 136) demonstrated that the inhibition obtained by dietary cholesterol was specifically deleted in hepatoma. This was shown in sev­ eral types of hepatomas in the rat (135), mouse (136), and man (137). The loss was localized to the tumor tissue and was not observed in the host liver. Also, it was characteristic of the malignancy and not merely of growth, since regenerating liver showed the inhibitory effect similar to normal liver (134). The inhibitory effect was present in the embryonic liver, in the neonatal stage and throughout the life cycle of the rat al­ though the degree of inhibition decreased with age (93). This provided the first evidence of a loss of regulatory mechanism in a malignant tissue. h. Environmental Factors. During acclimatization, an animal exposed to low environmental temperature directs its metabolism toward genera­ tion of extra heat instead of synthetic reactions. In a study testing for alterations in regulatory mechanisms in cold-exposed rats, it was found that the inhibitory effect by dietary cholesterol was not at all affected (2). Most of the experiments on the effects of dietary cholesterol were pre­ sumably carried out during the day at which time biogenesis of choles­ terol was the lowest. It is known that biogenesis increased severalfold at midnight due to diurnal rhythm (10, 88). Notwithstanding this enormous

CONTROL OF BIOGENESIS OF ISOPRENOID COMPOUNDS

179

increase in the biogenetic activity, excess cholesterol elicited a similar degree of inhibition at midnight (144)2. A*-CHOLESTENONE

Supplementation with Δ^-cholestenone was shown to depress cholesterol synthesis (25, 142, IBS). The results were similar to those obtained with dietary cholesterol. However, it is improbable that A*-cholestenone was the active regulatory metabolite under physiological conditions be­ cause the concentration required was high and toxic. 3 . BILE, BILE ACIDS, AND CHOLESTYRAMINE

It was recognized that removal of bile from enterohepatic circulation by bile fistula {14, 48, 58, 111) or by sequestering with cholestyramine {73, 148) increased the biogenesis of hepatic cholesterol severalfold. The bile acids, as metabolites of cholesterol produced in the liver, can be considered as the products of isoprene synthesis and potential inhibitors of the feedback type. Dietary supplementation of 0.5% cholic acid inhibited hepatic cholesterolgenesis markedly when tested with slices in vitro {43) or in vivo {12, 13) with acetate-^^C as the tracer. Mevalonate-2-^*C incorporation was only marginally decreased. These effects were identical with that of dietary cholesterol. Comparable inhibition was obtained with bile {113) and chelate, deoxychelate, taurocholate, and deoxytaurocholate {43) on addition to the liver homogenate system synthesizing cholesterol from acetate-^^C. This experimental approach was criticized because the ef­ fects may simply be due to the detergent action of the added compounds. However, detergents like Triton X - 1 0 0 did not show the inhibition at the level of homogenates {48), tissue slices or the whole animal {93), Hamprecht et al, {65) further found that HMG-CoA reductase in the microsomes was inhibited when chelate, deoxycholate, chenodeoxycholate, and taurine conjugates were added in vitro and feeding cholic acid also reduced the activity {63), Ogilvie and Kaplan {113) found that a major part of the inhibitory activity of the bile was associated with a protein factor. Migicovsky {107) reported isolation of an electrodialyzable peptide component from rat liver mitochondria capable of acting as an inhibitor of biogenesis of cholesterol. The evaluation of the eflScacy of bile, bile acids, or other components in regulation of hepatic cholesterol genesis must await fur­ ther experiments. 4. STARVATION AND REFEEDING

Deprivation of food for short periods of 1 - 2 days {25, 107, 108, 125, 161) suppressed the biogenesis of cholesterol from acetate-"C leaving

180

τ . RAMASARMA

only about 5 % of residual activity. Longer periods of starvation pro­ duced still further decrease in the incorporation of acetate-^*C, and also that of mevalonate-2-^*C ( 7 7 ) , into cholesterol. The defect was found to be localized in the microsomal fraction (25, 158). The amount of protein in the liver decreased under starvation, and this decrease was uniformly distributed in all the cell components, not specifically in microsomal fraction ( 7 7 ) . The observed decrease in enzyme activity was not, there­ fore, due to decrease in the microsomal content, but due to a loss in their capacity. The site of inhibition was shown to be HMG-CoA reductase by assay­ ing the enzyme {103, 121, 158). The specific activity of the enzyme de­ creased in microsomal fractions prepared from starved rats. It was con­ cluded that this was due to either decreased activity or content of the enzyme, not to the presence of any inhibitors in the preparations (103). The activity decreased very rapidly with a loss of 5 0 % of the initial activity in about 3 hours. On refeeding, the activity returned equally rapidly to the original level (121). The above decrease in starvation was not due to the lack of any par­ ticular component of the diet. Refeeding of starved animals with protein, carbohydrate, or fat increased the biogenetic activity (151). In another type of experiment, it was shown that deficiency of protein, carbohydrate, or fat in the diet had no infiuence on the biogenetic activity. These results suggest that caloric intake of any form will maintain endogenous bio­ genesis of cholesterol. 5. THYROXINE STATUS

The relationship between thyroxine status and biogenesis and concen­ tration of cholesterol had been a controversial subject. Hyperthyroidism was shown to stimulate hepatic cholesterol biogenesis tested by the incorporation of hydrogen from water (27, 89) or acetate-^*C (44) and also the activity of HMG-CoA reductase (56, 58). These experi­ ments were done in hypothyroid rats supplemented with thyroxine or triiodothyronine. In contrast to these findings, cholesterol biogenesis from acetate-^^C by liver slices decreased in the thyrotoxic condition obtained by dietary supplementing of thyroxine (0.018%, w/w) (125) and increased in the hypothyroid condition, obtained by treatment with iodine-^^^ in vivo (47) · The inverse relationship of thyroxine status and cholesterol had long been recognized. Thyroidectomy was shown to be accompanied by an elevation of concentration of serum cholesterol and of jS-lipoprotein, and this seemed to be due to decreased conversion to bile acids (20). In­ creased thyroid hormone secretion is known to reduce serum cholesterol.

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181

This was considered possible notwithstanding increased synthesis be­ cause of even greater increase in the catabolic processes which degraded and removed cholesterol {20, 27, 45). It was also shown that thyroxine stimulated at the cleavage and not at the initial stages of hydroxylations {109). In a comparative study of the effect of thyroxine status on the two end products of isoprene synthesis, cholesterol and ubiquinone, Inamdar and Ramasarma {76) found that biogenesis measured by the incorpora­ tion of mevalonate-2-i*C increased by about 50% into cholesterol and 200% into ubiquinone. This happened in the early time interval, after 5 days of thyroxine supplementation. At later time intervals, after 10 days and beyond, synthesis of sterols decreased under conditions of thyroxine excess produced either by subcutaneous injection or by dietary supple­ ment, but not with iodinated casein. Because the conditions employed in these experiments are dissimilar, direct comparisons are not possible. The results vary depending on the mode of administration, the form of thyroxine given, and the experimen­ tal method used for the determination of the biogenetic activity. More­ over, altered thyroxine status affects the caloric intake of the animal and possibly the redistribution of the metabolites, which in turn profoundly affect the biogenesis of cholesterol. The lack of attention to this aspect in these investigations has been correctly pointed out by Siperstein {134) · 6. ESTROGENS

Rats given estradiol-17)0 for 4-56 days showed progressive decreases up to 50% of the initial value in the hepatic biogenesis of cholesterol from acetate. By assaying the activities of the enzymes in the micrpsomal fraction, it was shown that HMG-CoA reductase and HMG-CoA synthetase were affected {110). Ovariectomy resulted in an increase in the biogenesis of cholesterol {86) and an elevation of plasma cholesterol and )8-lipoprotein {20, 114)- Administration of Enovid {1) and a number of estrogens {20) to male rats depressed the plasma concentration of cholesterol and lipoprotein (11). It was not possible, however, by any modification of the structure to retain the effect on cholesterol and re­ duce or abolish the endocrine functions of the estrogens. 7. α,ρ-CHLOROPHENOXYISOBUTYRATE (CPIB)

Administration of the drug, CPIB, was shown to reduce plasma choles­ terol in rats {149). For this activity of CPIB endogenous thyroxine seemed to be required, and Thorp and Barrett {150) proposed that CPIB might displace serum albumin-bound thyroxine and make it available in the liver. Avoy et al. {9) showed that the effect of CPIB on the syn-

182

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thesis of cholesterol was confined to the liver and was not obtained in other tissues. It was deduced that the most affected site on treatment with CPIB was HMG-CoA-mevalonate in view of the decrease of bio­ genesis of cholesterol from acetate, but not mevalonate, and lack of inhi);)ition of synthesis of fatty acids and ketone bodies. Recently, White (156) obtained further supporting evidence by measuring the microsomal activity of the enzyme, HMG-CoA reductase. It usually required some days before the effects on biogenesis appeared after CPIB was given orally at a level of 0.2-0.5% in the diet. However, these effects were obtained only when the drug was given to the intact animals, not when it was added in vitro in systems of liver slices or homogenates synthesizing cholesterol from acetate-^*C. Thus, the drug appears to act indirectly, possibly by affecting some hormone (s). The inhibition obtained was usually in the range of 50%, irrespective of the dose and time of administration of the drug, in contrast to the marked inhibition obtained with dietary cholesterol (120). The effects of depres­ sion of serum cholesterol and hepatic biogenesis were not observed in young suckling rats (66) or in rats exposed to low environmental tem­ perature (0-5°) for 5 days or longer (120). 8. UBIQUINONE

One of the most interesting effects produced by feeding small quanti­ ties of ubiquinone-9 to rats was the inhibition of hepatic synthesis of sterols from acetate (95). The site of inhibition appeared to be before mevalonate and after acetyl-CoA because neither the incorporation of mevalonate into sterols nor of acetate into fatty acids (of triglycerides and phospholipids) and ketone bodies was affected. The inhibition did not exceed 30-50%, despite long periods of feeding or increased doses of ubiquinone. It was not an unspecific lipid effect, because only the nat­ ural, major homolog of ubiquinone-9 gave the response (96). Also, the inhibiting effect of ubiquinone was observed in the liver, but not in the kidneys and the intestines. The concentration of ubiquinone increased exclusively in the liver, and in view of effects being similar to those ob­ tained with CPIB, including increased hepatic ubiquinone, it was conjec­ tured that ubiquinone might be acting as a regulatory molecule in the biogenesis of hepatic cholesterol (120). It must also be pointed out that some of the effects are small and variable and the conditions required for their manifestation are yet to be defined. The evidence at this stage is insuflScient to conclude that ubiquinone is functioning through a mecha­ nism of end-product regulation of a common enzyme in the pathway. It is possible that both CPIB and ubiquinone may be acting via another unknown intermediate as represented below:

CONTROL OF BIOGENESIS OF ISOPRENOID COMPOUNDS CPIB

183

ubiquinone —• hepatic cholesterol -> serum cholesterol ubiquinone

I CPIB

?

hepatic cholesterol -> serum cholesterol

9. X-IRRADIATION

Gould and co-workers (52) first demonstrated that exposure of rats to X-rays resulted in a severalfold enhancement of hepatic biosynthesis of cholesterol from acetate-^^C. This was confirmed by Bucher et al (25), who further showed that the effect was probably between acetate and mevalonate since incorporation of acetate, but not of mevalonate, into squalene and cholesterol in rat liver slices was stimulated. During X irradiation the animals were fasted and the biogenesis increased in these animals in contrast to large decrease in the control animals. 10. TRITON-WR 1 3 3 9

Intravenous injection of the detergent Triton-WR 1339 was capable of enhancing the biogenetic activity of cholesterol from acetate-^^C and of HMG-CoA reductase in normal, fasted, and cholesterol-fed rats {25, Jß, 88). The effect was obtained only in vivo when the animals were treated with the detergent, but not when it was added to the tissue slice system. Employing the isolated enzyme system. White and Rudney (158) showed that the incorporation of acetate into HMG-CoA increased slightly, and of acetate, acetyl-CoA, and HMG-CoA into mevlonate in­ creased 2 - to 3-fold. These effects required 1 6 - 2 4 hours after treatment with Triton with little or no effect in 4 hours. Serum cholesterol concen­ tration increased about 6-fold in 1 2 hours after Triton injection, coincid­ ing with increased biogenesis (49). 1 1 . CiRCADiAN RHYTHM

Biogenesis of cholesterol exhibits circadian rhythm. Incorporation of acetate-^*C into sterols and the activity of HMG-CoA reductase in mouse liver were shown to increase during the time between 8 : 3 0 AM and 8 : 3 0 PM (88), Following this observation several workers demonstrated the rhythm in rat liver (10, 33, 127,144) - The rhythmic activity, with a peak at midnight, was observed in the incorporation of acetate-^*C into sterols in liver slices as well as hepatic microsomal HMG-CoA reductase (64, 127) and this was considered to be independent of food intake since the rhythm persisted in starved animals (74) ^ Shapiro and Rodwell (128) demonstrated a severalfold increase in the activity of HMG-CoA reduc-

184

τ . RAMASARMA

tase at midnight compared to noon which could be prevented bytreatment of the animals with cycloheximide or actinomyin D. Sup­ plementation with cholic acid (65) and with cholesterol (128) were found to prevent the marked rise in activity, but the data do not support the conclusion that the rhythm was abolished, as claimed. Higgins et al. (69) showed that increased activity of HMG-CoA reductase was due to increased synthesis of the enzyme protein as tested by the direct measurement of incorporation of leucine-Ή into it. The diurnal variation in enzyme activity was considered to be due to a strange pattern of syn­ thesis of the enzyme for approximately 6 hours followed by complete cessation during the rest of the day (69). The cyclic increase and decrease in synthesis during a 24-hour period did not produce any change in cholesterol concentration of the liver or the serum (144)- This paradox would be resolved if the turnover of cholesterol in the blood had increased simultaneously. Evidence in favor of this was obtained by an indirect experiment using AY 9944 (imns-1,4(bis-2-chlorobenzylaminomethyl) cyclohexane dihydrochloride), an inhib­ itor of cholesterol biogenesis (40). Treatment with this inhibitor resulted in the output of 7-dehydrocholesterol into blood instead of cholesterol (89) and at midnight this output increased further, along with increased hepatic biogenesis (71). It was also found that the peak of activity depended on the food intake and that it could be shifted if the normal pattern of nocturnal food con­ sumption was altered to an enforced feeding schedule between 8:30 AM to 4:30 PM. Inhibition of cholesterol biogenesis by supplementation with cholesterol, CPIB and ubiquinone was also obtained at midnight, not­ withstanding the severalfold increase in the activity at midnight as com­ pared to the noon. In its circadian rhythm and short half-life, HMGCoA reductase, a membrane-bound enzyme, resembles the soluble enzymes, tyrosine transaminase and tryptophan pyrrolase, but it does not seem to share the property of being induced by Cortisol (144) 12. NOREPINEPHRINE

Bortz (19) found that injection of rats with norepinephrine resulted in a 2-fold increase in cholesterol biogenesis from acetate-^*C with a lag period of 12 hours. At earlier time intervals acetate-^^C incorporation into ketone bodies increased and into fatty acids decreased. Addition of norepinephrine in vitro caused no increase in cholesterol biogenesis. Mevalonate-2-C^* incorporation into sterols was also unaltered (75). The norepinephrine-induced increase was prevented by concomitant puromycin treatment. All these results imply a stimulation of the syn­ thesis of an enzyme before mevalonate formation.

CONTROL OF BIOGENESIS OF ISOPRENOID COMPOUNDS

185

13. ACTINOMYCIN D

In some of the above experiments protein synthesis inhibitors, includ­ ing actinomycin D, were used to test whether increased enzyme activity was due to synthesis of the enzyme-protein. But actinomycin D itself, at a concentration of 0.5 mg/kg body weight, stimulated the hepatic biogenesis of cholesterol from acetate-^*C, and not from mevalonate-2^^C, severalfold in as short period as 4 hours after administration. This effect in turn was abolished by supplementation with ethionine or puromycin or dietary cholesterol (38), After administration of actinomycin D, serum cholesterol concentration was elevated which appears to be due to increased synthesis and also to possible redistribution of cholesterol or its decreased catabolism—a picture akin to that of Triton treatment. C. jÖ-Hydroxy-j8-methylglutaryl-CoA Reductase Abundant evidence from incorporation studies with acetate-^*C and mevalonate-2-^*C under a variety of experimental conditions pointed to a primary regulatory effect on the formation of mevalonate and possibly on HMG-CoA reductase. The enzyme HMG-CoA reductase (mevalonate:NADP oxidoreductase (acylating CoA), EC 1.1.1.34) was purified by Knappe et al {91) and by Durr and Rudney {38) from yeast, and an assay system was devel­ oped using HMG-CoA as the substrate. Bucher, Overath, and Lynen {26) first measured the activity of the enzyme in rat liver microsomal fraction and found that the overall rate of cholesterol biogenesis from acetate in vitro was of the same order of that of the enzyme in micro­ somal fraction providing thus the evidence that this enzyme was the rate-limiting step. 1. MEASUREMENT OF ACTIVITY

The measurement of the activity of HMG-CoA reductase in liver microsomes had been carried out by many laboratories. All the methods employ incubation of ^*C-labeled HMG-CoA and NADPH generating system with microsomal enzyme system and measuring the radioactivity in mevalonate formed. The methods are being continuously improved by adding stabilizing agents or small alterations in the preparation of microsomal fraction. The activities varied from preparation to prepara­ tion and showed inconsistent concentration-activity relationships and apparent time lags. Nevertheless, the values in fasted {103, 121, 158), cholesterol fed {103, 128, 158) or Triton-treated {158) rats were claimed to vary sufiiciently from the controls to permit valid conclusions. How­ ever, most of the work was done with small numbers of animals.

186

τ. RAMASARMA

2. PURIFICATION OF THE ENZYME

The enzyme was extracted in a soluble form from acetone powders of microsomal fraction. The specific activity increased after removal of lipids (103). Kawachi and Rudney (90) were able to solubilize the enzyme with 0.25% deoxycholate and fractionated it between 20 and 35% saturation of ammonium sulfate, and then on DEAE-cellulose and hydroxyapatite columns followed by Sephadex G-200 filtration. 3. PROPERTIES OF THE ENZYME

The molecular weight of the purified enzyme was found to be 217,000226,000. The optimum pH for activity was 7.0. The activity increased about 2- to 3-fold on solubilization with acetone (103) or deoxycholate (90) showing that the enzyme existed in a cryptic state. Stimulation of activity observed when EDTA was added to the microsomal fraction was absent with purified enzyme. The KM values were for HMG-CoA, 6 X 10-^ Μ (8.1 X 10-'Μ for microsomes) and for NADP, 8.7 X 10"^ M. The activity increased 2-fold on addition of dithiothreitol. Sodium deoxy­ cholate inhibited the activity in microsomes, and more strongly the puri­ fied enzyme. Digitonin inhibited the activity, but cholesterol did not. Preincubation of the purified enzyme with 2 X 10"* Μ CoA-SH, acetylCoA, and acetoacetyl-CoA resulted in slight inhibition. 4. FEEDBACK OR REPRESSION MECHANISM

Attempts to show that the inhibition by cholesterol, or its products is by the feedback mechanism were unsuccessful. The inhibition obtained by feeding cholesterol seems to be due a decrease in the amount of en­ zyme protein. For the nocturnal increase in the activity, it has now been clearly established that puromycin (64) and cycloheximide (127) prevent the change, indicating the involvement of protein synthesis. Using the direct measurement of incorporation of leucine-^H into purified enzyme Higgins et al. (69) had observed a complete repression of the enzymeprotein synthesis for a period of 15 hours following a 6 hours phase of synthesis. All the evidence thus available suggests that the phenomenon of repression-derepression accounts for the alterations in levels of enzyme activity. However, the nature of the effector (s) still remains unknown. D. )8-Hydroxy-j8-methylglutaryl-CoA Synthetase White and Rudney (157) presented convincing arguments for con­ sidering the synthesis of HMG-CoA as a step for possible regulation

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187

under certain conditions. They also showed that cholesterol feeding de­ creased this step by 50% along with greater than 90% inhibition of HMG-CoA reductase (168), This regulation at the level of formation of HMG-CoA will become meaningful in cases where the formation of mevalonate becomes limited by the availability of HMG-CoA. At least one example may be cited in support of this possibility. Mukherjee and Bhose (110) reported that estradiol-17/? treatment reduced the synthesis of HMG from acetate-^*C by 85% where the decrease in HMGCoA reductase was about 50%. While the increase in the activity of HMG-CoA reductase was about 10-fold at midnight compared to noon, the increase of cholesterol syn­ thesis in vitro with liver slices (64) and in vivo (144) was only about 3-fold, which means that the increase in overall synthetic rate was less than the increase in enzyme activity. This implies that the reductase may not have been rate-limiting at midnight. These considerations prompt further studies to investigate the rate-limiting and regulatory properties of other enzymes under altered conditions. E. Control Points after Mevalonate 1. T H E SECONDARY CONTROL SITE

While the primary control point of mevalonate formation was affected first during the initial stages of cholesterol feeding, after prolonged feeding it was found that conversion of mevalonate to cholesterol was also variably depressed (26, 64, 96), The secondary control site was first recognized by Scaife and Migicovsky (126), who found that conversion of squalene to cholesterol in starved rats was decreased. In fact under this condition the authors considered that this site is more important target of control than the primary one. The indication for reduced con­ version of squalene to cholesterol is the enormous increase in the in­ corporation of mevalonate-2-^*C into squalene, accompanied by a decrease in cholesterol, without a marked change in that of total nonsaponifiable lipids. This occurred under conditions of cholesterol feeding (96, 100, 139), cholic acid supplement (93), norepinephrine and cortisone treatments (76, 119), and starvation (77, 126), Using squalene-^*C in­ corporation into cholesterol as the test system with cholesterol-fed rats, Bucher et al, (26) showed a corresponding decrease in the conversion by microsomes. Gould and Swyryd (64), however, found an apparent in­ crease in a homogenate system, and these authors themselves considered that this effect may be artificial and due to excessive solubility of the lipid substrate, squalene, in the cholesterol-rich homogenate. Similar in-

188

τ.

RAMASARMA

creased incorporation of mevalonate-2-^*C into squalene was obtained when oxygen was excluded in the in vitro system (25). This effect, there­ fore, appears to be due to a decrease in oxidative cyclization of squalene by the microsomal squalene hydroxylase. Under the above-specified con­ ditions, the decrease may have been obtained by way of excluding the availability of oxygen to the microsomal enzyme system. It is an unusual feature of the biogenetic pathway that both control steps are located in the microsomal systems, whereas most of the other enzymes are in the soluble cytoplasm. The choice of locating the regula­ tory enzyme systems in lipid-rich membrane site for the control of the biogenesis of the lipid end product seems most appropriate. 2. CONTROL A T T H E LEVEL O F PYROPHOSPHATE INTERMEDIATES

Using homogenates, Gould and Swyryd (54) observed a decrease in the incorporation of mevalonate-2-"C into farnesyl pyrophosphate and of farnesyl pyrophosphate-^*C into cholesterol in long-term cholesterol feeding extending up to 70-640 days. It is reasonable that such controls in the branched pathway might develop on prolonged treatment in order to divert the intermediates toward the synthesis of other products de­ pendent on the pathway. While it is not known where the branching occurs for synthesis of the side chain of ubiquinone, farnesyl pyrophos­ phate, or isopentenyl pyrophosphate are the potential intermediates. 3. INHIBITION B Y PHENYL ACIDS

It has been observed that phenylketonuria is invariably accompanied by mental retardation due possibly to abnormal metabolism of lipids, particularly cholesterol, in the brain (104). The high concentrations of phenylalanine occurring under conditions of this disease appear to have a specific role in decreasing cholesterol and thereby interfering with the process of myelination. Shah et al. {126) reported that prolonged experi­ mental hyperphenylalaninemia in immature rats led to decreased levels of brain cholesterol and decreased synthesis of digitonin-precipitable sterols in vitro from mevalonate-2-^*C by homogenates of the brain, but not of the liver. Brain levels of tyrosine increased in these animals but tyrosine had no effect on mevalonate incorporation into sterols. Phenyl­ alanine itself did not accumulate to inhibitory levels but the derived phenyl acids such as phenyl pyruvate, phenyl lactate, and phenyl acetate might account for the effect since in very low concentrations these showed marked inhibition of mevalonate incorporation. Of these phenyl pyruvate was the most potent in the in vitro system both with the brain and liver homogenates. The inhibition was also obtained with p-hydroxy-

CONTROL OP BIOGENESIS OF ISOPRENOID COMPOUNDS

189

phenyl pyruvate which is on the pathway of biogenesis of the aromatic ring of ubiquinone (118), It was further found that these phenyl acids, but not phenylalanine, blocked the decarboxylation of mevalonate-5pyrophosphate and prevented the formation of the prenyl intermediates (126). Thus, the inhibition shown by excess phenylalanine itself must be beyond the formation of prenols. Although in normal animals this type of inhibition may not be functional, it may assume significance and interfere with the synthesis of cholesterol and possibly ubiquinone under some pathological conditions. F. Effect on Serum Cholesterol Levels

It is well known that liver contributes the major proportion of cholesterol to the circulating blood plasma (60, 67, 72). Krum et al. (98) showed that dietary cholesterol equilibrates with pools of plasma and adrenal in the dog. Taylor et al. (IJß) estimated that 65-75% of serum cholesterol in human subjects fed on cholesterol to suppress hepatic cholesterol biogenesis, was derived from extrahepatic sources; but this should be considered a special case and does not rule out the possibility of liver being the source of serum cholesterol under normal conditions. The changes observed in the hepatic biogenesis of cholesterol may therefore be expected to result in corresponding changes in serum cho­ lesterol concentrations. A variety of conditions or supplements caused alterations in hepatic biogenesis of cholesterol and the activity of the rate-limiting enzyme, HMG-CoA reductase, but examination of the effects of these conditions on the concentrations of cholesterol and ubiquinone, the end products of the pathway, show striking variability. The following general observations emerge: (a) The liver concentration of cholesterol is the least affected, (b) Serum cholesterol is maintained in starvation and at midnight (compared to noon)—^the two conditions giving opposite effects of profound degree on biogenesis (type I and type III in Table I), (c) Serum cholesterol decreases about 15-30% under conditions where normally the biogenesis decreased by a maximum of 50% activity (type II in Table I). Only type II alteration of cholesterol biogenesis depresses cholesterol level in the serum. While in this type the site of inhibition seems to be before mevalonate, evidence in favor of HMG-CoA reductase being the sole control point is not unequivocal and changes in HMG-CoA syn­ thetase also were indicated. From these studies, it is apparent that the alteration in HMG-CoA reductase does not automatically produce a change in the serum cholesterol and two other considerations, viz., the production and recycling of bile acids and the turnover of serum cho­ lesterol as a source for steroidogenesis become important factors.

190

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ill. Ubiquinone A . Biogenetic Interrelationship of Ubiquinone a n d Cholesterol

The ratio of quantities of cholesterol/ubiquinone, the two end products of isoprene synthesis, has been found to vary by 12- to 1000-fold in rat tissues. Most tissues are capable of synthesis of both compounds. There are some indications that early embryonic cells may not synthesize ubiquinone but depend on an exogenous supply [80). In the case of ubiquinone, exchange between tissues seemed to be absent (82) whereas cholesterol of hepatic origin was shown to be the source for serum and for some steroidogenic organs (50, 98). Quantitatively, ubiquinone syn­ thesis is a small fraction of that of cholesterol. Agents which alter cho­ lesterol biogenesis, especially by acting at points in the pathway common to ubiquinone also, may be expected to have undesirable secondary effects on ubiquinone. Even in cases of severe depression of cholesterol bio­ genesis, a small fraction of mevalonate, about 1-^5% of initial, would be produced which then may be used for ubiquinone synthesis. In view of the small quantities of ubiquinone, even severe changes in isoprene synthesis do not seem to interfere with the second product. This is made possible by the presence of secondary control points at squalene and/or farnesyl pyrophosphate in the branch of the pathway leading to cho­ lesterol, which divert the available small pool of mevalonate, lowered on account of the inhibition of its formation, toward the synthesis of ubiquinone. This explains the limited decrease in the levels of ubiquinone under conditions of feeding cholesterol at 1% level or cholic acid in the diet. When cholesterol was fed at 5% level for long periods, liver ubiqui­ none was indeed decreased (100). In starvation, however, which has similar effects on cholesterol biogenesis, the concentration of ubiquinone in the liver decreased owing to simultaneous deprivation of the phenyl acids, the precursors of the ring moiety. Notwithstanding the marked decrease in synthesis, under any of the conditions, a deficiency of ubiquinone could not be obtained. The cells seem to preserve a minimum amount of ubiquinone, vital for oxidations, and this is achieved by lowering its catabolism and degradation (119). B. Effect of Inhibition of Cholesterol Biogenesis on Ubiquinone

In addition to those described in the preceding section, some drugs are known to affect cholesterol biogenesis. Steinberg and Frederickson (HI) found that a-phenylbutyrate affected the step of acetate activation, and Inamdar (75) found that an additional step after mevalonate was also affected. Holmes and Benz (70) showed that treatment of rats with )&-diethylaminoethyl-phenylpropylacetate hydrochloride (SKF-525A) in-

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hibited some step between isopentenyl pyrophosphate and squalene. Benzyl-iV-benzylcarbethoxyhydroxamate (W-398) was considered to inhibit at a site prior to mevalonate, but this drug was found, in this laboratory, to inhibit at two sites, one after mevalonate and another after squalene (75). In addition to these conditions, cold exposure, vitamin A deficiency, and protein deficiency, which were implicated in ubiquinone metabolism, were studied for their effects on concentration, synthesis, and catabolism of ubiquinone. While most of the conditions chosen de­ creased cholesterol biogenesis, the effects on ubiquinone were variable— some increased it, some decreased it, and some others had no effect (Table II). 1. AGENTS ACTIVATING UBIQUINONE SYNTHESIS

Supplements of thyroxine {15, 44), epinephrine {75), and CPIB {94) increased hepatic ubiquinone by increasing its synthesis at the inter­ mediate experimental stage. The large accumulation obtained in thyroxine {76) and CPIB {97) treatments, and in vitamin A deficiency {99) and cold exposure {3) was due to lowered catabolism in addition to increased synthesis. Paradoxically, CPIB, a drug known to inhibit mevalonate formation, produced a large increase in ubiquinone. An increased syn­ thesis occurring at an intermediate stage before the inhibition becomes manifest appears to be the reason {97), None of these agents were capable of activating ubiquinone synthesis in tissue slices when added in

vitro.

2. AGENTS INHIBITING UBIQUINONE SYNTHESIS

Other drugs, protein deficiency, and starvation caused a decrease in ubiquinone. These, therefore appear to act in a similar fashion as on cholesterol and must be affecting the common steps in the pathway. The exception is protein deficiency. The concentration and biogenesis of cho­ lesterol were unaffected when rats were given diets deficient in protein or deficient in fats or carbohydrates. Cholesterol biogenesis could be maintained by caloric intake in any form (151) whereas protein is re­ quired for providing the ring precursor of ubiquinone. 3. AGENTS W I T H O U T EFFECT O N UBIQUINONE

Inhibitors of cholesterol synthesis which inhibit after branching of the pathway should have no effect on ubiquinone. This was indeed found to be true in the case of cholesterol, cholic acid, cortisone, and norepi­ nephrine treatments, all of which inhibit at the squalene stage {75). Starvation and W-398 treatment decreased ubiquinone because they also

TABLE

II

INFLUENCE OF INHIBITION OF CHOLESTEROL BIOGENESIS ON UBIQUINONE

Effect on ubiquinone Condition, supplement

Possible site of inhibition of cholesterol biogenesis

Concentration

Thyroxine, 30 mg/kg diet

HMG-CoA-mevalonate

Increased (1δ, 41)

lodinated ca­ sein, 0.3% diet CPIB, 0.5% diet

HMG-CoA-mevalonate

Increased (4)

HMG-CoA-mevalonate

Increased (94)

— —

Increased (7δ) Increased (68)

Epinephrine Vitamin A deficiency

Cold exposure, — 0-5°C Isopentenyl P P SKF-525 A, 1% diet squalene 1. MevalonateW-398, isopentenyl PP 1%, diet 2. Squalene-cholesterol a-Phenylbuty- 1. Acetate-acetyl-CoA 2. Squalene-cholesterol rate, 1%, diet 1. ATP formation 2,4-Dinitro2. Mevalonatephenol, isopentenyl PP 0.3% diet 1. ATP formation Ethionine, 2. Mevalonate0.5% diet isopentenyl PP 1. No effect on Protein cholesterol deficiency 2. p-Hydroxybenzoate formation? 1. HMG-CoAStarvation mevalonate 2. Squalene-cholesterol 1. HMG-CoACholesterol, mevalonate 1% diet 2. Squalene-cholesterol 1. HMG-CoACholic acid. mevalonate 0.5% diet 2. Squalene-cholesterol Squalene-cholesterol Cortisone, 2 mg/day Squalene-cholesterol Norepi­ nephrine

Biogenesis

Increased (16)

Increased followed decrease at late stage (64) Increased followed decrease at late stage (76) Increased followed decrease at late stage (97) Increased (96) Increased followed decrease at late stage (99) Increased (3)

by

Decreased (76)

Decreased (76)

Decreased (76)

Decreased (76)

Decreased (76)

Decreased (76)

Decreased (76)

Decreased (76)

Decreased (76)

Decreased (76)

Decreased (83)

Decreased (84)

Decrea.sed (77)

Decreased (77)

No change (96)

Apparent increase (96)

No change (93)

Apparent increase (93)

No change (76)

No change (76)

No change (76)

No change (76)

by

by

by

CONTROL OF BIOGENESIS OF ISOPRENOID COMPOUNDS

193

have sites of inhibition in the common pathway (75), Cholesterol and cholic acid showed decreases after prolonged treatment. The circadian rhythmic variation in cholesterol biogenesis and HMGCoA reductase also showed no corresponding change in liver concentra­ tion of either cholesterol or ubiquinone. C . Secondary Control Point in Ubiquinone Synthesis

Exogenous ubiquinone also inhibited hepatic synthesis of ubiquinone from mevalonate without affecting that of hydrocarbons and sterols in the other branched pathway. A secondary site of inhibition appears to exist at the stage of synthesis of prenyl side chain presumably after isopentenyl pyrophosphate (95), The quantitative significance of this effect is yet to be understood, but this appears to exist under a variety of conditions where liver ubiquinone levels increased, such as cold ex­ posure (3), vitamin A deficiency (85), and thyrotoxicosis (76). D . Regulation of Aromatic Pathway

Biogenesis of ubiquinone involves two distinct pathways—one for the isoprene side chain and another for the ring. It is now well established that the ring is derived from p-hydroxybenzoate and the side chain from isopentenyl pyrophosphate. The intermediate steps of the pathway have not yet been deciphered in animals. In a photosynthetic anaerobe Rhodospirilhm rubrum the alkylation of the ring by the side chain was shown to occur early in the sequence, forming 2-polyprenylphenol (115), which then undergoes the substitu­ tions in the ring. Attempts to label with radioactive precursors or isolate nonaprenylphenol have not been successful (129, 155), Polyprenyl pyrophosphates of long chains had not been detected in animals, and their participation in ubiquinone synthesis is still a matter of conjecture (129, 155), The possibility of similar intermediates, but in enzyme-bound form, cannot be discounted. Despite the lack of delineation of the pathway, its pos­ sible regulation by ubiquinone was amenable for study by the effects on incorporation of the radioactive precursors. The interesting feature of ubiquinone biogenesis is that it is a con­ vergent branched pathway—^two pathways leading to one end product. The treatment of control mechanisms has thus far dealt with divergent branched pathways, where the two end products control the early step by any one of the four mechanisms listed by Stadtman (I40). In a con­ vergent pathway it is logical to expect the single end product, when occurring in excess, to control both pathways. In the pathway for the formation of p-hydroxybenzoate from tyrosine

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(Fig. 1), the enzymes tyrosine transaminase and p-hydroxyphenyl pyru­ vate dehydrogenase are already known. Studies in this laboratory demon­ strated in mitochondria the presence of an enzyme system which con­ verted p-hydroxycinnamate to p-hydroxybenzoate (117). The step of conversion of p-hydroxyphenyl lactate to p-hydroxycinnamate had been deduced from experiments on excretion pattern of phenyl acids in rats given orally different phenyl acids (18). The enzyme for this step is the only one yet to be demonstrated in vitro in the whole pathway. Two additional products are formed: p-hydroxyphenyl acetate from p-hydroxyphenyl pyruvate and p-hydroxyphenyl propionate from p-hydroxycinnamate. These along with p-hydroxybenzoate constitute the urinary excretion products of phenylalanine and tyrosine metabolism. In view of the inhibition of the incorporation of mevalonate-2-^*C into ubiquinone in livers enriched by exogenous, orally administered ubiquinone, the biogenesis of the second pathway of the formation of the ring also should be under similar control. Experiments in this laboratory showed that supplementation with ubiquinone had no effect on any of the enzymes in the pathway, nor on the urinary excretion of p-hydroxy­ benzoate (118). Small changes in the synthesis of ubiquinone will not be expected to make any change since the amount of p-hydroxybenzoate is far in excess of the requirement of ubiquinone synthesis. The reason is not understood for this excess production of this metabolite which can be considered the "gateway" for ubiquinone biosynthesis and has no other known function. What determines the amount of p-hydroxy­ benzoate utilized for ubiquinone ring is an unanswered question. One interesting feature of this pathway deserves comment. The catabolic removal of excess tyrosine by way of homogentisic acid may normally be considered sufficient to remove this amino acid if it occurs in excess. It is remarkable that the animal had developed enzyme systems to form three other phenyl acids which are excreted, of which only p-hydroxybenzoate has any known function, and that for only a frac­ tion of the total amount formed. It should, however, be recognized that in animals urinary excretion constitutes an important control mechanism which eliminates the need for metabolic control. IV. Bile Acids A . Alterations in the Production of Bile

The major pathway for the degradation of cholesterol in the liver is its conversion to bile acids. It was shown that removal of bile from enterohepatic circulation by way of ligature, fistula, or supplementing cholestyramine increased not only cholesterol biogenesis but also of bile

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acids in the liver. Biliary drainage increased the production of bile which implicated a controlled production when bile acids were recycled through portal reabsorption. Operation of negative feedback in the above process had been recognized by Bergstrom and Danielsson (14)- When bile fistulas were infused intraduodenally With sodium taurocholate at a. rate higher than 10 mg/hour per 100 gm weight of rat, the increase in the bile output was abolished (181), B. Site of Control of Biogenesis of Bile A c i d s

In rats with bile fistula, incorporation of acetate-^*C into cholesterol was eighteen times and that into cholest-5-ene-3jS,7a-diol, eight times greater than in control rats. Such large changes were not found with 7ahydroxycholest-4-en-3-one, cholest-4-en-3-one-7a,12a-diol, and taurodeoxycholic acid (32). The first and rate-limiting step in the oxidative conversion of cholesterol to bile acids had been recognized to be the intro­ duction of 7a-hydroxyl group (32, 102), Thus the above data indicate that under conditions of biliary drainage, where bile acid production in­ creased, the 7a-hydroxylation step must have increased in activity. Similar increased activity was observed on cholestyramine feeding {81), In another set of experiments, Shefer et al. {132) demonstrated the decrease in activity of the 7a-hydroxylation step when excess product was present. Intravenous infusion of taurocholate, about 11-14 mg/hour per 100 gm weight, in the bile fistula rat inhibited incorporation of acetate-l-^*C, mevalonate-2-^^C, and cholesterol-4-^*C into bile acids by about 90%, compared with the control values. In such experiments, 7a-hydroxycholesterol-4-^*C incorporation was inhibited by only about 10%. After an extensive survey of nine different hydroxylation reactions involved in the metabolism of bile acids, Johansson {81) concluded that 7a-hydroxylation of cholesterol is the major rate-limiting reaction under regulation. It was also found that under starvation 12a-hydroxylation was stimulated. C . Cholesterol 7 a - H y d r o x y l a s e

In view of its role as a regulatory step, the enzyme cholesterol 7ahydroxylase has gained importance, although the assay system for the enzyme is not yet perfected. Several features of this enzyme are already known by the study of Shefer et al. {131) and Danielsson et al. {32), Studies on this enzyme system were complicated because of the autoxidation of cholesterol in the presence of microsomal fraction and NADPH. The presence of EDTA minimized this process and allowed a study of the properties of the enzyme system {130), Thiol compounds, particularly

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glutathione and 2-mercaptoethylamine, together with an unidentified thermostable liver supernatant factor, were found to inhibit lipid per­ oxidation and stimulate cholesterol 7a-hydroxylase (22), The enzyme was found to be localized in the liver microsomes and required NADPH and molecular oxygen (31), The reaction was inhibited by carbon mon­ oxide, and this was reversed most efficiently by illumination with light of wavelength 450 nm (22). The enzyme was not sensitive to cyanide and therefore cytochrome P-450 was considered to be the terminal oxidase for this hydroxylation (21),

y . Steroid Hormones A . Steroid Biosynthesis in A d r e n a l s 1.

PATHWAYS

The adrenal steroid hormones are produced primarily in the cortex. Aldosterone is synthesized in zona glomerulosa, and Cortisol, androgens, and estrogens in zona fasciculata. The pathway for the formation of these steroid hormones is given in Fig. 1 (see 23,147). This appears to be the major pathway, if not the only pathway, although an alternate path­ way involving sulfated intermediates is likely to play an important role (122). There is considerable evidence to show that these steroid products are formed from cholesterol {δ9, 60) derived both from the steroidogenic tissues and the circulation. The first step is 20a-hydroxylation which appears to be the rate-limiting reaction. This is followed by a second hydroxylation at 22a-position and a subsequent desmolase action yield­ ing pregnenolone. The overall reaction is called "cholesterol side-chain cleavage.'' These reactions, along with other hydroxylase reactions in the path­ way, were found to be localized in mitochondria (28, 69). The hydroxyla­ tion reactions require N A D P H and molecular oxygen and are mixed func­ tion oxidases with cytochrome P-450 as terminal oxidase. The reactions leading from pregnenolone to other steroid products involve 17«-hydroxylations, 3)9-dehydrogenase, ll«-hydroxylations, 18«hydroxylation, 17-20 carbon cleavage, 17a-dehydrogenase, and 19-oxidative demethylation. Most of these seem to take place outside mitochondria {124). Of the products, only aldosterone and Cortisol (corticosterone in the rat in view of the absence of 17a-hydroxylation) are quantitatively the most important adrenal steroids, but these are excreted and removed from the gland and therefore do not seem to accumulate or need a sys-

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tern of end-product inhibition. The control of the rate of production of these is under the influence of pituitary and depends on the availability of cholesterol and the early rate-limiting step in the production of pregnenolone. 2. END-PRODUCT INHIBITION B Y PREGNENOLONE

A very significant observation was made by Koritz and Hall (92) on the control of steroidogenesis in the adrenal. They found that the con­ version of cholesterol to pregnenolone by an extract prepared from acetone-dried powder of mitochondria from rat adrenal cortex was in­ hibited by prenenolone added in vitro. The inhibition was specific to pregnenolone and was not obtained with any other adrenal steroid prod­ ucts such as progesterone, 21-hydroxy-4-pregnene-3,20-dione, 17a-hydroxyprogesterone, 17a-hydroxypregnenolone, pregnenolone, corticosterone, Cortisol, dehydroepiandrosterone, and 20a-dihydroprogesterone. The inhibition was limited to the first step of 20-hydroxyIation, since under similar conditions the conversion of 20a-hydroxycholesterol to pregnenolone was unaffected by added pregnenolone. These findings sug­ gest that control of steroidgenesis in the adrenal will depend on the accumulation and removal of pregnenolone from the mitochondrial site. 3.

EFFECT O F

ACTH

Adrenal production of Cortisol, (or corticosterone in the rat) is stim­ ulated by ACTH and the secretion of ACTH by pituitary is in turn con­ trolled by Cortisol (or corticosterone in the rat), but not by any of the precursors. The stimulation by ACTH was considered to be at some step between cholesterol and pregnenolone (143) in view of the absence of effect on pregnenolone conversion to Cortisol (112), On the other hand, ACTH may stimulate the availability of cholesterol in the adrenal, and some evidence is available in support of this. A direct stimulatory effect on accumulation of cholesterol in the adrenal (34) was observed when hypophysectomized rats were treated with ACTH along with aminoglutethimide (a-ethyl-p-aminophenyl glutarimide), a drug preventing further utilization of cholesterol by inhibiting the 20a-hydroxylation step (87), 4.

CONTROL O F ALDOSTERONE PRODUCTION

It is known that depletion of sodium selectively stimulated the secre­ tion of aldosterone without affecting Cortisol. Bledsoe et al, (17) had found that the change of high-sodium diet to low-sodium diet in human subjects treated with metyrapone (2-methyl-l,2-bis-(3-pyridyl)-l-propanone), an inhibitor of ll^-hydroxylase (101) resulted in stimulation

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of s e c r e t i o n of 11-deoxycorticosterone, but not 11-deoxyCortisol. This finding indicated that sodium depletion stimulated a l d o s t e r o n e p r o d u c ­ tion by an apparently exclusive activation prior to 11-deoxycorticosterone presumably localized to zona glomerulosa. Aldosterone and angiotensin in zona glomerulosa bear a similar i n t e r r e l a t i o n s h i p as Cortisol and ACTH in zona f a s c i c u l a t a . In experiments with dogs, Fabre et al (42) made an interesting ob­ servation on the effect of ubiquinone on aldosterone production. It was found that systemic venous infusion of ubiquinone-10 resulted in selec­ tive decrease in e x c r e t i o n of a l d o s t e r o n e and not of Cortisol in the dog. The effect was obtained only with the oxidized quinone aiid not with the corresponding quinol or chromenol. This is an interesting example of a distal product of isoprene pathway inhibiting selectively another product specifically produced in a region of the adrenal. It is not known how this effect is achieved by ubiquinone, but the two possibilities are reduced availability of cholesterol selectively to the zona glomerulosa or specific inhibition of some step after progesterone. 5.

ROLE O F

3',5'-AMP

The action of ACTH on steroidogenesis in adrenal cortex is known to be mediated by 3',5'-AMP (67). Corticosteroid production in the rat adrenal was stimulated by 3,'5'-AMP by increasing the activity of 11)0hydroxylation of progesterone (123). The effect was shown at the level of the enzyme lljß-hydroxylase in mitochondria, and also, 3',5'-AMP was found to have stimulatory effect on the conversion of cholesterol to preg­ nenolone by rat adrenal mitochondria (30). The mechanism of action of 3',5'-AMP is not clearly understood, but one of its functions seem to be the shifting of pregnenolone across the mitochondrial membrane making it unavailable for oxidation to progesterone. B. Regulation of Steroidogenesis in G o n a d s 1. TESTOSTERONE BIOGENESIS I N TESTIS

Dorfman and co-workers (154) had demonstrated that rat testis mito­ chondrial fraction was capable of forming neutral steroids from cho­ lesterol involving the typical cholesterol side-chain cleavage reaction. This reaction was enhanced on pretreatment of immature male rats with human chorionic gonadotropin. Hypophysectomy of rats decreased the cleavage reaction rapidly and the normal activity was restored on treat­ ment of these animals with luteinizing hormone, but not with prolactin.

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thyroid-stimulating hormone, or ACTH. Testosterone formation appears to be under specific control of gonadotropins {105). The rate-limiting step in the overall reaction seemed to be the first 20a-hydroxylation. The hormone-induced changes were found in the formation of 20a-hydroxycholesterol^ but not in the subsequent side-chain cleavage {105). The gonadotropin-induced stimulation of 20a-hydroxylase system was not accompanied by any increased protein synthesis and therefore represents modulation of the activity of this rate-limiting step. Cholesterol was shown to be the obligate precursor of testosterone {106). Inhibition of cholesterol biogenesis from acetate-^*C also showed corresponding inhibition in testosterone. The stimulatory effect of lutein­ izing hormone on the incorporation of acetate-^*C into testosterone in vitro was abolished on inhibition of cholesterol formation by a specific inhibitor, AY 9944. In addition to gonadotropin control, testosterone showed feedback type of inhibition of cholesterol side-chain cleavage reaction (86). Further control points after pregnenolone were also found in experiments with rat testicular microsomes. Inano and Tamoki {78) observed that human chorionic gonadotropin activated the step of conversion of pregnenolone to progesterone in immature rats. The same step was found to be inhibited competitively by 7a-hydroxy derivatives of testosterone, and its pre­ cursor, androstenedione. These compounds had no effect on other steps in the pathway such as 17a-hydroxylation, Ci7-C2o-lyase {79). The sig­ nificance of the multiple control points in this pathway remains to be elucidated. 2. PROGESTERONE BIOGENESIS I N CORPORA LUTEA

It is recognized that progesterone biosynthesis in corpora lutea is under hormonal control. The cholesterol side-chain cleavage reaction similar to that of adrenal is known to be present in corpora lutea {61, 74) With bovine corpora lutea slices the formation of progesterone was found to be stimulated by luteinizing hormone, interstitial cell stimulat­ ing hormone, and also 3',5'-AMP {62). Also, luteinizing hormone was found to increase the conversion of stored cholesterol to secretory prod­ ucts and the locus of action seemed to be beyond cholesterol and likely to be the first step of 20a-hydroxylation {40). Armstrong and Black (7) showed that while luteinizing hormone stimulated the net synthesis of progesterone, its biogenesis from acetate-l-^*C and production of lactate, only the first two effects were prevented by puromycin and not the stimulation of glycolysis. These results indicated that stimulation of glycolysis may be directly related to the 3',5'-AMP production and not a consequence of progesterone synthesis.

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The corpora lutea from the hypophysectomized rat when tested in vitro showed a decrease in the synthesis of progesterone and an increase in 20a-hydroxypregn-4-en-3-one thereby without altering the synthesis of "total progestin" (5). These changes are prevented completely by prolactin treatment while estradiol treatment was ineffective. Cook et al (29) using porcine corpora lutea showed inhibition of incorporation of acetate-"C into progesterone on incubation with pro­ gesterone, 5-pregnen-3)8-ol-2-one, 20a- and 20)S-hydroxy-4-pregnen-3-one, estradiol, and estriol. The major excretory estrogen in pigs is estrone and this, however, had no effect in the above system. Moreover, none of the steroid compounds influences the progesterone production from endog­ enous sources indicating the minor role they play in the control of progesterone synthesis in corpora lutea. The studies on alterations elicited by gonadotropic hormones gave information on the control of progesterone production possibly at the 20a-hydroxylation step, but ofifer no explanation of the basis for such altered activities at the enzyme level by any of the products. 3. METABOLISM O F CHOLESTEROL I N OVARIAN TISSUE

Treatment of immature rats with luteinizing hormone showed dramatic effects of decreased cholesterol stores and increased progesterone secre­ tion in ovaries (5). It was also found that added luteinizing hormone considerably stimulated the incorporation of acetate-"C into proges­ terone by ovarian tissue slices accompanied by decrease in cholesterol, particularly the esterified form. Armstrong (6) found that in hypophysectomized rats esterification of cholesterol by the ovarian tissue considerably decreased and the treat­ ment of these animals with prolactin or luteinizing hormone caused par­ tial reversal. Incorporation of acetate-"C into cholesterol increased in hypophysectomized rats, and this was considered to be due to "diminished feedback inhibition of this synthesis caused by the low levels of cho­ lesterol contained in these ovaries," but there is no evidence for the presence of this control mechanism in ovaries. These changes obtained could not be reversed by prolactin treatment. Sulimovici and Boyd {14S) studied the cholesterol side-chain cleavage reaction in enlarged ovaries of immature rats pretreated with pregnant mare serum gonadotropin and human chorionic gonadotropin. They found that the activity was localized in the mitochondrial fraction and exhibited the characteristics of a mixed function oxygenase requiring NADPH and molecular oxygen for activity, the main product being progesterone. Small amounts of succinate and glutathione stimulated the reaction while

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ascorbate showed inhibition. Addition of 3',5'-AMP increased the forma­ tion of pregnenolone from cholesterol by rat ovarian mitochondria at the expense of progesterone, an effect considered to be obtained owing either to dislocation of pregnenolone from the site of A^-3)S-hydroxysteroid dehydrogenase or the lack of NADPH. C , Pituitary a n d Cholesterol M e t a b o l i s m

In all the cases of production of steroid hormones the major site for control seems to be the first rate-limiting 20-hydroxylase. The only in­ termediate which may develop a pool and elicit feedback regulation appears to be pregnenolone, although such an effect was explicitly shown only in bovine adrenal mitochondria. The steroid products are usually excretory in nature and therefore do not seem to build high concentration, and none seem to have developed the direct product regulation, and the same effect seems to be achieved indirectly by controlling the output of pituitary hormones. Hypophysectomy invariably resulted in the decrease of steroid pro­ duction in the gonadal tissues and addition of one or the other gonado­ tropic hormones stimulated the activity. The locale of such tropic action seemed to be 20a-hydroxylase, and this is most likely mediated by 3',5'AMP. In all these conditions cholesterol dynamics—^the endogenous synthesis, the uptake from blood plasma and the esterification of cho­ lesterol in the steroidogenic tissues—also play an important role. Implicit in the turnover of circulating plasma cholesterol is its utiliza­ tion by other tissues. Whereas most tissues have the capacity for cho­ lesterol biogenesis, the endogenous pool partly or completely equilibrates with plasma cholesterol. In view of this, alterations of serum cholesterol may have some indirect influence in the cholesterol stores in some of the gonadal tissues. Hypophysectomy in rats was shown by Friedman et al, (49) to cause hypercholesterolemia. The higher serum cholesterol levels did not appear to be due to the lack of ACTH, gonadotropic hormones or thyroid hor­ mone since the replacement of these did not reverse the effect. Biogenesis of cholesterol in the liver considerably decreased, and hypophysectomized animals were discharging less cholesterol into the blood. Therefore, the increase in serum cholesterol can be explained only by its reduced utilization. The elevated serum cholesterol was completely reversed by giving physiological doses of thyroxine and growth hormone together in the hypophysectomized animals whereas the same doses in normal ani­ mals were without effect.

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VI. Concluding Remarks It is interesting to find that the control steps in the isoprene pathway are located in particulate systems of microsomes or mitochondria. This seems particularly appropriate because of the lipid nature of the products synthesized by the pathway. In fact for the very same reason study of end-product inhibition with isolated enzyme systems is complicated by the uncertain interactions between the lipid reactants and particulate enzymes. On the other hand, location in the lipid-rich membrane offers scope for regulation by modification of membrane structure or by displacement of the products thereof. The enzyme, HMG-CoA reductase is shown to exhibit latent activity on removal of the lipid by acetone or deoxycholate. This property of masking-unmasking of activity by the lipid may be used for fine control. Diverse types of controls seem to operate in cholesterol biogenesis. A classification of these into three types had been attempted. Of these types I and III may signify the two extremes of the same mechanism. Type II alteration seems to be more significant in the reduction of serum cholesterol and will merit intensive study in view of its relationship to the problem of atherosclerosis. Physiological control of the production of steroid hormones has been well studied. Attempts are being made to understand the processes at molecular level and there is further scope for investigation at the level of enzymes. The foregoing review has focused attention on the total isoprene path­ way and the control at various points as found in different zones, tissues, and animals. It offered examples of a variety of regulatory mechanisms, one of which, removal from the site of synthesis or discharge from the tissue and excretion from the animal, appears to play a critical role. REFERENCES

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τ . RAMASARMA

43. 44' 4δ. 46. 47.

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O n Allosteric Models J E F F R I E S WYMAN*

CNJl. Centre for Molecular Biology at the Institute of Biochemistry University of Rome, and Regina Elena Institute for Cancer Research Rome, Italy I. II. III. IV. v. VI. VII.

Some General Relations A Parent Model The MWC and Induced-Fit Models as Subcases . The Effect of a Second Ligand The Effect of Subunit Heterogeneity An Example from Trout Hemoglobin Conclusion References

.

.

.

209 211 214 218 220 221 225 225

The underlying idea of the allosteric interpretation of macromolecular behavior is that the various ligand binding sites of a macromolecule interact, not directly, but only indirectly as a result of ligand-linked conformational equilibria. Thus any cooperativity of ligand binding is the result of cooperativity present in the conformational equilibria of the macromolecule in the absence of ligand. This is the parent concept, but within its broad shadow lie many different types of physical model, and in any attempt to interpret equilibrium, steady state, and kinetic experiments on allosteric proteins, it is important to have a clear grasp of the nature of such models. A large part of what follows is devoted to a description and comparison of the two most current allosteric models, the MWC model (Monod, Wyman, and Changeux, 1965) and the inducedfit model (Koshland, Nemethy, and Imer, 1966; Haber and Koshland, 1967). I. Some General Relations As a helpful lead into the subject, we begin by considering the simple case of a macromolecule which exists in only one conformation and con­ tains r binding sites for a ligand X. We postulate nothing as regards the *The author acknowledges the support of a grant from the U. S. National Science Foundation. 209

210

JEFFRIES WYMAN

possible composition of the macromolecule in terms of subunits, and make no assumption about the sameness or independence of the r sites. The binding behavior of the macromolecule is described by the binding potential (Wyman, 1965) JI

=

RT In(l

+ Klx + · · · K,.xr) =. RT In N

(1)

and the total amount of X bound by it is given by

x=

dJI=

dp,z

dJI dinN RTd In x = d In x

(2)

Here the polynomial in parentheses, denoted by N, is the binding polynomial of the macromolecule, and JLx and x denote, respectively, the chemical potential and activity of X; the K's which occur in the polynomial are the overall equilibrium constants for the reaction of the macromolecule with 1, 2, ... r molecules of X. It is evident that the relative amounts of the various macroscopic forms M, MX, . . . MX r are given by the successive terms of the binding polynomial N. We now pass on to the case where the same macromolecule, with its r binding sites, exists, as an at least partially allosteric system, in 8 + 1 conformations, which, in the absence of ligand X, occur in the mole fractions vo, VI, • • • V s corresponding to the mole ratios 1, II' .. · /s. In this case, by a simple extension of the concept of the last paragraph, we see that in the presence of ligand at activity x the relative amounts of all the macroscopically different forms is given by the elements of the (8 + 1) X (r + 1) matrix

!.

1

!,Kalx

KOlx

K o,.xr IlK l,.xr

•••

!.K.,.xr

of which the general element of index ij is liKijXi. Here again nothing is assumed about the presence of subunits and there is no restriction on the values of the K's beyond the fact that every K iO = 1. This matrix, which is characteristic of the macromolecule, may be considered in relation to the expression for its binding potential. On the basis of a different and more general analysis, this may be written as (Wyman, 1967) JI = RT In

l

B

JlieJ!,IBT

(3)

i=-O

where JIi is the binding potential of the macromolecule in conformation i,

211

ON ALLOSTERIC MODELS

or, alternatively, since dividing the sum difference to X, as

JI

= RT In

~

by the constant

Vo

makes no

,

LheJI,

/R 7'

(3.1)

i-O

When each JI i in Eq. (3.1) is expressed in terms of the corresponding binding polynomial N, as in Eq. (1), the result is

,

JI

=

RT In

Lloll + K ..

x

1

i-O

+ · · · K ..,xr]

(4)

Comparison shows that the (8 + 1) (r + 1) terms of the summation, which is nothing but the full binding polynomial of the macromolecule, are identical with the elements of the characteristic (8 + 1) X (r + 1) matrix. We see therefore that, quite generally, the relative amounts of the various forms of an allosteric molecule, as indeed of any macromolecule, are given by the various terms of its full binding polynomial. It may of course happen that each JIi in (3.1) is itself of the same form as (3.1), as will be the case when the allosteric macromolecule is composed of allosteric subunits (see Section IV below), but this does not invalidate the general principle, and, since the exponential and logarithm are inverse functions, only results in an enlargement of the polynomial.

II. A Parent Model In reality both the MWC model and the induced-fit model, at least as commonly interpreted, are only special cases of a more general parent model, which is itself a rather special case. The underlying assumptions of this model are the following. The macromolecule is composed of a set of identical subunits. Each of these subunits has a single site for a given ligand and each one can exist in only one or other of two different states (or conformations) A and B characterized by the binding constants K A and K B • The macromolecule itself exists in a variety of polymeric conformations, which are uniquely determined by the state of the subunits-one for A" one for BAS-I, another for B 2 A,-2, and so on. All subunits are independent of one another in their reaction with ligands, and of course the whole system is in thermodynamic equilibrium. This means that the model is completely allosteric, all site-site interactions being indirect and mediated by the ligand-linked conformational equilibria. In this special case, since each subunit contains a single ligand binding site, and since the number of polymeric conformations is the same as the number of subunits, 8 becomes equal to r and the cha~acteristic matrix becomes square. At the same time, owing to the fact that the sites

212

JEFFRIES WYMAN

in any polymeric conformation bind independently of one another, the polynomial N i corresponding to conformation i in which i subunits are in state Β and r - i are in state A, becomes (1 +

Κχχ)^'{1

+

KBXY

Thus the full binding polynomial of the macromolecule is given by r

Ν

=

2

Λ·(1

+

KAX)^KI

+

KBXY

(5)

i-O

From this it follows that element ij of the characteristic (r + 1) X (r + 1) matrix, whose elements give the relative amounts of all the different forms realized in the presence of X, is*

^'"^ LI

- m)\0 - m)!(r - ΐ - j + m)\

where ; denotes the number of molecules of ligand bound per macro­ molecule and m is a parameter. It is to be noted that by summing the elements in appropriate ways we can use the matrix to obtain not merely the relative amounts of the individual forms, but also of any desired combination of them as a function of x, e.g., the fraction of the mole­ cules combined with 0, 1, . . . r molecules of X regardless of confor­ mation, or the fraction of molecules in each conformation regardless of the distribution of X. The use of the characteristic matrix helps to clarify a number of points such as the concept of the "switchover point" introduced by Hopfield, Shulman, and Ogawa in their analysis of the ligand binding process in hemoglobin (Hopfield, Shulman, and Ogawa, 1971).t Elquation (5) may be developed to read as an ordinary polynomial (a simple power series) in χ—each term would then correspond to the sum of the elements in a row of the matrix. If in such a development the ratio of the coeflScient of x^^^ to that of x^*(l < ; < r - 1) is statistical or less than statistical, then the polynomial will be completely factorable and the macromolecule will therefore behave as if it contained a set of r independent sites. The binding curve will be the same as, or less steep than, a simple titration curve, and the system will show no cooperativity —^the value of η in a Hill plot will be everywhere equal to or less than * l n employing expression (6), it should be recalled that 01 = 1, and that the factorial of a negative quantity is to be taken as infinity. t A s an alternative and more geometrical way of representing such multiple equilibria we might introduce an r dimensional cube, of which the number of i dimensional subelements (comers, edges, "faces") is given by the ith term of the expansion of (2 + 1)".

ON ALLOSTERIC MODELS

213

unity. (Any allosteric system is of course cooperative in the sense that the total work of liganding is diminished as a result of the ligand-linked conformational changes. This is a distinction which should be kept in mind.) On the other hand, if the ratios of successive coeflScients are not all statistical (or less than statistical), then the polynomial will not be completely factorable and the ligand binding of the macromolecule will not be describable in terms of a set of r independent sites—it will be necessary to invoke positive (cooperative) interactions between some at least of the sites. The value of η at any given point of a Hill plot may or may not be greater than 1. In the extreme case where the ratio of every coeflScient to the preceding one is greater than statistical, the value of η will be everywhere greater than 1, and we may describe the system as being completely cooperative. All this is brought out by the following considerations. Suppose that the probability of a subunit, in the absence of ligand, being in state A is ρ and that of its being in state Β is g = 1 - p, independently of the state of the r - 1 other subunits. Then vo, which is the same as the probability of all r subunits in the polymer being in state A, is p""; on the same basis

It follows that the fi in Eq. (5) have the statistical values

l , r 5 . ...-;i-_f5Y, ..

(8)

and Ν becomes simply Ν =

(1 +

Kj,x)

+ I (1 +

KBX)

= [1 + (PKA + qKB)xfp-'

(9)

The constant factor p'^ makes no difference to X and may be omitted. In this case, therefore, the polymer binds like a simple monomer with binding constant ΡΚΛ. + QKB, which is an average value of the two K„ the weighting factors being ρ and q. If the ratios of successive /'s are all less than statistical, then the polynomial in (5), again factorable, can be written in the form

Ν = [(1 + Kj^x) + 6,(1 + Kj^xMl + K^x) + h{l + K^x)] • • • [(1 + KAX) + 6,(1 + Kj^x)] ( g A + hKB)x'\ = (1 + fex)(l + 6i) · · · (1 + br)

1 -L ( g A + brKB)x (10)

l + br

214

JEFFRIES WYMAN

where the b^s are real positive numbers. The polymer therefore behaves as if it contained r different and independent sites, each characterized by a binding constant Ki = [K^ + hiKB)/{l + 6»). It will be seen that 1/(1 + 6i) and 6 i / ( l + fci) correspond respectively to the probabilities ρ and q introduced above.* III. The M W C and Induced-Fit Models as Subcases In the MWC model it is assumed not only that the state (conforma­ tion) of a given subunit is subject to influence by that of its neighbors, but that the interaction is so strong as to require all subunits to be always in the same state, so that only two polymeric conformations are permitted. This steric interaction has been interpreted as representing a requirement of symmetry conservation (Monod, Wyman and Changeux, 1965). Mathematically it means that only the first and last columns of the characteristic matrix remain, and that the polynomial resulting from the development of Eq. (4) becomes simplyf N=

{l + K^xY

+ fril

+ Kj^xY

(11)

In the case of the induced-fit model, no such drastic steric constraint is invoked. On the other hand, it is assumed that the values of the constants are such that the macromolecules in any given stage of ligand binding, e.g., the macromolecules MX^, exist essentially all in only one polymeric conformation, namely that in which the subunits combined with ligand are in the high affinity state, while the others are in the low *If we were to admit complex factors, it is clear that they would have to occur in pairs of complex conjugates, one for 6 = 6 ' + ib" and the other for 6 = 6 ' - Ϊ6", in order to give a product with real coefficients. It can be shown that such a pair of factors would correspond to a pair of positively interacting sites. By extending this principle we could, formally at least, factor any binding polynomial. Another remark about the binding polynomial and the associated matrix con­ cerns their relation to symmetry. It can be shown that symmetry of the binding curve, X vs In a; (or the corresponding Hill plot), requires a corresponding sym­ metry of the binding polynomial represented by the following relation between its coefficients where Xy^ is the activity of the ligand required to half-saturate the macromolecule. It follows from this that the curve showing the amount of each form MX* vs In χ is the mirror image of the corresponding curve for its opposite, MXr-i, reflected about the line χ = Xi/i. Since each Ki is the sum of the coefficients of in any row of the matrix, the above relations therefore impose a set of restrictions on these co­ efficients. In a cooperative system the extreme forms will be predominant; in an anticooperative system, the middle forms. t T h i s becomes the same as the expression (1 + x)*^ + L ( l + οχ)*^ originally given in the paper by Monad, Wyman, and Changeux (1965), when we choose the standard state of the ligand so that KA^ I and identify fr with L and KB with C.

215

ON ALLOSTERIC MODELS

affinity state. This means that as liganding proceeds the system passes down the principal diagonal of reaction scheme 1, where A represents a subunit in the low affinity state (A) and Β a subunit in the high affinity state (B). i = 1

ί = 0' 0 j = 1 i = 2

BA,_i BA,.i BAr_iXi

A, Α,Χ

i =

i = 2

•

SCHEME 1

The condition for this is that for any degree of ligand binding i.e., in any row ; of the reaction scheme, all terms except that corresponding to i = ; be negligible. Now it follows directly from (6) that as the ratio KB/KA increases indefinitely, the frequencies of the various forms in the reaction scheme approach those given in Table 1. Thus all terms below the principal diagonal become negligible regardless TABLE I FREQUENCIES OF VARIOUS FORMS

j

\

0

1

2

3

0

1

Λ

/a

1

0

flKBX

SfzKBX

2

0

0

3

0

0

0

j

0

0

0

id - 1)

iji - m - 2) 2-3

0

216

JEFFRIES WYMAN

of the values of the /i. On the other hand, those above the diagonal will be negligible if and only if i i - m

fi«fj

(12)

for all values of ; and i > j . But this condition can always be satisfied if we make the ratio of each /i to the following one sufficiently large. Let ai and ocj be factors which relate /< and fj to their statistical values given byEq. (8). Then the inequality (12) becomes simply

a - j)lir -

a\q)

^^^^

By making p/q suflSciently large, this relation may always be satisfied for any value of the ratio α^/α» not equal to zero. Subject to this con­ straint, ai and aj can be given any values we please. Provided condition (12) is satisfied, then all terms above as well as below the principal diagonal may be forgotten and the binding poly­ nomial becomes simply Ν = 1 -h fiKBX

+

f2KW +

· · · frKix^

If the a^s are such that the fs all have their statistical values, or that the ratios of successive fs are all less than statistical, then the poly­ nomial will be completely factorable and the system will show no cooperativity. In the opposite case where the ratios of successive /'s are all greater than statistical, the binding curve will be everywhere steeper than a simple titration curve (n > 1) and the system will exhibit a degree of cooperativity determined by the values of the as. In both models the cooperativity, or better perhaps the apparent cooperativity, as judged from a Hill plot, is dependent on the choice of the fSj all the interactions between sites being indirect and mediated by ligand-linked conformational changes. This of course is the essence of the allosteric concept. The MWC model represents an extreme choice where all the mixed polymeric conformations are excluded, all fs except fr and / o = 1 being set equal to zero. This model shows complete co­ operativity, in the sense that the ratio of every term to the preceding one in the developed polynomial is greater than its statistical value so that η is everywhere > 1 . This is apparent when we write the polynomial in the form

1 + ^^^+···ϊ!(Γ§1)!+···^^'

(14)

217

ON ALLOSTERIC MODELS

where the bars denote averages (e.g., K* = v o K a + v r i f e ) and take ac­ count of the relation > K ^ R * In contrast to the MWC model, the induced-fit model, though coopera­ tive in the sense that the work of liganding is always less than it would be in the absence of the conformational changes, nevertheless in terms of a Hill plot may be either cooperative or anticooperative, i.e., the value of η may be either less than or greater than unity, depending on the choice of the fs. It is easy to show that in the case of the MWC model the total apparent free energy of interaction realized per site in completely saturating the molecule with ligand X, which is given by the spacing of the asymptotes, is ΔίΊ.ο.., = RTIn

i^oK^+^rK^yfr'+^Ki-')

(15)

This may also be written as Δ ί Ί total =

RTln^4t*

and on the basis of the statistical relation just introduced is always positive. On the other hand, for the induced-fit model AFitotai = i e r i n - ^

(16)

and may be either positive or negative. It is significant that in this case depends only on the fs and not at all on the values of the ÜL'S,

A F I total

* A proof of this is the following. Let Xi, X2, . . . Xn be any set of positive numbers.

Villi.y?i

XjXj

•^-ΣΣ'·'"'"£'"·"' Provided ί > 2, all pairs of parentheses in the double sum have the same sign. When i = 1, the second parenthesis vanishes. When ί < 0, the two parentheses are of opposite sign. It follows that for our case where ί > 2 x'>^x

> ^ 0

>. ..

(xy

218

JEFFRIES

WYMAN

ΚΑ being assumed to be negligible in relation to KB. This constitutes a significant difference between the two models and is closely related to the question of the shape invariance of the binding curves, which has been the subject of frequent discussion. It will be seen that in the case of the MWC model shape is determined by the two parameters KB/Kj^ and fr] in the case of the induced-fit model it is determined by the r param­ eters, /, but it is unaffected by the X's. This leads us to consider the effect of a second ligand. IV. The Effect of α Second Ligand Suppose that in the general case considered in Section I the macro­ molecule, in addition to the r sites for ligand X also contains t sites for a second ligand Y. Then the characteristics (s + 1 ) X (r + 1 ) matrix is replaced by a three-dimensional (s + 1 ) X (r + 1 ) X (t + 1) array of elements corresponding to the binding polynomial 1 +

8

r

X

X

t

X fiKii^'y"

(17)

t»0 y=0 k^Q Let us see how this carries over into the case of the two special allosteric models we have been considering, the MWC model and the induced-fit model. Assume that in both cases each subunit carries, in addition to the site for X, a second site for Y and that the binding constant, L, of this site is, like if, different in the two states (conformations) A and Β of the subunit. Then the binding polynomial for the MWC model becomes Ν

=

(1 +

^ ^ ( 1

+

UyY

+

Λ(1 +

ΚΒΧΠΧ

+

L^vY

(18)

and that for the induced fit model r Ν

=

2

Λ-^Ul

+

LBVYX'

(19)

It will be seen that in either case the effect of changing the activity of the second ligand is the same as that of changing the /^s and hence is bound to change the shape of the binding curves. Now in many allosteric systems the binding curves appear to be invariant in shape for changes in the activity of the control ligand, the effect of the control ligand being simply to shift them back and forth along the In χ axis. This seems to be the situation in the case of the Bohr effect in human hemoglobin (Antonini and Brunori, 1 9 7 1 ) . How can it be explained? The most obvious and plausible explanation would be that the control effect arises locally within the subunits. Then of course Eqs. ( 1 8 ) and

ON ALLOSTERIC MODELS

219

(19) would no longer be applicable, and the problem would disappear. If .such a local effect were direct, not allosteric, there would be no diflSculty in accounting for it, but if the effect is allosteric, as there are strong grounds for believing in the case of the Bohr effect of hemoglobin, then the situation is not so simple. We submit the following proposal. Suppose that in the low aflSnity form (conformation) A, the subunit in reality exists in two sub conformations Al and A2 characterized by binding constants XAI and KA2 and LAI and LA2 for ligands X and Y , respectively, and that in the absence of both ligands the mole fractions of the two subconformations are VAI and VA2. Then the binding poly­ nomial of the subunit in state A is Ν = . A i ( l + Kj^ixKl +

LKIV)

+ vUl

+ 15:Α2Χ)(1 + LA22/)

(20)

When this is expanded and "normalized" by dividing by the sum of all terms independent of X , we obtain the expression

where LA =

VMLAI

jjt

VAIKAILAX

_

+

VK2Li,2 +

^22)

^Α2^Α2^Α2

This shows that the subunit in the overall conformation A has a binding constant R A for X which varies with the activity of the control ligand as a result of local, i.e., intrasubunit, conformational changes in accord­ ance with the simple expression (21). As seen from the definitions (22), the constants R A , L^, and in (21) are simply averages of the various subconstants KAI ' * · LA2, since VAI and VA2 = 1 — VAI are frequencies or probabilities. An expression of exactly the same form as (21) applies to the subunits in the overall conformation B. Now if we assume that the difference between states A and Β involves only the X binding properties and that the two K B in state Β are simple multiples of those in state A, then it will be seen that for either of our models (and indeed for any purely allosteric model) the Κ A and K^ which describe the system are simple functions of the activity of ligand Y given by

This would provide nicely for the shape invariance of the X binding curves. It is the same as the expression used in the past at a more phenomenological level to describe the alkaline Bohr effect of hemoglobin.

220

JEFFRIES WYMAN

One can push this idea a little farther. We know from linkage relations (the effect of a ligand on an equilibrium constant) that in the absence of both ligands the ratio of molecules in subconformation A2 to those in Al is given by VA2/VAI; in the presence of saturating amounts of Y , but in the absence of X , it is VK2LA2/VAILXX] in the presence of saturating amounts of X but in the absence of Y it is VA2-KA2/VAI-KAI; in the presence of saturating amounts of both ligands it is VA^K^ZL^Z/VAIKAILAI, Let us assume that in the absence of X the molecules are essentially all in subconformation Al, regardless of the presence or absence of Y , and that when saturated with X they are all essentially in subconformation A2, again regardless of the presence of Y . This means that ΙΆ1

—> 1,

VA\LAI

y> VAiLiAii VAJKAI

^

VA\KA\,

VAIKATJ^KI

^

VAIKAILAI

(24)

In this extreme case, which represents an essentially complete subformational change induced by ligand X (say oxygen in the case of hemoglobin), the constants in (22) become simply RA

=

ΪΆΙΧΑΙ,

LA = LAI,

LA = LA2

(25)

If, under these extreme conditions, instead of one there were two or more independent binding sites for Y in each sunbunit, the single factor (1 + L"y)/{1 + Uy) in Eq. (23) would be replaced by a product of two or more such factors, one for each Y binding site. This is exactly what has been invoked more phenomenologically to explain the total Bohr effect of hemoglobin, including both the alkaline and acid part. V. The Effect of Subunit Heterogeneity In both the MWC model and the induced-fit model, it is assumed that the subunits are all alike. The question arises as to what sort of modi­ fication is required when they are not all alike, but have different ligand binding constants. This is not an idle question, for there are a number of cases of enzymes and respiratory proteins where there is reason to believe that the subunits fall into classes having different properties. Thus in human hemoglobin, recent studies indicate that the oxygen bind­ ing properties of the a and β chains, as they exist in the tetramer, are significantly different; in respect to their redox properties the chains are almost certainly different, and it appears that as a result of different local oxidation Bohr effects they become progressively more different as the pH is lowered. As we shall see in Section VI, recent studies on trout hemoglobin show that one of its components consists of two kinds of chain whose oxygen binding properties, like the redox properties

ON ALLOSTERIC MODELS

221

of the a and β chains of human hemoglobin, are different and become progressively more different at acid pH, an effect which serves an important physiological function. The general case is complicated, requiring as it does the introduction of different values of the binding constants JKA and KB for each of the different classes of subunit. Without, however, attempting a general formulation it is clear that in the case of the MWC model the effect of this will be to change the binding polynomials of each of the two permitted polymeric conformations from the simple forms (1 K^x)'' and (1 + KBX)"" to the noncooperative forms (1 + Κ^χχ) (1 + Κ^χ) . . . and (1 + K^Bpc) (1 + K^x), . . . In general this may be expected to result in a reduction in the cooperativity of the whole macromolecule. A similar result might also be expected in the more complex case of the inducedfit model. Each case of this kind is probably better treated individually. For example, if we adopt the two-state model for hemoglobin, the elements of the characteristic matrix will be the same as the terms of the expanded form of the suitably modified binding polynomial given by Eq. (11), namely (1 +

Κκ^ηι

+

κ^^γ + U{1 +

KBaXm

+

KB^Y

VI. A n Example from Trout Hemoglobin What appears to be an almost perfect example of the principles of the two preceding sections is to be found in trout hemoglobin, which has been extensively studied in recent months by my colleagues in both Rome and Camerino. So far, results are limited to two more or less preliminary papers (Binotti et ah, 1971; Giovenco et at, 1970), but the work is going on and a more complete account will be given soon. Trout hemoglobin contains four different components, which can be readily separated by the modern techniques of protein fractionation and are designated as I, II, III, and IV in accordance with their relative speeds of migration in electrophoresis. The relative amounts of I, II, and IV are, respectively, approximately 20, 10, and 65%, component III being present as only a very small minority. Up to now, work has been concentrated on the two major components, I and IV, both of which exist under ordinary conditions as tetramers. Component I contains two types of chain, as shown by the two bands observed on electrophoresis of the denatured material. So far, however, it has proved impossible to separate the chains in the native state, and the tetrameric molecule gives no evidence of dissociation into dimers under any of the experimental conditions that have been tried. As­ sociated with this is the fact that the molecule fails to hybridize. It

222

JEFFRIES WYMAN

Y

0.5 L

logpO,

FIG. 1. Oxygen dissociation curve of component I of trout hemoglobin at 20° in 0.2 Μ phosphate buffer. Protein concentration Z-5 mg/ml. O , pH = 6.8; Δ , pH = 7.2; • , pH = 7.6. The smooth curve corresponds to η = 2.6.

contains no C terminal histidine or tyrosine. As for functional properties, it is highly cooperative in its oxygen binding ( n ^ 3 independently of pH), but shows no Bohr effect (see Fig. 1). The oxygen aflSnity, as well as the cooperativity, is only very slightly if at all dependent on temper­ ature and the maximum value of the enthalpy of oxygenation cannot exceed 3000 cal per mole of oxygen, as compared with about 12,000 for human hemoglobin; thus the corresponding free energy change, including the contribution due to cooperativity, must result primarily from entropy effects. Component IV likewise consists of two types of chain; however, un­ like Component I, it can be readily digested by the carboxypeptidases, and the results indicate that the C terminal residue of one type of chain is probably arginine and that the other type of chain ends with tyrosine and histidine. Like component I, this component shows no tendency to dissociate and fails to hybridize. But its most interesting properties involve its liganding behavior with oxygen and carbon monoxide. At alkaline pH (8-9) it shows high cooperativity, like component I (n = 3), but unlike component I it has an oxygen aflSnity which is dependent on temperature in accordance with a heat of oxygenation of about 12,000 cal. As the pH is lowered to 5 or 6 there is a profound change in the liganding behavior. As shown in Fig. 2, the value of η drops to 1 or less, and the oxygen affinity becomes so low that it is impossible to

223

ON ALLOSTERIC MODELS

1

2

1.5

2.5

log PO,

FIG. 2. Oxygen dissociation curves of component IV at different pH values and at 20°. Experimental conditions as in Fig. 1.

complete the binding curve, even with the use of pure oxygen at atmo­ spheric pressure; at pH 6 the pi/2 may be estimated as 1000 times or more greater than at pH 8.5. How is this behavior to be interpreted? In my view the most plausible picture is the following. The Bohr effect is local to the chains, as in the case of human hemoglobin, but, in contrast to the case of hemoglobin in its reaction with oxygen, it is grossly different for the two types of chain. At alkaline pH the chains are essentially equivalent; however, owing to the large difference in the two Bohr effects, they become widely different at acid pH, and this, in conformity with the allosteric principles described above, leads to a large decrease in η and the develop­ ment of a biphasic type of binding curve with a very high value of pi/zWe have looked at this behavior of component IV in terms of mechanism; it is also worthwhile to look at it teleologically. The trout, like many other fish, has the problem of controlling his up and down position in the water. A mechanism for this is provided by the swim bladder, a gas chamber whose volume determines the buoyancy of the fish. But clearly, in the absence of some further provision, this by itself

224

JEFFRIES WYMAN

would place the fish in a position of unstable equilibrium; if he were to descend, the increase of hydrostatic pressure would diminish the volume of the chamber and hence reduce his buoyancy—^just the opposite of what is required for him to keep his balance. To overcome this he must be in a position to secrete gas into the bladder, as need arises, against what may be a considerable pressure if he is at any significant depth, and in any case it will be necessary to replace the gas which is constantly going into solution. Clearly component IV provides a mechanism for doing this. As the hemoglobin passes through the gills it takes up oxygen under conditions where its oxygen aflSnity is high; when it passes through the rete mirabile, the vascular organ adjacent to the swim bladder, where the pH is low, its oxygen aflSnity is greatly reduced and the oxygen dissociation pressure becomes very high, potentially far above 1 atmo­ sphere. Thus the hemoglobin, exploiting to the maximum the principle of the Bohr effect, acts as a pump to force oxygen into the bladder against the opposing hydrostatic pressure. The question might be asked: What is the source of the energy expended in this process? The answer is clear: it is the movement of proton, carried by the blood, from a region of high activity in the rete mirabile to one of low activity in the gills and other tissues. The metabolism of the fish maintains the pH gradient, and we may suppose that the blood flow through the secreting organ, or perhaps the local pH in the rete, is subject to nervous or hormonal control in accordance with the needs of the moment. And what of the function of component I? This would seem to be the more prosaic one of assuring the normal transport of oxygen to the tissues. But why, in contrast to component IV and to the mammalian hemoglobins, should this molecule have been so designed as to have no heat of oxygenation? It is tempting to answer this question in the following way. It is of course essential for the actively swimming trout that oxygen transport should not be cut off or seriously reduced in cold water. In mammals body temperature is maintained constant within narrow limits, in spite of changes of temperature of the surroundings; their hemoglobin therefore operates always at constant temperature. In contrast, in the poikilothermous fish there is no such regulation of body temperature, and to make up for its lack there has been developed in the course of evolution a highly specialized form of the hemoglobin molecule which is insensitive to temperature. There is every reason to believe that the trout does not stand alone. Tuna fish hemoglobin behaves in much the same way (Rossi Fanelli and Antonini, 1960), and one may anticipate that other fast swimming fish will be found to show the same remarkable molecular adaptation. This remains to be explored in further experiments.

O N ALLOSTERIC M O D E L S

225

VII. Conclusion The fish hemoglobins represent only a very special case, special even among the hemoglobins. They were introduced simply as a possible example of the concepts of Sections IV and V. But although other respiratory proteins and enzymes behave quite differently, a very large number of them undoubtedly fall together within the class of allosteric macromolecules. In this discussion we have attempted to clarify the common features of all such systems as represented in the various models which have become current, starting with the "parent model" of Section II, of which both the MWC model and induced-fit model are special cases. But it should not be forgotten that other models are possible within the framework of the allosteric concept. Of the relations we have developed, only those of Section I are of general applicability. They bring out the usefulness of the concept of the binding polynomial and the associated characteristic matrix in describing any allosteric macro­ molecule. In conclusion, perhaps an apology is due to the reader for the some­ what abstract character of this discussion, for certainly the interest of the molecular biologist lies, and rightly so, in physical mechanism rather than in formalism. Apart from the fact that a precise mathematical formulation provides a powerful touchstone for the evaluation of any physical interpretation, a defense might be offered on the basis of a more positive and deeper philosophy. If it be true, as so persuasively maintained by G. H. Hardy (1967) in his charming little essay "A Mathematician's Apology," that mathematical objects are the most real elements of our experience, enjoying as they do a timeless and space­ less existence of their own which by far transcends that of the localized and transitory physical world around us, then surely it is in order to see how far we can go in displaying our physical models as special incarnations of these universal objects. REFERENCES

1. Antonini, E., and Brunori, M. (1971). In "Hemoglobin and Myoglobin in Their Reactions with Ligands," Res. Monogr.: Frontiers Biol. (A. Neuberger and E. L. Tatum, eds.), North-Holland Publ., Amsterdam. 2. Binotti, I., Giovenco, S., Giardina, B., Antonini, E., Brunori, M., and Wyman, J. (1971). Arch, Biochem. Biophys. 142, 274. 3. Giovenco, S., Binotti, I., Brunori, M., and Antonini, E. (1970). Int. J. Biochem. 1, 57. 4. Haber, J. E., and Koshland, D . E., Jr. (1967). Proc. Nat. Acad. Sd. U. S. 58, 2087. 6. Hardy, G. H. (1967). "A Mathematician's Apology." Cambridge Univ. Press, London and New York. 6. Hopfield, J. J., Shulman, R. G., and Ogawa, S. (1971). 7. Mol. Biol. 61, 425.

226 7. 8. 9. 10. 11.

JEFFRIES WYMAN Koshland, D . Ε., Jr., Nemethy, G., and Imer, D . F. (1966). Biochemistry Monod, J., Wyman, J., and Changeux, J.-P. (1965). / . Mol. Biol. 12, 88. Rossi Fanelli, Α., and Antonini, E. (1960). Nature (London) 186, 895. Wyman, J. (1965). J. Mol. Biol 11, 631, Wyman, J. (1967). J. Amer. Chem. Soc. 89, 2202.

5, 365.

Regulation of Uridylic Acid Biosynthesis in Eukaryotic Cells MARY ELLEN JONES

Department of Biochemistry School of Medicine University of Southern California Los Angeles, California I. General Pathway for Uridylic Acid Biosynthesis . . . . II. Genetic Control of Enzyme Levels in Fungi A. Genes for the First Two Enzymes of U M P and Arginine Biosynthesis B. Genetic Control of the Feedback Inhibition Site for the CPSaseump-ATCase of Fungi C. Metabolite Control of Gene Expression in Fungi . . . D. Summary and Comparison of Gene Regulation in Fungi with Bacteria III. The Eukaryotic Enzymes A. The Carbamyl Phosphate Synthetase-Aspartate Transcar­ bamylase Complex of Fungi and the Carbamyl Phosphate Synthetase-Aspartate Transcarbamylase-Dihydroorotase Com­ plex of Mammals B. Dihydroorotase C. Dihydroorotate Dehydrogenase D . The Orotidylic Enzymes E. Summary References

227 229 229 233 233 234 235

235 250 251 253 258 261

I. General Pathway for Uridylic Acid Biosynthesis In all organisms that have been studied in any detail, the biosynthesis of uridylic acid requires the six enzymes shown in Fig. 1 (132), namely, carbamyl phosphate synthetase (CPSase)* (1, 3, 50, 64, 73, 90, 91, 97, 119, 124, U9, 163); aspartate transcarbamylase (ATCase) (2, 8, 28, Jß, 63, 90, 96, 97, 115, 116, 118, 120, 127, 139, 146, 163); dihydroorotase (DHOase) {16,86,90, 94,104,1S9,167,170); dihydroorotate dehydrogen* Abbreviations used in this article: ATCase, aspartate transcarbamylase; CAA, carbamyl aspartate; CAP, carbamyl phosphate; CPSase, carbamyl phosphate synthetase; DHO, dihydroorotate; DHOase, dihydroorotase; DHOdehase, dihydrooratate dehydrogenase; PP-R-P; ribose-l-pyrophosphate-5-phosphate; OA, orotic acid; OMP, orotidylic acid; OMPdecase, orotidylate decarboxylase; OMPppase, orotidylate pyrophosphatase. 227

228

MARY ELLEN JONES

Ο DHOase

HN^^CH, H^OOH

H^COGH

Dihydroorotlc acid

© OMPppase

1 Ribose-5'-P

DHOdehase

HN^

Ittbose-5'-P Orotidyllc acid

Orotic acid

Uridylic acid UTP CTP TTP

Nucleic acids

FIG. 1. Enzymatic reactions required for uridylic acid synthesis and structure of the intermediates of the pathway.

ase (DHOdehase) {88,77, 90,107,109,110,139,153,154,167); orotidylate pyrophosphatase (OMPppase) and orotidylate decarboxylase (OMPdecase) {5, 24, 29, 53, 63, 72, 77, 118, 139, 152, 159, 169)* This pathway is usually regulated either by metabolite control of CPSase and/or ATCase activity {1, 2, 4, 8, 28, 37, 46, 64, 97, 115, 118, 119, 123, 163, 171), or by control of the rate of gene transcription so that the amount of enzyme present increases or decreases in response to the pyrimidine and purine nucleotide pools in simple bacteria {1, 6, 53, 63, 64, 114, ^18, 124, 172) and fungi {25, 72, 86, 87, 113, 162, 163). In multicellular organisms, feedback regulation can occur {149-151) and variations in enzyme ac*The known exception is Lactobacillus leichmanii, in which carbamyl phosphate is synthesized by the deimidation of arginine {60). It is possible that the same situation holds in Streptococcus jaecalis, i.e., that carbamyl-P is provided by phosphorolysis of citrulline derived from arginine {68).

URIDYLIC ACID BIOSYNTHESIS IN EUKARYOTIC CELLS

229

tivity are observed that may represent control of enzyme biosynthesis {U, 15, 19, 21, 22, 39, Iß, U, 50, 62, 66, 69, 70, 81, 84, 117, 120, 125, 126, IJfl-lIß, 146, 148, 155, 160, 173), although definitive proof, i.e., a direct measurement of an increase or decrease in the rate of change of enzyme protein, cannot yet be obtained because pure enzymes are not yet available. The studies using prokaryotic organisms have illustrated that a variety of regulatory patterns can be observed {1, 2, 6, 8, 28, 63, 70, 118, 124, 1^7, 172), and it is possible that when suflScient detail becomes available that a number of patterns will also be found to occur in eukaryotic organisms. It is only in the last five years that a sufficient number of studies have been made with eukaryotic organisms, and at the moment a pattern different from those seen in prokaryotes seems suggested. It appears possible that enzyme complexes may be extremely important in the control of pyrimidine biosynthesis in nucleated cells.* It is even possible that all six enzymes of this pathway may be part of a single, rather fragile complex in vivo. This manuscript will describe the somewhat limited facts available and try to relate data that seem to be important for further study. II. Genetic Control of Enzyme Levels in Fungi In eukaryotes, the only definitive genetic studies have been made in simple organisms, namely the fungi: yeast {72, 74, 76, 86, 87), Neurospora {31-34, 36, 56, 95, 106, 111, 128-130, 147, 162, 164, 165, 169), or Coprinus {45, 57), A . Genes for the First Two Enzymes of U M P a n d Arginine Biosynthesis

Genetic studies have shown that these three fungi possess two CPSases as indicated in Fig. 2. The CPSase which normally provides carbamyl phosphate (CAP) for arginine biosynthesis, C A P a r g , is coded for by two structural genes, arg'2 and -3 in Neurospora, while arg'12 is the Neurospora structural gene for ornithine transcarbamylase. In the pyrimidine pathway a single polar structural gene, pyr-3 in Neurospora and ura-2 in yeast, codes for both CPSase and ATCase. Many mutants lack both CPSase and ATCase activities; however, single mutants can be produced •There is evidence from J. Wild and W. Belser (see 161), detailed in Section III of this paper, that an enzyme complex containing DHOase, OMPppase, and OMPdecase exists in Serratia marcescens. O'Donovan and Neuhard (118) as well as Haywood and Belser (63) and Wild and Belser (161) suggested that such com­ plexes may be important for coordinate repression-derepression of unlinked genes of this pathway in bacteria (6, 63,162).

230

MARY ELLEN JONES repression

ATP

arg-2

Ii

HCOa' Glu-NHa ^

Ornithine + CAPare

+ +

arg-J

pyr 3a ura2

arg-12

) - Arginine

-Citrulline

'

\\ pyr 3d pyr 6 pyr 1 pyr 2 pyr 4 APUMP _ > CAA- -OMP —— Ρ ^ ——»-t.ÄÄ :*-DHO r -OA CAPuMP ura 5 ura 3 -UMP + ^^^z. 3 ^^^^^^a4 ^xj^ 1 Aspartate pyr 3

repression

FIG. 2 . Genes for carbamyl phosphate synthesis and utilization and for uridylic acid synthesis in Neurospora and yeast, indicating sites of metabolite control. Abbreviations used are: Glu-NHa, glutamine; CAParg, carbamyl phosphate synthesized by the carbamyl phosphate synthetase (CPSase) which is repressed by arginine, whose mutational loss causes arginine to become a required nutrient; CAPump, carbamyl phosphate synthesized by the CPSase which is repressed by uracil, whose mutational loss causes uracil to become a required nutrient; CAA, Lcarbamyl aspartate; DHO, dihydroorotate; OA, orotic acid; OMP, orotidylic acid; arg-2j argS, arg-12, Neurospora structural genes for CPSase (arg-2 and arg-3), which provides carbamyl phosphate for arginine biosynthesis, and for ornithine trans­ carbamylase {arg'12); pyr-l, etc., Neurospora genes for pyrimidine biosynthetic enzymes; ura-l, etc., yeast genes for pyrimidine (uracil) biosynthetic enzymes.

lacking only one of these two enzymes (Fig. 3). For example, in Neuro­ spora, mutants lacking only CPSase are called pyr-Sa while those lack­ ing only ATCase are called pyr-Sd (Figs. 2 and 3). The remainder of the genes for UMP biosynthesis are unlinked and are numbered as shown in Fig. 2 {pyr denotes the Neurospora genes, and ura denotes the yeast genes). In Neurospora, the carbamyl phosphate produced by each of the two synthetases is normally utilized only for its intended product, i.e., arginine or uridylic acid. Thus, a mutant having a defective arg'2 or arg-S gene will not grow in the absence of arginine, but it does not require uridine, whereas a mutant possessing a defective pyrSa gene requires uridine for growth but not arginine. This stringent flow of CAP into only one of the two biosynthetic pathways has been called "channeling" by Davis (54). Williams, Bernhardt, and Davis {163) have quantitated the amount

URIDYLIC ACID BIOSYNTHESIS

IN

EUKARYOTIC

1 ATCase-

CELLS

231

CPSase-

pyr-3gene 1 ATCase"

CPSase-

\

pyr-3d region | pyr-3a region direction of translation

Mutagen used Number of mutants Single mutation Double mutation ATCase' CPSase"

ATCase' CPSase'

UV light

20

1

6

Nitrosoguanidine

13

1

54

Acridine ICR-170 26

12

0

FIG. 3 . Schematic representation of the pyrS gene of Neurospora showing areas leading to loss of carbamyl phosphate synthetase (CPSase) and/or aspartate trans­ carbamylase (ATCase) activity, the direction of polar gene translation, and the type and number of mutants produced by various mutagens (23, 36, 129, 130, W, 164).

of CAP produced by the two CPSases by measuring the in vivo CAP levels in wild-type Neurospora under different growth conditions and in mutants lacking one or both of the two C P S a s e s . When both synthetases are missing (Table I, experiment 11) there is no detectable CAP present. In wild-type Neurospora, the CAP pool is 6 nmoles/gm dry weight if the cells are grown in minimal medium. If the CPSasOump is repressed (experiment 2) by growth on uridine then 5.4 nmoles of CAP are present. This figure can be interpreted to indicate that CPSascarg produces nearly 90% of the normal pool of CAP. In agreement with this interpretation is the datum of experiment 3 which shows that growth on arginine (repression of CPSase^rg) lowers the CAP pool to 0.7 nmole. Therefore, the pyrimidine biosynthetic pathway seems to utilize only a small percentage of the total CAP produced in wild-type cells grown in minimal medium. This figure (0.7 nmole) also agrees with the value of 0.4 nmole CAP/gm dry cell weight observed in mutants lacking CPSasearg (experi­ ment 5) or CPSasearg and OTCase (experiment 6). In addition, the CAP (3.5 nmoles) present in pyr-S mutants that cannot make UMP (ex­ periments 8 and 9) is consistent with experiment 2 which indicates that the biosynthesis of arginine consumes nearly 10 times as much CAP as pyrimidine biosynthesis. Davis had shown that overflow of CAP from one pathway to the other could occur if a double mutant was produced which lacked the

232

MARY ELLEN JONES TABLE I CHANNELING OP CARBAMYL PHOSPHATE IN Neurospora AND Loss OF CHANNELING BY DOUBLE MUTATIONS (34, 163)

arg-S and S

arg-lg

> CAPARG

A T P + Glu-NH, + H C O 3 -

pyr-Sa

• citruUine

pyr^Sd > CAPUMP

A T P + Glu-NHs + H C O 3 -

• carbamyl aspartate

Additions to media

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Mutant

For growth

Wild type Wüd type Wild type Wüd type arg-3 arg-3 orfir-5, pyr'3d pyr'3a pyr-3a and 3d pyrSa, arg-l^^ arg-S, pyr-Sa

— — — Arginine Arginine Uridine Uridine Uridine None Uridine and arginine

For enzyme repression None Uridine Arginine Uridine and arginine

—

— — — —

— —

Amount of - in vivo CAP (nmoles/gm dry weight) 6.0 5.4 0.7 0.2 0.4 0.4 6.6 3.8 3.0 30-38 0.0

transcarbamylase of the alternate pathway (see experiments 7 and 10). For example, the arg-S mutation produces a cell which requires arginine for growth and only accumulates 0.4 nmole CAP (experiment 5), but if a second mutation is placed in pyr-Sd gene so that ATCase is also defective, the double mutant only requires uridine for growth and the CAP pool rises to 6.6 nmoles because the CAP produced by CPSasCump can no longer be utilized for primidine biosynthesis. Arginine synthesis now occurs and the mutant no longer requires arginine for growth. Experiment 10 shows that a very large pool (35 nmoles) of CAP col­ lects from CPSasCarg in a pyr-Sa, arg-W mutant which lacks a CPSasCump and has a defective O T C a s e whose activity is low but suflScient to allow growth in minimal medium even in the absence of the pyr-Sa mutation; however, the presence of the pyr-Sa mutation allows the demonstration of CAP overflow from CPSasOarg into the pyrimidine pathway so that uridine is no longer required for growth. Davis had suggested that this channeling might be due to the fact that CPSasCump and A T C a s e were part of a single enzyme, and although CPSasCarg and O T C a s e were not similarly linked together, they might be physically separated from the CPSasCump-ATCase protein {S4). Davis (SS) has recently found that O T C a s e is in the mitochondria while the

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CPSaseump-ATCase is not. Therefore, his suggestion may be correct for Neurospora; indeed, it has been estimated from the specific activity of the homogeneous CPSaseump-ATCase enzyme (54) that wild-type Neurospora contains 2 nmoles/gm dry weight of this double enzyme (the number of CAP binding sites/mole enzyme is not known), so that the 0.4 to 0.7 nmole of CAP present in the CAP pool of the wild-type cell grown in an arginine-rich medium or of the arg-3 mutant may all be enzyme bound to the CPSasCump-ATCase. The double enzyme containing CPSase and ATCase activities does not guarantee channeling, however. Yeast (see Fig. 2) also contains the single structural gene, ura-2y for both enzymes, and a similar enzyme complex has been partially purified {97, 100); but yeast CPSase mutants do not show the channeling phenotypically {87), i.e., a mutant lacking only CPSasCump or CPSasCarg will grow as well in minimal medium as in a medium containing uracil or arginine, respectively. Only when these mutant cells are grown in a medium which represses the remaining CPSase, i.e., if a CPSasCarg- mutant is grown in a medium containing uracil, is growth rate depressed unless arginine is also added. B. Genetic Control of the Feedback Inhibition Site for the CPSaseump-ATCase of Fungi

This double enzyme is feedback-inhibited by UTP {99, 163), Both the CPSase and ATCase {37, 78, 99) activities of yeast are affected, but in Neurospora only the CPSase activity is inhibited by ÜTP {162). Not only can mutants be obtained which lack one or both of the enzyme activities (the three mutant types of Fig. 3), but also these three mutants can be obtained possessing or lacking feedback inhibition sites: there are therefore six known mutant types in yeast {86), Unfortunately, as will be discussed later, the genetic data available do not as yet allow one to decide whether the two catalytic sites plus the regulatory site belong to three separate polypeptide chains or to two, or even only to one polypeptide chain {86), C. Metabolite Control of G e n e Expression in Fungi

Jund and La Croute {72, 86) studied the regulation of the synthesis of the pyrimidine enzymes in yeast. Since none of the enzymes has been puri­ fied to homogeneity, the actual amount of enzyme protein could not be measured; however, each thesis presented, i.e., induction or derepression, was checked in several ways, including the amount of enzyme activity present as the gene dosage changed, so the variations in enzyme activity would seem to be changes in gene product, not in activation or in­ hibition of the enzyme activities. La Croute has found that the ura-2

234

MARY ELLEN JONES

enzyme activities, CPSase-ATCase, are repressed and derepressed by a nucleotide end-product. It is not known if the product is a uridine or cytidine nucleotide. Enzymes 3, 4, and 6, determined by genes ura-1, and ura-3, respectively, are all inducible. LaCroute was able to show directly by the use of special yeast mutants permeable to CAA and DHO that increases in the activity of these enzymes was indeed due to induction and that end-product levels do not control these genes (86). The normal inducer is not known, but the induction may be sequential; however, high levels of CAA or its products can induce DHOase, DHOdehase, and OMPdecase, while high levels of DHO can induce DHOdehase and OMPdecase. It was assumed that perhaps OMPppase might be induced in parallel with OMPdecase. These two enzyme activi­ ties are closely related in mammalian cells, both genetically (14^) and physically (5, 52, 78, 138), and appear to be physically related in Brewer's yeast (29), However, Jund and La Croute (72) have recently isolated OMPppase mutants and found that this gene is relatively un­ responsive to end products and, therefore, shows little change in activity under conditions for repression, derepression, or induction. On the other hand, either the gene or the stability of the enzyme may be controlled, for enzyme activity increases slightly in cells grown on azauracil as well as in a mutant resistant to 3-fluoroorotic acid (for-l mutant). The latter gene lesion decreases the incorporation of orotic acid, cytosine, and adenine into nucleic acids and increases the specific activity of OMPppase and OMPdecase while leaving ATCase, DHOase, and DHOdehase activities unaffected. In addition, CO2 itself affects the activities of the enzymes of this pathway in Neurospora as well as the enzymes of the arginine and purine pathways (25). D. S u m m a r y a n d Comparison of G e n e Regulation in Fungi with Bacteria

The genetic control of pyrimidine biosynthesis in simple eukaryotes differs from that seen in the bacteria that have been well studied (70, 118) in two major ways. First, most bacteria possess a single enzyme, usually the glutamine-utilizing CPSase for the source of carbamyl phos­ phate for both arginine and UMP biosynthesis. Therefore, in bacteria the end-product repression of the single structural gene for this CPSase is controlled by both arginine and pyrimidine nucleotide. In addition, end-product inhibition by UMP and activation by ornithine is indicative of the fact that the product of this single enzyme serves both pathways. In contrast, when two enzymes are present to provide carbamyl phos­ phate for each pathway, as in the fungi, then repression occurs only for

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the structural gene for the particular CPSase which normally serves the given pathway. This is true even in yeast, where tight "channeling" of carbamyl phosphate to its "normal" route is not observed {87, 98, 100), as well as in Neurospora, where tight channeling is found. In addition, end-product inhibition can also be specific for the pathway the synthe­ tase is to serve, as is the case with the CPSaseump of Neurospora and yeast, which is not affected by ornithine. Second, in many bacteria all of the six enzymes are under repressionderepression control, except for the pseudomonads, where no genetic control is apparent {63). Although the nature of the corepressor and the degree of repression or derepression is different for CPSase, ATCase, and the remaining four pyrimidine enzymes, repression is the unitary type of control, which ensures that all the enzymes either increase or decrease in response to an accumulation of pyrimidine products. Also, the range from full repression to full derepression for ATCase can be very large, about 200-fold {6). In contrast, the fungal control is more con­ servative, with only a 5-fold difference between full repression and derepression; however, both CPSasCump and ATCase are presumably coordinately controlled since they share a common gene. The other enzymes, except for OMPppase, are induced by a pathway intermediate which is either a precursor of the substrate or the substrate for enzymes 3, 4, and 6; therefore, these enzymes would tend to increase on dere­ pression of the CPSasCump-ATCase gene, or if a secondary substrate such as PP-R-P were limiting, or when a normal inhibitor of the terminal enzymes, such as P P i , OMP, or UMP, accumulates.

III. The Eukaryotic Enzymes A . The Carbamyl Phosphate Synthetase-Aspartate Transcarbamylase Complex of Fungi a n d the Carbamyl Phosphate Synthetase-Aspartate TranscarbamylaseDihydroorotase Complex of M a m m a l s 1.

HISTORY

Although the first carbamyl phosphate synthetase known, CPSase I, (see Eq. 1 of Table II) was found in the mitochondria of mammalian and ureotelic vertebrate liver {47, 105), it became obvious that this enzyme could not be the only synthetase in mammalian cells and that a source of CAP had to exist in nonureotelic animals. The principal reason for seeking a second CPSase was the necessity for de novo pyrimidine

236

MARY ELLEN JONES TABLE II THE CARBAMYL PHOSPHATE SYNTHETASES OF THE ANIMAL KINGDOM«

Type I II

IIP

Reaction catalyzed Ac-glu • NH2CO2PO3«- + 2 A D P 3 - + 2H+ Glu-COirNHa + HCOs- + 2 A T P - + H2O -> NH2CO2PO32- + 2 A D P - + GIU-CO2- + 2H+ Ac-glu GIU-CO2-NH2 + HCO3- + ATP*- + H2O > NH2CO2PO3''- + ADP«- + GIU-CO2NH4+ + HCOj- + 2ATP*-

References 47, 106 3, 91

166

« Ac-glu, iNT-acetylglutamate; GIU-CO2-NH2, glutamine; Glu-C02~, glutamic acid. * The number of moles of ATP required is not known.

biosynthesis in nonhepatic and/or nonureotelic tissues (69), particularly certain rapidly dividing tissues, such as pigeon liver (13), fetal mam­ malian tissues (79, 81, 117), and cancer cells (71, 85). The case was most clear for certain cells like the Ehrlich ascites cell which synthesized pyrimidine nucleotides in vitro from HCOi" (49, 85), ammonia and as­ partate (89, 134) with no known or readily demonstrable CPSase (13, 71, 85), In addition, the discovery that CAP is not a normal blood constituent (55) made it essential that all mammalian tissues capable of de novo pyrimidine synthesis in vivo (other than liver, small in­ testinal mucosa, and perhaps kidney) (71, 131, 136), all of which have CPSase I, contain another synthetase. Lowenstein and Cohen (96) had already shown that most of these tissues lacking CPSase I had ATCase activity which tended to parallel the rate at which cell division was known to occur. Salzman et al. (134), using cultured HeLa cells, have shown that N-3 of the pyrimidine ring was derived from the amide Ν of glutamine, not from ammonia; therefore, the new synthetase should utilize glutamine as a nitrogen source. CPSase II (Eq. 2 of Table II) was difficult to demonstrate in mam­ malian cell homogenates because of its low activity and because it had limited stability in homogenates, but its existence was demonstrated in rapidly dividing cells, namely intact Ehrlich ascites cells (49, 50) and slices of hematopoietic spleen (65, 149). The discovery that the enzyme could be stabilized in sucrose homogenates if Mg-ATP was added (50, 149) or if 30% dimethyl sulfoxide or 5% glycerol was used as sol­ vent (150) allowed the wide distribution of the enzyme in mammalian tissues to be established (150,173). The activity of mammalian CPSase II seems low, particularly when compared to the large amount of CPSase I in adult rat liver which is

XJRIDYLIC ACID BIOSYNTHESIS I N EUKARYOTIC CELLS

237

necessary and not excessive for the CAP required for urea biosynthesis (69, 135), However, the activity of CPSase II found in several mam­ malian cells, though possibly rate limiting, is suflScient to supply the CAP required for pyrimidine biosynthesis of the given tissue (50, 66, 67, 149), Its presence has also been confirmed in a few nonureotelic animal tis­ sues, such as fetal rat liver and adult pigeon liver (50), chicken liver (122), and frog eggs (90), as well as in plants (119, 120), yeast (97, 100), and Neurospora (95, 162, 163), The enzyme will use ammonia as a nitrogen donor; however, since the Km for glutamine is near 10"^ Μ while that for NH4^ is nearly 100-fold larger, and since the intracellular concentration of glutamine in multicellular animals is usually much higher than that of ammonia, it seems logical to assume that glutamine is the normal N-donor. Indeed, in the experiments by Salzman et al, with intact HeLa cells, this is the case (134) · The specificity of the enzyme, and therefore of uridylic acid biosyn­ thesis, for glutamine is also evident in intact Ehrlich ascites cells, for these cells incubated in vitro are inhibited by glutamine analogs whether the nitrogen source is ammonia or glutamine. The glutamine analogs do not inhibit CPSase I (50), The most effective analogs are chloroketone (122), 0-carbamyl-L-serine and diazonorleucine (50); azaserine is usu­ ally a markedly less effective inhibitor of CPSase II. The initial binding of all inhibitors is competitive with glutamine, so the analogs can be relatively ineffective unless their concentration is high relative to the glutamine concentration of the tissue slices or homogenate. In fact, in crude extracts of many mammalian tissues, one does not need to add glutamine or ammonia as a substrate to have full enzyme activity; rather, it is usually necessary to remove glutamine from most homogenates, to demonstrate that either glutamine or ammonia are required as substrates. Recently, a third synthetase, CPSase III of Table II has been dis­ covered in snails (156), This enzyme combines the characteristics of CPSase I and II and may represent the evolutionary "intermediate" in the conversion of a CPSase II enzyme into a CPSase I enzyme. T&e snail CPSase III, like CPSase II, utilizes glutamine as its preferred nitrogen substrate but shows an absolute requirement for the allosteric cofactor, acetyl glutamate, as does CPSase I. The final evolutionary change to convert an enzyme like the snail CPSase III to the ureotelic CPSase I is the loss of the ability to utilize glutamine as a substrate. The ability of CPSase II to use glutamine is a function of a special glutaminase subunit [MW = approximately 45,000 (103, 158) ]; it will be of interest to see whether the snail CPSase III also possesses a glutamine subunit, which the rat liver CPSase I apparently does not (48).

238

MARY E L L E N

JONES

2. STRUCTURE O F T H E COMPLEXES

α. Fungi. As genetic studies in Neurospora and yeast had suggested, the CPSasCump (which is a glutamine-dependent enzyme and, therefore, a C P S a s e II) of these fungi copurifies with ATCase. The ratio of ATCase activity/CPSase II activity is about 2 fqr the highly purified yeast enzyme (100), and 10 for the highly purified Neurospora enzyme (163). It is possible that the channeling observed in Neurospora is aided by the relatively greater ATCase activity of the Neurospora complex, which may fix CAP more effectively; other more subtle differences in structure of the two fungal enzymes may be even more important for channeling. The CPSase of the yeast and of the Neurospora enzyme complexes is inhibited nearly 100% by 10"^ Μ UTP (99, 162). However, differences in the detailed structure of the two enzymes are again appar­ ent, for UTP has opposite effects on the molecular weight of the yeast and Neurospora enzymes (Fig. 4, note that FB indicates the feedback inhibition site) (100, 163), and while the ATCase of yeast is inhibited by UTP (37, 75, 99), the ATCase activity of the Neurospora complex is not (162). UTP, glutamine, and Mg2+ are required for the yeast enzyme to have a molecular weight of about 8.1 X 10^ (100). In this form the enzyme is Neurospora enzyme:

-CPSase 630,000

-"^^

"^CPSase 385,000

Yeast enzyme: UTP + Gin or FB

-ATCase -vJPSase 810,000

Mg + Gin "

UTP + Gin

j—ATCase ^

*"A_cpsase 380,000

FB—Λ '

^ΖδΟ^,Βοο" -CPSase

50" for 5 min or DEAE chroma­ tography ATCase 140,000

FIG. 4 . Apparent molecular weights, and interaction between the feedback ( F B ) regulation and catalytic sites of the fungal CPSase-ATCase complexes (97, 99, 100, 163).

URIDYLIC ACID BIOSYNTHESIS

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most sensitive to UTP and possesses both enzyme activities. On removal of either UTP or Mg ion the larger structure is lost, and now both enzymes cosediment in a sucrose gardient as though they had a molecular weight of 3.8 X 10^ The feedback inhibition of the CPSase activity is unaltered, but the ability of UTP to inhibit ATCase is only about onethird to one-half that observed previously. Removal of UTP, glutamine, and Mg ion liberates a portion of the CPSase activity from the ATCase activity. The CPSase activity remaining is still fully inhibitable by UTP. Heat treatment at 50° destroys in parallel the CPSase activity and the feedback inhibition of CPSase but leaves an ATCase whose molecular weight is only 1.4 X 10^ and this activity is completely in­ sensitive to UTP (97, 99). These data suggest, but do no prove, that in yeast the single gene for these two enzymes might code for at least two polypeptide chains, one polypeptide possessing ATCase activity whose molecular weight is 1.4 X 10*^ (or some integer of this figure), and at least one other polypeptide chain containing the CPSase active site as well as the feedback regulation site. Obviously the CPSase feedback unit could be composed of several different polypeptides {103, 158), a glutaminase subunit, an ammonia-dependent CPSase subunit, and a regulatory subunit. If there are at least two separate polypeptides, the fact that mutations throughout the entire gene length can effect both enzyme activities or the feedback site could be explained if there were critical regions in both polypeptide chains designating amino acid residues essential for the separable polypeptide chains to interact with one another. A mutation at these interaction sites could simultaneously alter both enzyme activities or the feedback inhibition site. It is of considerable significance that during derepression in yeast the ATCase activity becomes less sensitive to feedback inhibition [76), This could indicate noncoordinate synthesis of a nonregulated ATCase polypeptide, or instability of the peptide (s) containing the C P S a s e activity and its feedback regulation site. Since there is no tight chan­ neling of C A P produced by the two C P S a s e s of yeast, additional noncoordinate ATCase synthesis could result in deflection of some of the CAP produced by CPSasearg into pyrimidine biosynthesis. h. Mammals. In mammals, CPSase II and ATCase sediment together in sucrose gradients {59, 70, 137) from a dimethyl sulfoxide-glycerol ex­ tract, and they copurify and are eluted from hydroxyapatite together {59). The molecular weight found when the two activities are together varies, perhaps with the particular conditions or the molecular weight markers used by the laboratory studying the enzymes. Shoaf and Jones {137) found a value of 7.5 to 8.5 X 10^ for the protein peak containing the two enzyme activities, whereas Hoogenraad et al {59) obtained a

240

MARY ELLEN JONES

GS

C PSase ATCase DHOase

I

6 H I

α

Φ (Λ

17

19 BOTTOM

FIG. 5. Cosedimentation of the CPSase, ATCase, and DHOase activities of Ehrlich ascites cells in a 5 to 20% sucrose gradient. The homogenate was prepared in 5% (v/v) glycerol, 30% (v/v) dimethyl sulfoxide, and 0.05 Μ Tris with a final pH of 7.5 (137). The linear 12-ml gradient was centrifuged at 5° for 20 hours at 40,000 rpm in a Beckman-Spinco L2-65B preparative ultracentrifuge using the SW 41Ti rotor. OS represents the peak for Eschenchia coli glutamine synthetase added as an internal marker (kindly provided by Dr. Earl R. Stadtman). The apparent molecular weight determined from the relative migration of the CPSase-ATCaseDHOase peak with respect to the OS peak ranges between 750,000 and 850,000 in various experiments.

molecular weight of 6 X 10^. Shoaf and Jones found that this peak also contained DHOase activity (Fig. 5); therefore, the first three enzymes of pyrimidine biosynthesis appear to form a single complex (137, 138). Such a large protein complex undoubtedly contains subunits; at the present moment, since it is only partially purified, one cannot estimate either the number or variety of the subunits. The unusual solvent 30% dimethyl sulfoxide-5% glycerol was initially used by Tatibana and Ito [150), who found that this solvent stabilized the mammalian CPSase activity for a reasonable period. If the dimethyl sulfoxide concentration is lowered, the 8 X 10^ dalton complex dissociates and CPSase activity

URIDYLIC ACID BIOSYNTHESIS

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CELLS

is slowly lost (70). However, one can find some CPSase activity remain­ ing after the 20 hours required for a sucrose-gradient separation (Fig. 6). The remaining CPSase activity is associated with a protein that travels as though it had a molecular weight of about 1.7 X 10^ The recovery of the ATCase and DHOase activities is better; these two activities remain associated with one another, but they now appear in two protein peaks whose mass is estimated to be approximately 3.5 X 10° and 6.5 X 10° daltons. The picture is even more complicated, for a mammalian tissue ho­ mogenate can contain at least three ATCase fractions. Koskimies et al

19 BOTTOM

FIG. 6. Dissociation of the CPSase-ATCase-DHOase complex of Ehrlich ascites cells. The conditions for this experiment have been described (137). The complex of Fig. 5 was concentrated by centrifugation at ΙΟΟ,ΟΟΟβτ for 4.5 hours at 0°C. The pellet was resuspended in the same medium except that the dimethyl sulfoxide concentration was reduced to 10%. This material was layered over a 12-ml, 5 to 20% linear sucrose gradient, containing the glycerol-dimethyl sulfoxide-Tris medium, and was centrifuged for 15 hours at 40,000 rpm in a SW 41Ti rotor in a BeckmanSpinco L2-65B preparative ultracentrifuge. L D H indicates the migration of rabbit muscle lactate dehydrogenase, and CATase represents the migration of beef liver catalase; these enzymes were added to the homogenate as standard molecular weight markers.

242

MARY ELLEN JONES

(83), preparing homogenates in 0.25 Μ sucrose containing Mg-ATP and albumin to stabilize the ATCase (and CPSase) observe two peaks of ATCase activity in the 100,000 g cell supernatant of a variety of tissues. The apparent mass of these proteins is 6 X 10^ and 9 X 10^ daltons. The ratio of ATCase activity in these two peaks is apparently constant for a given tissue from an animal at a given developmental age, but this ratio varies from one tissue to another and is not the same for a given tissue in a fetal animal vs the same tissue from an adult (83). The sig­ nificance of the two forms is not known as yet, nor is it known whether they both contain CPsase activity. When a liver supernatant was di­ gested for 15 minutes at 37°C with either 0.0005% trypsin or 0.03% papain, very little ATCase activity was lost, but the enzyme activity now sedimented as if it had a molecular weight of 8 X 10* and the apparent Km for carbamyl phosphate had increased about 20-fold. These data do not help describe the construction of the large complex, but they may indicate that the ATCase activity resides in a small compact poly­ peptide structure which resists proteolysis when the larger complex is exposed to these two proteolytic enzymes. Inagaki and Tatibana (62) found three forms of ATCase activity in hematopoietic spleen. Table III shows the results of their simple cell fractionation. It is clear that quite a large fraction of the total ATCase activity of the homogenate is not recovered in either the 13,000 g or the 100,000 g supernatants. From the 100,000 g supernatant the authors were able to separate two ATCase subfractions by purification on hydroxyapatite. One of these forms had an apparent s value of 19 S (ca. 6 X 10^ daltons) in a sucrose density gradient while the second was larger and gave an extremely broad peak. The smaller enzyme was converted to the larger enzyme (as judged by the elution pattern from hydroxyTABLE III DISTRIBUTION OP ATCASE ACTIVITY IN MOUSE SPLEEN (62) Activity"

Fraction Homogenate'' 13,000 g Supernatant 105,000 g Supernatant 105,000 g Pellet

Normal mouse

— —

15 0.7

Acetylphenylhydrazinetreated mouse 40 25 16 3

« Micromoles C A A formed in 20 minutes at 37°C at pH 9.2/gm wet weight of spleen. * Homogenate prepared by addition of 9 volumes of ice-cold 0.25 Μ sucrose containing 0.01 Μ potassium phosphate buffer (pH 7.4) and 2 mM mercaptoethanol.

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apatite) after storage at 0°; once again total enzyme recovery was low. As indicated in Table III, there is a small portion of the enzyme that comes down with the microsomal (100,000 gf) pellet. This activity is of some interest, despite its apparently small contribution to the total ATCase activity of the homogenate, since it increases markedly in the spleen of mice treated with acetylphenylhydrazine. Bottomley and Lovig (10) had previously observed that ATCase was not present enth^ely in the soluble supernatant of mammalian tissues. In addition, Spors and Merker (145) in histochemical studies find ATCase localized at the cytoplasmic edge of ribosome-coated membranes. It is possible therefore that all these forms of ATCase may not be equally active in the cell and that some of the characteristics one sees in vitro are not truly repre­ sentative of in vivo function or physical character of the enzyme. c. Plants, Only a few studies have been made in plants, and it is not known whether plants possess only one or two CPSases. The CPSase of Alaskan pea seedling (Pisum sativum) is a CPSase II (119), which appears from its regulatory properties, discussed below, to resemble the CPSase II of E, coli (4) in that ornithine increases its activity. This could indicate that plants, unlike other eukaryotes, possess a single CPSase which serves both the pyrimidine and arginine biosynthetic pathways. The molecular size and intracellular location of this synthe­ tase are unknown, nor is it known whether this CPSase cosediments with either ATCase or OTCase. Recently the ATCase of Phaseolus aureus (mung bean) has been partially purified (IW), The enzyme activity was associated with a protein having a molecular weight between 83,000 to 93,000 and had been purified away from a CPSase II activity which resembles the enzyme found in the Alaskan pea. It has been re­ ported that germinating lettuce seedlings homogenized in 0.25 Μ sucrose contain two ATCases (116); one sedimenting with the "mitochondrial" pellet represented one-third of the total activity of the homogenate and was quite labile; the second ATCase, representing two-thirds of the activity of the homogenate, remained in the soluble supernatant and was more stable. In a number of plant extracts prepared with 0.1 Af a single ATCase was found, which was in the supernatant fraction of the homogenate (14^). d. Invertebrates, A new type of CPSase, CPSase III of Table II, occurs in invertebrates (156, 157), This enzyme utilizes glutamine as the nitrogen source and requires iV-acetyl glutamate as cofactor (156), The enzyme utilizes ammonia extremely poorly. The activity in the absence of acetyl glutamate is about 10% of the activity in its presence. The majority of this enzyme is localized in the mitochondria of the land snails Strophocheilus, Otala, and Helix, and the planarium Bipalium (9),

KHCO3

244

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JONES

In the earthworm, Lumbricm, CPSase III is also in the soluble super­ natant. It is thought that CPSase III is concerned principally with arginine biosynthesis in Strophocheilm, for ornithine transcarbamylase and CPSase III are both localized in the mitochondria while ATCase is in the soluble supernatant. There is, however, no known CPSase in the supernatant. Invertebrates also have a CPSase similar to CPSase I which can occur either in the mitochondria or the soluble supernatant fraction of a homogenate (9). There is a suggestion that CPSase II may be present in the soluble supernatant of Otala, Helix, and Bipalium, for this cell fraction utilizes glutamine as an Ν donor, but the stimulation of the CPSase activity of this cell fraction by acetyl glutamate is very small or nonexistent in the case of Helix. 3. METABOLITE CONTROL O F C P S A S E - A T C A S E ACTIVITY

a. Fungi. Most of the detailed studies have been made on the yeast enzyme complex. Only one nucleotide, UTP, inhibits the CPSase and ATCase activities of this complex. ATP does not activate either enzyme by itself, but it can annul the UTP inhibition as though it could be bound in place of UTP, but without producing a conformational change in the catalytic sites. Unlike the E. coli CPSase, the yeast enzyme is not influenced by ornithine. Despite the fact that tight channeling is not observed in the intact yeast cell, the ATCase of the CPSase-ATCase complex preferentially utilizes the CAP synthesized by the CPSase of the complex [98). Lue and Kaplan demonstrated that added ^^C-CAP did not dilute the ^^C-HCOa" converted to CAA until its concentration reached 1 mikf. In addition, CAP formed as a product of the CPSase activity of the CPSase-ATCase complex preferentially went to the ATCase of the complex when E. coli OTCase and ornithine were added unless the added OTCase exceeded the ATCase activity of the complex When the OTCase activity was 5-fold greater than the ATCase activity of the CPSase-ATCase complex the amounts of CAA and citrulline formed were equal. The feedback inhibitor, UTP, stabilizes the yeast and Neurospora complexes. As mentioned earlier, it stabilizes the feedback inhibition site for the yeast enzyme (97) and it prevents inactivation of the coldlabile Neurospora enzyme at 0° (162). In addition, it can affect the molecular size of both of these proteins (see Fig. 4). The substrate curves for both carbamyl phosphate and aspartate with the ATCase activity of the yeast CPSase-ATCase complex are hyper­ bolic. The addition of UTP does not affect the Km values; rather, it affects only F^ax (75). The inhibitory effect of UTP can be overcome by the addition of ATP (75).

URIDYLIC ACID BIOSYNTHESIS

IN

EUKARYOTIC

CELLS

245

b. Mammals. UTP is also the major feedback regulator of the mam­ malian CPSase II activity of the CPSase-ATCase complex (67, 93,149). However, with the mammalian enzyme UTP reduces the binding of ATP to the CPSase. The normal ATP curve is sigmoidal (50, 93, 173), and UTP increases the sigmoidicity. It appears that ATP may be both a sub­ strate and an allosteric activator for this CPSase (93). Some inhibition of the mammalian CPSase II has been observed with dUTP, CTP, dCTP, CMP, and dCMP (93). Both ATP (50, 93) and UTP (93) stabilize the mammalian CPSase II, but ATP is more effective than UTP, and mixtures of the two give an intermediate stabilization rather than an additive effect, a result suggesting that these nucleotides com­ pete for a common site. A very important observation has recently been made by Tatibana and Shigesada (151). They find that the mammalian CPSase II is stimu­ lated 2- to 3-fold by 20 μΜ PP-R-P. This regulation provides another interconnection between purine and pyrimidine biosynthesis. The ATCase activity of the mammalian CPSase-ATCase complex, like that of the Neurospora enzyme, does not seem so far to be signifi­ cantly affected by nucleotides (18, 30, 59, 126). However, these studies have been made only under a limited number of conditions. Bourget and Tremblay* (11) have recently found that orotic acid synthesis from either bicarbonate or CAP by rat liver slices is inhibited by uridine while the synthesis of orotate from CAA was not affected by the presence of uridine. This observation suggests that, regardless of where ATCase is positioned within the cell, it can be inhibited by a uridine product, per­ haps UTP, as is the CPSase II. This result might also indicate that the lowering of the PP-R-P pool decreased both CPSase and ATCase ac­ tivities. It is still possible therefore that the mammalian enzymes may respond together to inhibitors and activators. The CAP saturation curve for the rat liver tissue slices is sigmoidal, with half-saturation being observed near 2 mili CAP. Addition of uri­ dine moved the 50% saturation point to 5 mili CAP. Since the for the ATCase of the CPSase-ATCase-DHOase complex is near 10"« Μ in two studies (59, and Shoaf and Jones, unpublished results) and the curve is hyperbolic, it is hard to know whether the sigmoidal curve ob­ served by Bourget and Tremblay is related directly to the saturation of the intracellular ATCase or if the sigmoidal CAP saturation curve re­ flects the added effect of a number of processes, one of which might be permeability. In summary, studies on the feedback control of the mammalian en*Dr. Tremblay has recently informed the author that his laboratory is having some difficulty in reproducing the experiments with rat liver of reference 11. How­ ever, in a few other tissues, such as mammary gland, uridine does inhibit the incorporation of bicarbonate into orotic acid.

246

MARY ELLEN JONES

zymes free or in complex are not complete. Certainly, the CPSase is strongly regulated by UTP [and to a lesser extent by other pyrimidine nucleotides (93)] and PP-R-P. The regulation of ATCase is open for further studies, which now require that an attempt be made to relate the kinetic parameters to physical state of the enzyme under the con­ ditions of assay. c. PZanis. O'Neal and Naylor (119) have purified a CPSase II from Alaskan pea and Ong has studied this enzyme in mung bean extracts (IW). The enzyme diifers from the CPSase II of other eukaryotes in that its activity is increased by L-ornithine which acts as if it were a positive effector (119, 120), L-Ornithine also protects the enzyme against inactivation (119, 120). In addition, the most effective inhibitor was UMP although other nucleotides also inhibited (119, 120). In any given series of nucleotides of the same base the inhibition decreased with phosphorylation, i.e., monophosphate > diphosphate > triphosphate. Al­ though these characteristics are not identical to those of the CPSase II isolated from E. coli, they more closely resemble this enzyme than they do the eukaryotic enzymes listed above. In particular, the stabilizing and activating effects of ornithine suggest that this enzyme may also serve the arginine pathway. This enzyme is the only CPSase that has been reported in plants (119,120). Kleczkowski (82) has found that glutamine was a better substrate than ammonia for citrulline synthesis by pea seedling extracts. The first studies on the metabolite regulation of a plant ATCase were those of Neumann and Jones (115), who found that the ATCase of the soluble supernatant fraction of a germinating lettuce seedling was in­ hibited by pyrimidine nucleotides. By far the most effective inhibitor was UMP; UDP was less effective than UMP, and UTP was less so than UDP. This progression is similar to the CPSase of Alaskan pea, and it will be interesting to see whether the plant kingdom in general possesses a type of regulation different from that seen in the animal kingdom. Ong (120) has recently reported a partial but extensive puri­ fication of this enzyme from the mung bean, Phaseolm aureus, and has observed the same inhibition pattern, i.e., UMP > UDP > UTP. The inhibition is competitive with CAP but noncompetitive with aspartate. The substrate curves are normally hyperbolic, but the CAP curve be­ comes sigmoidal in the presence of UMP, which is reminiscent of an inhibition pattern seen with crude extracts of some pseudomonads and Azotobacter species (8, 116). Despite the fact that the pea CPSase II and the mung bean ATCase both show the same nucleotide inhibitor specificity, there is no indication as yet that they form an enzyme com­ plex. As mentioned earlier, the effect of ornithine on the plant CPSase

URIDYLIC

ACID B I O S Y N T H E S I S I N E U K A R Y O T I C CELLS

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and the fact that two groups have observed only a single enzyme in two different plant sources suggests that a single enzyme serves both the pyrimidine and arginine pathways in this kingdom. 4. CHANGES I N TOTAL CELLULAR ACTIVITY O F CPSASE I I , ATCASE A N D D H O A S E I N MAMMALS

As mentioned above, the tissue distribution of the arginine and py­ rimidine biosynthetic enzymes differ in mammals. The first two enzymes of the arginine pathway are limited to liver, intestinal mucosa, and per­ haps kidney. CPSase II and ATCase are widely distributed (67, 96, 160, 173) in both normal and cancer tissues. In addition, the amount of these enzyme activities per cell or tissue wet weight increases or decreases as the rate of cell division (44, 160, 173, 174) or rate of growth of a tissue increases or decreases, as observed with rat liver after partial hepatectomy or at the end of fetal development and after birth (60, 69, 81, 161). It cannot be stated whether the three activities always parallel one another over a short time scale, but they obviously do so over a longer interval. A similar tendency for ATCase and DHOase levels to parallel one another and to parallel the growth rate of tissues has also been recorded (44, -^4^)· These data fit well with the idea that the CPSase II-ATCase-DHOase complex found in the Ehrlich ascites cell may be an important entity in most mammalian cells. However, changes in the levels of these enzymes may not be extremely tightly coupled, for Bresnick et al (19) found an increase in ATCase and DHOase levels in rats given a diet containing 1% orotic acid. The activity of the two enzymes increased more or less simultaneously, but when orotate was removed the DHOase activity returned to normal in 2 days, a rate that was twice as fast as the decrease of the ATCase activity. This experi­ ment of Bresnick might indicate that regulation of the mammalian en­ zymes differs significantly from that observed in yeast. If the increased activity of these two enzymes was due to increased synthesis of ATCase and DHOase, it is possible that DHOase synthesis was induced because orotate is an inhibitor of DHOase (17) and therefore the CAA pool might have been increased. In the yeast model, ATCase synthesis would be expected to decrease since it is under repression-derepression control, and Bresnick found that the pyrimidine nucleotide pool was increased. However, if there are several forms of ATCase in the cell and if all forms are not equally important functionally, then one does not know the true significance of the observed changes. Bresnick's observation that orotic acid feeding causes an increase in the ATCase and DHOase activity levels of rat liver has a parallel in human tissue, for Smith (142) found that a child with untreated oroticaciduria, a situation resembling

248

MARY E L L E N J O N E S

the feeding of orotic acid, had high ATCase and DHOase levels in the erythrocytes. Uridine therapy, which reduced the amount of orotic acid produced and excreted by this child, led to normal ATCase and DHOase levels. It would seem, therefore, that these enzyme activities may change in mammals in the same direction as does the tissue orotic acid concentration. Ito and Uchino (66) have studied changes in CPSase II activity and in the rate of ^ * C 0 2 incorporation into C-2 of the uracil ring of acidsoluble uridine nucleotides of cultured lymphocytes during their trans­ formation into blast cells by phytohemagglutinin. The specific activity of the enzyme in the 100,000 g supernatant increased nearly linearly be­ tween 1 2 and 4 8 hours, preceding somewhat the increase of " C O 2 in­ corporation into the acid-soluble nucleotide which occurred after 2 4 hours. By mixing extracts from untreated and treated cells they could demonstrate that there was no activator in the treated-cell extracts nor an inhibitor in normal cell extracts, and indeed either puromycin or actinomycin D halted the increase in enzyme activity caused by phyto­ hemagglutinin addition. This work then is the first clear demonstration that under a controlled situation demanding an increased de novo py­ rimidine nucleotide biosynthesis to yield net RNA formation, the syn­ thesis of CPSase II may be increased.

5. CHANNELING O F CAP

I N MAMMALS

Tremblay {12, 112) and his co-workers have addressed themselves to the question of whether the mitochondrial CPSase I of liver, intestinal mucosa, and kidney could provide CAP for pyrimidine biosynthesis. Natale and Tremblay [112) clearly demonstrated that CAP could move out of the mitochondria and be trapped by an excess of added E. coli ATCase. More recently Bourget et al (12) have studied the incorporation of H ^ ^ C O a " into RNA in rat liver slices and have tried to differentiate between the relative contribution of the cytoplasmic CPSase II and the mitochondrial CPSase I toward this synthesis. A summary of their data is given in Table IV. When ATP is minimal, there is a blank incorpora­ tion of 2.7 nmoles of bicarbonate. If endogenous ATP is available, the bicarbonate incorporation increases to 6.5 nmoles. This incorporation could be due to endogenous CPSase II (or CPSase I), particularly since added glutamine or ammonia [either will serve as a substrate for CPSase II (50, 150, 173)] do not significantly increase the incorporation. Liver has a high glutamine concentration of about 5 mM (102). Since the Km for glutamine for CPSase II is near 10"^ M, one seldom has to add glutamine to homogenates to see full activity; in fact to see a glutamine effect with homogenates one usually has to separate the high

URIDYLIC ACID BIOSYNTHESIS IN EUKARYOTIC CELLS

249

TABLE IV« EFFECT OF ATP SUPPLY, ACETYL GLUTAMATE (AC-GLU) AND AMMONIA OR GLUTAMINE ADDITION ON THE INCORPORATION OF I^C-BICARBONATE INTO RNA OF SURVIVING RAT LIVER SLICES (12) Additions affecting Ν source for C A P NH4+ and Available ATP

None

Glutamine

NH4+

Ac-glu

Ac-glu

1. Minimal, i.e., DNP* and oligomycin added 2. Endogenous 3. Enriched a. ATP, DNP''and oligomycm added b. As 3a but atractyloside added also

2.7

—

—

—

—

6.5

6.3

7.6

13.1

12.5

21.3

20.9

37.9

63.0

—

—

—

—

26.2

—

« Dr. Tremblay has recently informed the author that his laboratory is having some difficulties in reproducing the data of Table IV, i. e., the tissue slice studies on the role of CPS-I in pyrimidine biosynthesis (12); however, the transport of CAP across the mitochondrial membrane has been confirmed repeatedly (112). * Values are nanomoles of bicarbonate incorporated per gram of liver in 1 hour at 37°C, D N P = dinitrophenol.

and low molecular-weight compounds by dialysis or Sephadex chroma­ tography. However, Bourget et al. observed that the addition of acetyl glutamate (10 mM) doubles the amount of bicarbonate incorporated into RNA. Therefore CPSase I can provide a significant amount of CAP for RNA synthesis. When excess ATP is added the rate with no Ν donor added or with glutamine added increases to 21 nmoles CO2 (which can be due to CPSase II). Ammonia alone and, particularly, ammonia plus acetyl glutamate raise this level further. The addition of atractyloside, which would end the flow of ATP into the mitochondria, reduces the rate even in the presence of ammonia and acetyl glutamate to a value of 26 nmoles, suggesting once again that the 21 nmoles observed in the absence of an Ν source are due to a nonmitochondrial enzyme, perhaps CPSase II. In summary, Bourget et al, have demonstrated that a very significant overflow of CAP from the liver mitochondria to the pyrimidine pathway can occur. However, the more significant question of whether this occurs normally has not been answered by their study. Shigesada and Tatibana have studied tissue concentrations of acetyl glutamate (136), They find acetyl glutamate in liver and small intestine. The normal level in liver mitochondria is between 1 and 2 X 10"* Af, which is about the con­ centration for CPSase I. This might explain why Bourget and Tremblay find CPSase I activated by acetyl glutamate, even though freshly pre­ pared mitochondria have been reported to show no dependence on acetyl glutamate (26). This low value for the normal range also suggests that

250

MARY ELLEN JONES

the levels Bourget and Tremblay may have reached by the addition of 10 mM acetyl glutamate to the rat liver slices may be much greater than those one can ever obtain in vivo {136). In addition, the level of ammonia added by Bourget et al. is as high as that observed in am­ monia intoxication and about ten times the concentration (0.7 mikf) in normal rat liver {20). Indeed, there is a direct evidence that in a situation of semichronic ammonia intoxication, as is found in human mutants with a defective OTCase {92), orotic acid excretion does increase. There presumably is overflow of CAP from the mitochondria to the pyrimidine pathway in these mutants. Bourget et al. mention that azaserine did not inhibit the bicarbonate incorporation into RNA as evidence against the importance of CPSase II for RNA synthesis in rat liver. Unless they establish that the liver slices are low in glutamine or know that the azaserine added and entering the cells is at a concentration at least 10-fold or more greater than the glutamine concentration, they could not expect to see inhibition by this analog, which is the least potent of the glutamine analogs for the rat CPSase II (4P, 50). In the one case where azaserine was a moderate inhibitor of the CPSase II of intact cells {Jß), the cells required the addition of glutamine or ammonia as a nitrogen donor. Despite the facts that (1) an assessment of the normal channeling of CAP formed by CPSase I remains to be carried out and (2) lack of channeling would apparently serve pyrimidine nucleotide synthesis only in the liver, intestinal mucosa, and perhaps kidney, excess pyrimidine synthesis in these tissues may play a role in providing pyrimidine bases or orotic acid for salvage by nonhepatic tissues. As Levine et al. {93) have pointed out, the salvage pathway is known to be insuflftcient for patients with oroticaciduria, so that the salvage pathway seems to be an aid, but not a solution, to the pyrimidine requirements of the entire ani­ mal; however, it could be important for specific tissues. Udenfriend and co-workers {58), for example, have preliminary studies which suggest that in neural tissues the de novo and salvage pathways are of equal importance. The overflow of CAP from the mitochondria of liver, in­ testinal mucosa, and kidney could also be of importance for the CAPglucose phosphotransferase activity of the microsomes {101) of liver, small intestine, and kidney. B. Dihydroorotase 1. CONDITIONS RESULTING IN CHANGES IN ENZYME LEVELS

This enzyme has not been extensively purified from any source; only preliminary kinetic studies with homogenates or partially purified ex­ tracts have been undertaken. As mentioned in Section II, A above, the

URIDYLIC ACID BIOSYNTHESIS I N EUKARYOTIC CELLS

251

structural gene for DHOase in yeast is unlinked with any of the other genes of the pathway; the enzyme is induced by either CAA or DHO, and the CPSase-ATCase protein responds to repression or derepression by a uridine metabolite {72, 86). In Neurospora, conditions leading to the derepression of the CPSase-ATCase complex do not result in any change in the DHOase level {162). In vertebrates, on the other hand, DHOase levels are increased or decreased with ATCase during normal fetal development of the chick {44), in the rat in response to orotate feeding {19), or in parallel with the growth rate of various cancer cells {148). The same nearly parallel increase of the two enzymes is observed in the erythrocytes or leukocytes of patients with pernicious anemia and in the nucleated erythrocytes of patients with Di Guglielmo's syn­ drome {140). 2. METABOLITE OR ANALOG CONTROL

In mammals, this enzyme is inhibited by millimolar levels of pyrimidine-related bases, ribosides, and mononucleotides {17). The nucleoside and mononucleotides tend to be somewhat better inhibitors than the free bases of the compounds tested, uracil, cytosine, and azauracil, and better than orotic acid. Of particular interest is the fact that orotic acid is a rather potent inhibitor of the enzyme both in homogenates {17) and in the intact Ehrlich ascites cell {49). In homogenates, 5-fluoroorotic acid is an even more effective inhibitor than orotic acid. Carbamyl aspartate analogs also inhibit, but levels near or above 10 mM are needed to give 50% inhibition {144). 3. PURIFIED ENZYME

The only partially purified DHOase has been obtained from pea plants {104)' The plant enzyme was purified 320-fold and was stable; enzyme from other sources has been reported to be unstable {86). The plant enzyme had an apparent molecular weight of 110,000 as judged by Sephadex gel filtration, and a for CAA of 6 mM and for DHO of 1.5 mikf. The equilibrium constant at pH 6.0 was 0.8. The pH optimum for the enzyme obtained from various sources is usually below 7.0 for the dehydration reaction, while the pH optimum for the hydration reaction is above 8.0. C. Dihydroorotate Dehydrogenase 1. NATURE OF T H E BIOSYNTHETIC E N Z Y M E

The enzyme from Zymobacterium oroticum, which is a flavoprotein requiring DPN+, is homogeneous and has been well studied; this and other similar enzymes do not appear to be biosynthetic {118), The

252

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single exception to this conclusion may be the only DHOdehase observed in extracts of wheat embryos, which utilizes DPN+ (77). In Eschenchia coli and a pseudomonad, the biosynthetic enzyme is membrane bound (109, 153, 154) · In a Pseudomonas strain two enzymes occurred after growth on orotate, the biosynthetic membrane-bound enzyme and an induced TPNH-requiring enzyme. In Neurospora a single DHO-oxidase has been identified, located in the mitochondria (38), from which it can be extracted and purified (107). The purified enzyme couples with quinone electron acceptors. The identity of the mammalian biosynthetic enzyme is not known. Miller and co-workers (108,110) have shown that beef liver mitochondria contain a DHO-oxidase resembling the Neurospora enzyme. However, DHO can be converted to orotate by three membrane fractions of the liver cell, namely, the "nuclear" pellet (a 600 to 700 g pellet), the mito­ chondrial pellet, and the microsomal pellet or the mitochondrial super­ natant solution (110, 167). On the other hand, the conversion of DHO to OA by homogenates of leukocytes (139) or of Ehrlich ascites cells (138) occurs only with the "nuclear" pellet if the homogenizing fluid is 0.5 Μ sucrose (139) or 0.1 Μ phosphate buffer, pH 7.4, containing 5% glycerol (138). Electron acceptors, such as ferricyanide and dichlorophenolindophenol, which are good electron acceptors for the mitochondrial enzyme, do not stimulate the conversion of DHO to OA in leukocyte or ascites cell homogenates, nor does the addition of DPN+. What the natural electron acceptor is in these tissues is not known. A preliminary experiment in which the 600 of dimethyl sulfoxideglycerol supernatant from Ehrlich ascites cells was incubated with "CHCO3", aspartate, and PP-R-P resulted in nearly 100% conversion of bicarbonate to uridine nucleotides with essentially no intermediates accumulating. This is what happens in the intact ascites cell (49), so that it would seem as though the activity extracted from the 600 g pellet by dimethyl sulfoxide and glycerol might be the biosynthetic enzyme of this cell and perhaps of the leukocyte also. It is of some interest that the erythrocyte, lacking membrane structures, contains all the pyrimidine biosynthetic enzymes except the enzyme for the conversion of DHO to OA (139). When the unfertilized frog egg was homogenized with an equal volume of 0.2 Μ phosphate buffer, pH 7.5 containing 20% glycerol, 5 mM magnesium ATP, and 5 mM mercaptoethanol followed by centrifugation at 100,000 g for 30 minutes, an enzyme capable of convert­ ing DHO to OA was in the supernatant solution, for DHO was converted to a small amount of orotic acid, larger amounts of UDP, and a trace of UTP (90). It has been suggested that the use of an oxidase for the reaction rather

URIDYLIC ACID BIOSYNTHESIS I N EUKARYOTIC CELLS

253

than a dehydrogenase would be advantageous because it would be an irreversible step; this seems desirable in view of the reversibility of the preceding reaction catalyzed by DHOase. In summary, the biosynthetic enzyme in bacteria is membrane bound (109, IBS, 1B4). Since only one enzyme has been observed in Neurospora, it is assumed to be the biosynthetic enzyme; it is localized in the mito­ chondria. In mammals, the identity of the biosynthetic enzyme remains to be established. Liver cells have enzyme activity in three cell fractions, but two nonhepatic tissues contain activity only in the "nuclear" pellet. 2 . METABOLITE CONTROL

As the above discussion indicates, so few studies have been made with the possible biosynthetic enzyme that very little is known about their regulation by metabolites. As mentioned in Section II, C, the yeast enzyme is induced by CAA or DHO (72, 86) and is also increased in cells grown on 6-azauracil [which after its conversion to azauridylic acid would be expected to inhibit OMPdecase (121), thereby reducing the uridine nucleotide pool of the wild-type cell and causing the ac­ cumulation of intermediate(s) prior to UMP]. Growth of yeast on 6 azauracil not only induced DHOdehase, but also increased ATCase (by derepression) and DHOase and OMPdecase (by induction). OMPppase activity increased only slightly. Similarly, growth of mammalian cells in culture on azauracil has been reported to have increased ATCase (S9), OMPppase, and OMPdecase (84, 12B) levels with no apparent change in DHOase and DHOdehase levels (12B, 168). D. The Orotidylic Enzymes 1. FUNGI

The genes for OMPppase and OMPdecase are unlinked in baker's yeast (72) and Neurospora (2S), and OMPppase levels do not change in response to the level of the pyrimidine pool or to the pool levels of prior intermediates, whereas OMPdecase can be induced by CAA, DHO, or OA (72, 86). Indeed, these two enzymes from baker's yeast have been separated from one another and extensively purified by Umezu et al. (1B9). OMPppase activity was separated by DEAE-cellulose chro­ matography into two fractions which had molecular weights of 3 2 , 0 0 0 and 3 9 , 0 0 0 , while OMPdecase activity resided in a single protein whose apparent molecular weight was 5 1 , 0 0 0 . The OMPppase reaction was freely reversible, and the values were as follows: orotic acid, 3 3 μΜ; PP.R-P, 6 2 μΜ; P P i 2 2 0 μΜ; and OMP, 8.5 μΜ. Since OMP binds relatively tightly, it inhibits the forward reaction; however, of the other

254

MARY ELLEN JONES

nucleotides tested, none that contained as bases U, C, G, or A were inhibitors. OMPdecase was inhibited by nucleotides in the order: GMP > GDP > CMP > CDP (but not by UMP) if their concentration was 1 mAf. The Km for OMP was 5 μΜ. Therefore, OMPppase might be inhibited by product accumulation if OMPdecase were inhibited by cytosine or guanine nucleotides. 2.

MAMMALS

a. Enzyme Isolation Studies. There are many experiments which sug­ gest that the two orotidylate enzymes exist as a complex in mammals (δ, 52, 78, 138). The most definitive study is that of Appel (δ), who has purified these enzymes to homogeneity from bovine brain and finds that they copurify, have the same degree of heat instability, and are simultaneously protected against heat denaturation by nucleotides. It is particularly significant that 6-azauridylic acid, an inhibitor generally found to be specific for OMPdecase, prevents the heat denaturation of both enzyme activities. Nucleotide inhibition of the OMPdecase activity of the complex was competitive with OMP; the order of effectiveness was 6-aza-UMP » CMP > UMP > CDP > UDP > CTP. The Km for OMP was 3 μΜ, and the Ki values for the inhibitors tested were between 0.1 and 3 mikf. The liver OMPdecase has a similar Ki value for 6-azaUMP, but apparently differs from the brain enzyme, for it is inhibited by UMP but not by other nucleotides {29). These two enzymes also copurified from thymus tissue (78) and red blood cells (52). After a 500-fold copurification, the two activities in thymus preparations could be separated by electrophoresis; however, considerable OMPppase activity was generally lost by this procedure. This observation taken together with AppePs heat denaturation studies suggests that the two activities are part of a single protein complex of at least two nonidentical peptides. The complex may well confer stability to the OMPppase activity. In addition, it has recently been observed that these two enzymes, extracted from Ehrlich ascites cells (138), cosediment in a sucrose gradient (apparent molecular weight of 100,000 to 120,000). b. Genetic Studies and Changes in Cellular OMPppase and OMPdecase Specific Activity Caused by Pyrimidine and Purine Analogs. The only mutants known in the animal kingdom are human beings who excrete orotic acid. The majority of these human mutants have very low levels of both enzymes utilizing orotidylic acid (7, 40, 42, δ1,61,126,133,143), but one mutant described by Fox et al. (4^) has only very low OMP­ decase levels with elevated OMPppase levels. The OMPppase levels decrease to normal on uridine therapy. Uridine or cytidine therapy also

URIDYLIC ACID BIOSYNTHESIS I N EUKARYOTIC CELLS

255

decreases the amount of orotic acid excretion in all patients and improves growth and survival and other symptoms of insuflScient pyrimidine bio­ synthesis, such as the megaloblastic anemia that these patients exhibit. These genetic diseases are carried in a recessive manner so that the mutants are rr while the parents are heterozygotes, Rr. Studies which have been made so far that givf some insight on the regulation of the levels of these enzymes include: (a) changes in the welfare of the patients; the level of orotic acid excretion by the patients as well as preliminary studies on the levels of these enzymes in erythro­ cytes and leukocytes (7, Jfi, Jfi, SI, 61,133,143); (b) changes in enzyme activity levels in fibroblasts grown in culture which were derived from the patients, their parents, and control subjects {84, 125); and (c) studies on the levels of enzyme activity in human beings treated with allopurinol {43).

These studies have led to three diverse suggestions concerning the mutants, each reflecting to a degree the progress made in studies on regulation of metabolic pathways in bacteria {6, 172), fungi (86), and mammals {135). The first suggestion by Smith {14^), covering only the larger group of mutants which have low levels of both enzymes, was that these two enzymes were coded on a single messenger RNA whose synthesis was controlled by an operator gene that was defective. This would explain, assuming two discrete proteins catalyzed these sequential enzyme reactions, why both enzyme activities were simultaneously low. Uridine therapy was successful because it provided sufiicient end product which was needed for proper growth, and it simultaneously repressed (or in­ hibited) the synthesis of the early enzymes (or intermediates) of the pathway, so that orotic acid excretion decreased even though it did not become normal. This explanation became less necessary with AppePs discovery that the two enzymes may normally exist in mammals as a complex (5). Such enzyme complexes could, for example: (a) be essential for enzyme activity; (b) improve (or slow down) the rate of the enzyme activities markedly so that if complex formation were defective the enzymatic rates would be slow; (c) stabilize the enzyme activities either toward chemical denaturation or against proteolysis, among other possibilities. Therefore, the existence of enzyme complex would obviate the need for the messenger RNA coding for these enzymes to be under the control of an operator gene. All that would be required for the mutants simul­ taneously lacking both enzyme activities is that an amino acid residue be changed at a site such that the conformation of both enzymes of the complex and their activities were simultaneously altered. There would presumably be a limited number of crucial mutation sites, but since the

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surfaces essential for complex formation would be dependent on a number of amino acid residues, there could presumably be several amino acids which could affect complex formation and might therefore affect both enzyme activities simultaneously. Obviously, we need to know a good deal more about this enzyme complex. Is it composed of a single poly­ peptide chain? The data of Kasbekar et al make this seem unlikely (78). Are the. separated polypeptides that make up the complex active enzymes, or is complex formation necessary for enzyme activity? Once again, limited data suggest that a distinct polypeptide contains OMP­ decase activity, but we do not know whether the separated polypeptide has the same kinetic characteristics as the intact complex. The OMPppase peptide (s) may be unstable in the absence of the OMPdecase poly­ peptide (s), (5, 78), When enough is known about the normal complex we will need to compare the mutant and normal enzymes. The efficacy of uridine treatment in reducing the excretion of orotic acid could be due primarily to feedback inhibition by UTP of the CPSase II activity of the CPSase II-ATCase-DHOase complex {67, 93, 119), Such inhibition would decrease the production of orotic acid. However, it is possible that there is repression of CPSase II synthesis; changes in the level of this enzyme in animals or in cells grown on uridine have not been investigated. It seems probable that orotic acid production is increased in the untreated patients due to increased levels of ATCase and DHOase (19, H2) and perhaps DHOdehase (19), ATCase and DHOase are not increased by a repression-derepression mechanism in mammals, according to Bresnick's studies on orotic acid feeding (iP). It is important therefore to know whether CPSase II levels change in the same direction and tend to parallel the ATCase-DHOase changes under similar circumstances, or if the levels of the first enzyme are controlled independently. It is even possible that increased levels of UMP may stabilize or help increase the levels of the two orotidylate enzymes. Krooth has found that pyrimidine analogs, namely azauridine, azaorotic, or barbituric acids, placed in the medium surrounding fibro­ blasts from patients with either type of oroticaciduria (84, 125) increase the low enzyme or both enzyme activities 10- to 50-fold. The same effect, somewhat reduced in the magnitude of increase obtained, is seen in fibroblasts cultured from the heterozygous parents and from normal individuals. This increase in activity is apparently not due to the presence of a dissociable activator or inhibitor in the extracts. Since the increase in enzyme activity occurs even when sufficient cytidine is present in the medium to permit optimal growth, the effect cannot be due to repression-derepression control. Krooth can observe a similar increase in the activity of these enzymes if DHO is added to the growth

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medium of the mutant cells. He suggests that these enzymes are induced much as the yeast enzymes are, except that in the human cells no increase is observed in DHOase or DHOdehase (168), which are induced in yeast by DHO or its products or by growth on azauridine (72). Fox and his co-workers (43) have recently measured the levels of the orotidylate enzymes in the erythrocytes of humans receiving the drug allopurinol. Humans ingesting this drug sometimes excrete elevated amounts of orotic acid and orotidine, and exhibit elevated levels of OMPppase and OMPdecase (43). The explanation for these observations appears to be a stabilization of the two orotidylate enzymes as judged by comparing the decrease in the levels of these two enzymes in aging red blood cells from normal individuals, where the two enzymes decrease in parallel over a 3-fold range, whereas in patients treated with allo­ purinol the decrease is again parallel for both enzymes but is only half as rapid. Fox et al hypothesized that the stabilizing agent is the ribonucleotide of oxypurinol because allopurinol itself can only be converted to a nucleo­ tide by hypoxanthine-guanine phosphoribosyltransferase. Since the effect of allopurinol on the orotidylate enzymes and orotic acid and orotidine excretion is seen in patients lacking this enzyme, a metabolite other than allopurinol mononucleotide must be the direct agent. Since the orotidylate enzymes bind and are stabilized only by orotic acid or pyrimidine mono­ nucleotides (5) it is logical to assume that the protective allopurinol metabolite is a nucleotide. Indeed, Fox et al could demonstrate that erythrocytes lacking hypoxanthine-guanine phosphoribosyltransferase could form an inhibitor of OMPdecase from oxypurinol and PP-R-P, presumably via OMPppase. OMPppase converts not only orotic acid to OMP, but forms nucleotides of xanthine and uric acid where the ribose-P moeity is attached to N-3 of the pyrimidine ring of these bases (52), and it is presumed that oxypurinol can be similarly converted to a nucleotide (in this cases the equivalent Ν atom is numbered N-7) by OMPppase. In summary, the two orotidylate enzymes appear to exist as an enzyme complex in mammalian cells. This complex is seen in sucrose gradient fractionations of the crude homogenate (138), where it appears to have a molecular weight of about 120,000. The complex exists through extensive purification procedures (5, 52) and may confer equal heat stability on the two enzymes (5). In addition, nucleotides which can bind to the complex appear to increase the in vivo stability of these two enzymes (43). Pyrimidine analogs, such as azauridine, which is converted to a nucleotide intracellularly, can increase the level of these two enzymes in cells grown in culture (84, 125). It remains to be ascertained whether

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this reflects merely a decrease in the normal degradation of the enzymes or increased enzyme synthesis. More important, however, may be the role this complex may contribute to the overall flow of metabolites through the pathway and to the catalytic efiiciency of the individual enzymes themselves. These aspects remain to be studied. 3.

PLANTS

The two orotidylate enzymes have been demonstrated in extracts of several plant species (77, 166), but they have not been purified or studied in detail. The OMPdecase is inhibited by 6-azauridylic acid, but OMP­ ppase activity is not affected. It is not known whether the two enzyme activities would copurify as a complex. E. Summary In fungi (34, 87) and the vertebrates {69) there can be a complete separation of the arginine and pyrimidine biosynthetic pathways be­ cause each pathway can derive CAP from a specific carbamyl-phosphate synthetase. In some organisms like Neurospora the two pathways normally do not share CAP pools, but in other organisms, such as yeast, CAP can fiow one "pool" to the other. Plants appear to differ from other eukaryotes in that a single synthetase has so far been observed, with feedback regulatory characteristics {116) typical of the bacterial synthetase (4), which can serve both pathways {124), Critical experi­ ments have not been carried out, however, to test whether a second synthetase exists in plants. The other prominent phenomenon in eukaryotes is the appearance of enzyme complexes. In fungi {100, 163), one such complex for the pyrimidine pathway exists consisting of the first two enzyme activities, namely, CPSase-ATCase. These two enzymes are coded for by a single polar gene. It is not known with certainty whether this gene codes for one or several polypeptide chains, although the studies from Kaplan's laboratory suggest that more than one type of polypeptide chain is in the complex. In mammals two enzyme complexes may exist. The first one appears to contain the first three enzymes of the pathway, CPSaseATCase-DHOase {137), while the second one, the two orotidylate enzymes, OMPppase and OMPdecase {6, 78, 138), No complexes have been described in plants. Gene regulation of enzyme biosynthesis occurs in yeast {5, 86), The CPSase-ATCase complex is controlled by repression-derepression, while DHOase, DHOdehase, and OMPdecase are controlled by induction.

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OMPppase activity is relatively constant and seems to show little gene regulation. There are no direct data on gene regulation of enzyme bio­ synthesis in mammals. However, parallel changes in CPSase-ATCase levels, ATCase-DHOase levels, ATCase-DHOase-DHOdehase levels, and OMPppase-OMPdecase levels have been recorded under various situations (see Section III, D ) . Whether these reflect coordinate gene regulation or are dependent on some other phenomenon (such as stabili­ zation of the enzymes if they are in a functional enzyme complex) is not known. Certainly, the levels of the orotidylate enzymes can be varied by conditions that are most readily related to induction (84, 125) or stabilization of the enzymes against cellular degradation (43). A striking phenomenon in one mammalian cell, the Ehrlich ascites cell studied in vitro, is the total lack of a net accumulation of any intermediate between ^^C-HCOs^ and the uridylic nucleotides, unless orotic acid (which inhibits DHOase) is added (49). When orotic acid was present, only the product (CAA) of the enzyme (ATCase) preceding DHOase accumulated, and it appeared in an amount nearly equivalent to the amount of uridine nucleotides normally produced in the absence of orotic acid. Although this experiment could be explained by a ratelimiting first step, with appropriately low binding constants of each of the succeeding enzymes for their substrates, it might also indicate that the enzymes of the entire pathway exist as a complex such that when bicarbonate enters the complex it is not normally freed until UMP is formed, or the series of reactions catalyzed by the complex is blocked by the inhibition of an enzyme, or unless a defective enzyme (or enzymes) exists, as in oroticaciduria. The existence of enzyme complexes in this pathway in eukaryotes has only recently been observed, so that time has not permitted a true assessment of their role. Although at this moment these enzyme com­ plexes and enzyme-induction seems to make the eukaryotic regulation distinctly different from the regulation of this pathway in prokaryotes, recent results of Wild and Belser (161), who have been studying pyrimidine biosynthesis in Serratia marcescens, may tend to reunify both groups. These investigators have found that the DHOase, OMP­ ppase and OMPdecase activities of Serratia form a complex of 70,000 daltons. This complex is dissociated by chromatography on DEAEcellulose such that the OMPppase and OMPdecase activities remain in a complex having an apparent molecular weight of 40,000 but DHOase is now free. When DHOase is removed from the DHOase-OMPppaseOMPdecase complex, the OMPppase activity of the remaining OMP­ ppase OMPdecase complex is only 60% of its original level. If the large

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enzyme complex is reformed by addition of DHOase to the OMPppaseOMPdecase complex, the lost OMPppase activity is recovered. This effect is observed in pyr-C mutants, i.e., mutants which require DHO for growth and have low DHOase (6-28% of wild-type cells) activity. Extracts of these mutants all have reduced OMPppase activity (21-68% of wild-type cells), which can be elevated by adding DHOase extracted from other cells. As in mammals, the OMP enzymes copurify but can be separated by acrylamide gel electrophoresis (5, 78), This treatment causes the loss of 80% of the OMPppase activity and 30% of the OMPdecase activity. These results resemble the loss of OMPppase activity observed on electrophoresis of the mammalian enzymes (5, 78), However, Wild and Belser recombined the two OMP enzymes (separated by electrophoresis) and obtained a total recovery of the lost activities. Obviously, the DHOase-OMPppase-OMPdecase complex plays a significant role in the normal functioning of the pathway in the wildtype Serratia cell. Wild and Belser suggest, as had Haywood and Belser iS3)j that coordinate changes in enzymes whose genes were widely separated on a chromosome might depend, if an enzyme complex exists, on only one operator site for one enzyme and the subsequent incorporation of the other "coordinate" enzymes into the complex. The permutations possible seem at times to become very diflScult for the scientist trying to purify enzymes in the native state from organisms with multiple enzymes synthesizing a single product, in the presence of natural low or high molecular weight activators and inhibitors. In ad­ dition, the enzymes may be more or less active, depending not only on the degree of denaturation of sites essential for catalytic activity and for substrate and effector binding, but also on whether the enzyme has been purified in such a manner that it has remained as a part of a complex fully reflecting its native regulatory characteristics, or has been freed from such a complex. Care in the planning, execution, and description of experiments as well as a sensitivity to the unexpected or difiicult-to-explain discovery should help us to understand the great amount of delicate structural detail that has functional significance in the construction of the biosynthetic enzymes and other metaboliteregulated proteins, and in understanding the control mechanisms in vivo. There are many other metabolic pathways in which enzyme complexes are important (for example, see ^4, 166), Many of these enzyme complexes are in eukaryotes {41, 54), but they are also very important in prokaryotes (for example, see 27), It would be particularly helpful if we could visualize more enzymes within the cell. It is possible that a single protein may serve somewhat diverse functions in the cell depend­ ing on whether it is part of, or is separated from, an enzyme complex.

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ACKNOWLEDGMENT The author would like to acknowledge the very helpful criticism in addition to the editorial assistance of Dr. Luisa Rai j man, which has markedly improved this manuscript. REFERENCES 1. 2, S. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 16. 16. 17. 18. 19. 20. 21. 22. 23. 24. 26. 26. 27. 28. 29. 30. 31. 32. 33. 34. 36. 36. 37.

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157. Tramell, P. R., and Campbell, J. W., Comp. Biochem. Physiol. 40B, 395 (1971). 158. Trotta, P. P., Burt, M. E., Haschemeyer, R. J., and Meister, Α., Proc. Nat. Acad. Sei. U. S. 68, 2599 (1971). 159. Umezu, K., Amaya, T., Yoshimoto, Α., and Tomita, K., J. Biochem. 70, 249 (1971). 160. Weber, G., Queener, S. F., and Ferdmandus, J. Α., Advan. Enzyme Regul. 9, 63-95 (1971). 161. Wild, J., Ph.D. Thesis, The University of California at Riverside, California (1972). m. Wüliams, L. G., and Davis, R. H., J. BacteHol. 103, 335 (1970). 163. Williams, L. G., Bernhardt, P. H., and Davis, R. H., Biochemistry 9, 4329 (1970). 164. Woodward, V. W., Proc. Nat. Acad. Sei. U. S. 48, 348 (1962). 165. Woodward, V. W., and Davis, R. H., Heredity 18, 21 (1963). 166. Woolcott, J. H., and Ross, C , Biochim. Biophys. Acta 122, 532 (1966). 167. Wu, R., and Wilson, D . W., J. Biol. Chem. 223, 195 (1956). 168. Wuu, K.-D., and Krooth, R. S., Science 160, 539 (1968). 169. Yan, Y., and Demerec, M., Genetics 52, 643 (1965). 170. Yates, R. Α., and Pardee, A. B., J. Biol. Chem. 221, 743 (1956). 171. Yates, R. Α., and Pardee, A. B., J. Biol. Chem. 221, 757 (1956). m. Yates, R. Α., and Pardee, A. B., / . Biol. Chem. 227, 677 (1957). 173. Yip, M. C. M., and Knox, W. E., / . Biol. Chem. 245, 2199 (1970). 174. Young, J. E., Prager, Μ. D., and Atkins, I. C , Proc. Soc. Exp. Biol. Med. 125, 860 (1967).

Flip-Flop Mechanisms and Half-Site Enzymes M I C H E L LAZDUNSKI Centre de Biologie Molecuhire du CJ^Jtß,, Marseille and Department of Biochemistry, Faculty of Sciences, Nice, France I. Introduction II. The Flip-Hop Mechanism. Description of a Model, the Alkaline Phosphatase of Escherichia coli A. A Characteristic Property of the Enzyme: Negative Cooper­ ativity and Reactivity of Half of the Sites B. The Catalytic Properties in Vitro and in Vivo . . . . C. The Flip-Flop Mechanism of the Alkaline Phosphatase of Escherichia coli D . A Comparison with Intestinal Alkaline Phosphatase HI. Half-Site Enzymes A. Malate and Alcohol Dehydrogenases B. Muscle Glyceraldehyde-3-Phosphate Dehydrogenase . . . C. i8-Galactosidase D . Escherichia coli Methionyl-tRNA Synthetase . . . . E. Acetoacetate Decarboxylase and Transaldolase . . . . IV. Half-of-the-Sites Reactivity and Flip-Flop Mechanisms for Allosteric Enzymes A. Main Features of Allosteric Enzymes with Flip-Flop Mechanism B. Cytidine Triphosphate Synthetase C. Aspartate Transcarbamylase V. The Functional and Evolutionary Advantages of Half-Site En­ zymes and Flip-Flop Mechanisms References Note Added in Proof

267 268 269 274 276 283 283 284 290 292 293 295 296 296 298 300 302 305 309

I. Introduction Most intracellular enzymes are polymeric proteins (63), Many of them are already known to be, or will probably prove to be, oligomeric enzymes, that is enzymes made up of identical subunits (94), For that reason a search for the functional meaning of the subunit structure of polymeric enzymes is of primary interest. Kinetically speaking, there are two main types of enzymes with quaternary structures: those with cooperative ν versus [S] profiles, designated as allosteric enzymes, and those with "classical" Michaelis-Menten kinetics. This last class of enzymes is much larger than the first one. Allosteric enzymes which are characterized by a sigmoidal response 267

268

MICHEL LAZDUNSKI

to increasing substrate or inhibitor concentration have been extensively studied in recent years. Monod and his collaborators (94) were the first to show that cooper­ ative kinetics for allosteric enzymes necessitate polymeric structures with indirect interactions between distinct binding sites. In the concerted model of these investigators, the interactions are mediated by a structural change, the allosteric transition (94). Other related models have been described in recent years (69, 71) to explain allosteric behavior or more generally the conformational aspects of enzyme regulation. However, while the theoretical tools for analyzing allosteric behavior are now available, hemoglobin remains thus far the only ex­ ample of an allosteric protein for which a stereochemical meaning can be given to the allosteric transition (97). For more details concerning allosteric enzymes, the reader is referred to several recent excellent reviews (6, 61, 69, 109, 120), While there is an obvious functional mean­ ing for the subunit structure of allosteric enzymes, the most immediate interpretation for "classical" Michaelian kinetics with polymeric enzymes is that the different active sites on each constitutive monomer function independently. This interpretation has been retained up to now but fails to give a functional meaning to oligomeric structures. There is an everincreasing literature describing negative (or anti) cooperativity for substrate binding by Michaelian polymeric enzymes. Negative (or anti) cooperativity certainly indicates that constituent protomers do not behave as independent entities in oligomeric structures. To try to resolve the apparent paradox between anticooperative binding and classical Michael­ ian kinetics, a new interpretation of the functional interrelationship between distinct active sites on identical monomers which are part of oligomeric enzymes with Michaelian kinetics has been recently proposed. It has been called the flip-flop mechanism (80). The theoretical aspects of negative cooperative interactions between distinct binding sites will not be treated here. The reader is referred to very detailed and interesting descriptions published by Koshland and his group (28,69,70,71). In this review, our interest will be centered on kinetic cooperativity which is the basis of the flip-flop mechanism and on the analysis of the most recent literature concerning half-site enzymes (that is enzymes with an apparent reactivity of only half of their sites). II. The Flip-Flap Mechanism. Description of α Model, the Alkaline Phosphatase of Escherichia coli Studies carried out with the alkaline phosphatase of E. coli will be described in some detail. This chapter will serve to illustrate the essential properties of a flip-flop mechanism.

FLIP-FLOP MECHANISMS AND HALF-SITE ENZYMES

269

A . A Characteristic Property of the Enzyme: Negative Cooperativity and Reactivity of Half of the Sites The alkaline phosphatase is a dimeric enzyme with a molecular weight of 86,000. The demonstration that there is only one structural gene for the protein indicates that it is made of identical subunits (ΙΟδ, 112). It is a zinc-metalloenzyme [100). Studies concerning the Zn^^ {79), the Co^^ {118), the Cd^^ {78), the Mn^^ {24, 78), and the Cu^^ {73) phosphatase have now demonstrated that there are four essential metal binding sites in the enzyme. They belong to two different families. There are two tight and two loose metal-binding sites {78). The metal atoms play a fundamental role in the mechanism of the alkaline phosphatase. Schwartz {116) and Engström {36) have shown that the enzyme mechanism involves the intermediate formation of a phosphoryl phos­ phatase. The critical site of phosphorylation in the active center is a serine residue. The phosphatase mechanism can be schematized as follows E + ROPO;- ;=±ES- • E

(S)

• Ε + PO4H2PO;- -f ROH

The properties of the enzyme that will lead to the final mechanism will be successively analyzed. Phosphorylation of the strategic serine residue can be performed either with the substrate ROPO^~ or with the reaction product PO4H2-. We will first center our attention on equilibrium situations that describe phosphorylations with inorganic phosphate. Figure 1 shows the extent of phosphorylation of the enzyme by high

FIG. 1. pH dependence of phosphorylation of the active centers of the alkaline phosphatase of Escherichia coli with [**P]orthophosphate (10 mM). Zn**-phosphatase at 0°C ( • ) . Co** phosphatase at 23*'C ( # ) . π/Εο = moles of ['diphosphate covalently bound {π) per mole of enzyme (Eo). (77, 80).

270

MICHEL LAZDUNSKI

concentrations of orthophosphate at different pH^s. The extent of phos­ phorylation is higher at acidic pH; this observation was first made many years ago by Engström (36) and by Schwartz (116) and also recently confirmed by Wilson and his group (84) and by Applebury and Coleman (5). The Zn2+ alkaline phosphatase incorporates two covalent phos­ phates per mole of enzyme at acidic pH while it incorporates no covalent phosphate at alkaline pH. The noncovalent binding of inorganic phosphate to metallophosphatase at alkaline pH was studied by means of a variety of techniques (24j 77, 78, 80,119), such as equilibrium dialysis, gel filtration, spectrophotometry, and electron paramagnetic resonance (EPR) studies. The characteristic feature of the binding is anticooperativity. Our equilibrium studies have shown the absorption of two phosphates per mole of enzyme. One site binds phosphate tightly, and its saturation occurs at low phosphate concentration; the saturation of the second site is then much more diflScult, and much higher concentrations of phosphate are necessary. A typical representation of this anticooperative behavior is presented in Fig. 2. Noncovalent complexes containing 2 moles of orthophosphate per mole of Zn2+ phosphatase have also been isolated (77). They are stable only at high protein concentrations; they dissociate at lower concentrations into 1:1 complexes which will also dissociate on further dilution to regenerate the free enzyme. Complexes containing

FIG. 2. Left: Scatchard plots for the binding of orthophosphate to the Zn^* phosphatase. 7 =• mole binding ratio Pi^dimer; c = equilibrium concentration of orthophosphate. 25**C, 0.4 Μ NaCl. X , pH 8.0; O , pH 8.8. Right: Variation of pKi with pH (Ki dissociation constant of the enzyme-orthophosphate complex). Δ , Kinetic determinations; # , pKi was calculated from the slope (l/Ki) of the y/c - f plots for the saturation of the first (tight) site. (77).

FLIP-FLOP MECHANISMS AND HALF-SITE ENZYMES

271

only 1 mole of orthophosphate per mole of Co^* or Cu2+ phosphatase have also been prepared (77, 78). It was of interest to know whether this anticooperativity also exists in the formation of covalent derivatives at acidic pH. Figure 3 presents the concentration dependence for the covalent phosphorylation of the active centers of the Zn2+ and Co^^ phosphatases with orthophosphate at pH 4.2 and 5.0. The experimental data are in accordance with the calculated curves for the phosphorylation of 2 different sites with 2 distinct Michaelis constants K i and -Kg. At pH 4.2, the values of these constants are 25 μΜ and 1 mM for the Zn^^ phosphatase, and 25 μΜ and 0.5 mM for the 00^+ phosphatase. and K2 differ by a factor of 40 for the Zn2+ enzyme and a factor of 20 for the Co^^ phosphatase. The

5

4 -log[Pi](A/)

1.0 h

5

4

3

-log[P|](A/)

FIG. 3. The ["P] orthophosphate concentration dependence of the covalent phos­ phorylation of the Zn'* and Co** phosphatases at 20°C (80). (A) At pH 4.2, ionic strength 0.05. Δ , O , Zn** phosphatase; X, Co** phosphatase. The solid and broken lines which pass through the experimental points are calculated curves assuming two active sites with different aflSnities for orthophosphate. (B) Behavior of the Zn'* phosphatase at pH 5.0, 20^*0; ionic strength 0.05 (a), 0.15 (b), 0.5 (c).

272

MICHEL LAZDUNSKI

anticooperative character of the covalent phosphorylation is even more dramatic at pH 5.0. At an ionic strength of 0.05, the values of the Michaelis constants are Ki = 10 μΜ and Kz = 7.0 mM; Κχ and K2 differ by a factor of 700. These data indicate that the anticooperative character of the covalent binding does exist and that it increases with increasing pH. Figure 4 provides the kinetic evidence for anticooperativity. The diphosphorylated derivative E [E] > [aldehyde] ). The results showed mainly that saturation of the two binding sites for the coenzyme and for the aldehyde (benzaldehyde or azoaldehyde) gives rise to the formation of a complex containing 1 mole of NAD+, 1 mole of NADH, 1 mole of alcohol and 1 mole of aldehyde per mole of enzyme E - NADH, aldehyde

t - NAD, alcohol

This complex accumulates at pH 8.75 under steady-state conditions; its decomposition is the rate-limiting step of the enzyme mechanism. Typical results obtained by Bernhard and his group are presented in Fig. 10. Similar data were obtained by Dunn and Bernhard (35) for the transient kinetics of the reduction of p-nitro-N,N-dimethylaniline. The turnover number of this substrate analog is 2.4-fold greater than that of the "physiological" substrate, acetaldehyde. Aromatic aldehydes and transient kinetics were excellent tools in this case for demonstrating the half-site reactivity of alcohol dehydrogenase. The analysis of the transient kinetics for the reduction of benzaldehyde has been extended recently by Luisi and Favilla (wide pH range, different ionic strengths, and temperatures) (90). The action of horse liver alcohol dehydrogenase on a number of slowly reacting aromatic aldehydes and of aliphatic aldehydes has been studied by these investigators. The interested reader will find in Luisi and Favilla's paper an evaluation of the potential of. transient kinetics for identifying a flip-flop type mechanism. Only the main conclusions of these authors will be described in this review. The flip-flop mechanism proposed by Luisi and Favilla is presented below E(R,S)

1

E(R,S) (I)

Ie.

E*(R,S)

-+1

E(R,S) (II)

lei

E(O,P)

-+1

E(R,S)

t

(III)

ler

E(O,P)

-+1

E*(R,S)

~

E*(O,P)

-+1

with reciprocation

kd

I

E(O,P)

(IV)

(V)

FLIP-FLOP MECHANISMS AND HALF-SITE ENZYMES

'2> 0.6

an

)(

2

!0.5

i'0.5

)(

en

-:n

jO.4

~ 0.4

c:

c:

~0.3 CD E

~

0.3

o

E ~

0.2

.; O.t

o u

en

0.4

~

'0

CD

~ 0.2

c:

u

o

°

289

CD

.0 20

3.0 4.0 5.0

[NADH]_xt0 5

2

00

.0 2.0 3.0 4.0 5.0 [Azoaldehyde] 0 X 10 5

FIG. 10. (A) The variation in the moles of reactant consumed in the rapid initial step as a function of the concentration of [Ni\DHlo for the system LADHbenzaldehyde-NADH. Conditions: Eo (concentration of sites with one site per subunit) 11.6 p.N; benzaldehyde; 44.5 p.N, 25°C, pH 8.75 (8). (B) The variation in the moles of reactants consumed in the rapid initial step as a function of the concentration of the azoaldehyde for the system· LADHa.zoaldehyde-[NADH1; Eo, 8.75· p.N; NADHo, 0.11 mM; 25°C, pH 8.75 (8). c/J gives the stoichiometry relationship between the number of equivalents of NADH (or aldehyde) bound and the number of equivalents of enzyme-NADH (or enzyme-aldehyde) binding sites. Pmu is a measure of the stoichiometry relationship between the moles of reactants consumed in the initial step (when all sites are saturated in all ligands) and the enzyme normality. From Bernhard et ale (8) (by permission of the American Chemical Society). .

where Rand S represent NADH and aldehyde, respectively, and 0 and P represent NAD+ and alcohol. The different steps of this mechanism can be independently studied using different aldehydes (aromatic or aliphatic) and different conditions of pH (90). The first step (ka ) is a slow process which was identified as the structural modification brought about by coenzyme binding. Intrasubunit cooperativity, in this case, is of the same type as for malate dehydrogenase. Binding of the coenzyme I "activates" one of the two subunits (E*) and permits the transformation of Rand S into 0 and P. Intersubunit cooperativity is represented by the transformation (k d ) of species (V) into (III). Formation of products in the "second" subunit, species (V), induces dissociation of products from the first one. Products are then replaced by substrates Rand S, and species (III) is formed again. In this sche'me intersubunit cooperativity means that exclusion of products from one subunit is coupled with the oxidation-reduction reaction that takes places in the other one. This scheme is in accord with the compulsory order of substrate binding described by the Theorell-Chance mechanism (1S7, 1S8). The postulated asymmetry of the two subunits after binding of the coenzyme is consistent with the observations of Branden and his group (13). These

290

MICHEL LAZDUNSKI

workers observed that the dimeric alcohol dehydrogenase in the absence of coenzyme has a 2-fold axis of symmetry. This symmetry is lost in crystals of dehydrogenase containing bound coenzyme. B. Muscle Glyceraldehyde-3-Phosphate Dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a tetramer (MW, 144,000) composed of four identical subunits (56). It catalyzes the following reaction involving three substrates. Glyceraldehyde 3-phosphate + NAD+ -f- Pi ^ 1,3-diphosphoglycerate + N A D H + H+

The oxidative phosphorylation of D-glyceraldehyde 3-phosphate takes place in two steps (137). Ο RCHO + NAD+ + E—SH -> R—(ϊ^—S—Ε -f N A D H + H+ Ο Ο -S—Ε

R—h—OFOl- + E—SH

In the first step the aldehyde reacts with the enzyme active site to form a thiol ester intermediate. The 3-phosphoglyceroyl enzyme is deacylated in the second step to produce the acyl phosphate. The enzyme presents classical steady-state kinetics with all of its substrates (including both oxidation forms of the coenzyme) (137), but the active sites on each subunit are not independent. Both NAD+ and NADH binding show negative cooperativity (12, 28, 32), As could be expected from its tetrameric structure, the enzyme binds four molecules of coenzyme. However, the binding constants differ greatly. The first two molecules of coenzyme are bound very firmly—^too firmly, in fact, to allow a determination of the dissociation constants. The third molecule is bound less tightly than the first two, and the fourth molecule, still less tightly. While coenzyme binding data clearly imply the existence of intersubunit interactions, they do not clearly show that GAPDH is a half-site enzyme. An indication that this is so is to be found in the results of Givol (45), who observes that an active-site-directed reagent, ρ,ρ'difluoro-m,m'-dinitrophenylsulfone, could bind covalently to only two of the four subunits. GAPDH might then possibly be a double dimer (αα)2. This conclusion corroborates the independent evidence obtained by using an active-site-specific acylating reagent (91,92). Since a thiol ester acyl enzyme is an essential intermediate in the catalytic mechanism, Bernhard and his group (91, 92) have chosen to study the effect of covalent thiol modification on the properties of rabbit muscle GAPDH. The tetrameric enzyme contains four particularly re-

FLIP-FLOP

MECHANISMS AND HALF-SITE ENÄYMES

291

active thiols. Chemical modifications have shown that they react with substrates to form the intermediate thiol ester derivative (55, 91, 92), There is no preferential alkylation by iodoacetate or iodoacetamide among the four essential thiols {91, 92, 132), Four moles of alkyl groups are required to inactivate the enzyme and the percentage of inhibition is directly proportional to the degree of alkylation. However the four thiol active sites which appear to be identical and independent for "non­ specific" thiol alkylating agents, do not appear to be identical and inde­ pendent in catalytic reactivity. The 3-phosphoglyceroyl enzyme can be formed readily by incubation of GAPDH with 3-phosphoglyceroyl phosphate. The 3-phosphoglyceroyl enzyme is a true enzymensubstrate complex and the fact that it is fairly stable suggests that this derivative accumulates under steady-state conditions (deacylation would then be the rate-limiting step of the whole process). Only two of the four essen­ tial thiol groups of the tetrameric GAPDH were acylated in the 3-phosphoglyceroyl enzyme {91), More extensive studies done using a pseudo substrate )S-(2-furyl) acryloyl phosphate, as an acylating reagent also showed that the reaction is complete when only two acyl groups are incorporated per mole of tetrameric enzyme {92), Moreover, in accord­ ance with these results, Keleti {60) has shown that there is a transient release of only 2 moles of NADH in the action of the fully saturated GAPDH [E (NADi4] on glyceraldehyde 3-phosphate. All these results demonstrate very clearly that GAPDH is a half-site enzyme. The functional unit of the tetrameric structure is the «2 dimer. Analysis of the kinetics and of the stoichiometry of acylation by )&-(2-furyl) acryloyl phosphate indicates that the two protomers ( « 2 dimers) of GAPDH are identical and independent in the catalytic mechanism when the NAD+ concentration is high enough to saturate the four coenzyme sites {92), Muscle GAPDH is obviously an excellent candidate for a flip-flop mechanism. The relationship between the negatively cooperative binding of the coenzyme and its effect on the kinetics is diflScult to appreciate. Although GAPDH from different sources all behave as half-site enzymes, the type of cooperativity for coenzyme binding differs considerably. Yeast GAPDH is also a tetramer showing half-of-the-sites reactivity for acylation by /?-(2-furyl) acyloyl phosphate {92). However, in contrast to the GAPDH from most mammalian muscle sources and from lobster, it binds NAD+ with much higher aflSnity and with a sigmoidal binding isotherm (positive cooperativity) at alkaline pH {62), Coenzyme dis­ sociation constants are such that the muscle enzyme is fully or nearly fully saturated with NAD+ [E (NAD+)4] under the usual conditions for steady-state kinetics.

292

MICHEL LAZDUNSKI

C. ^-Galactosidase B. coli j8-galactosidase has a molecular weight of 520,000 and consists of 4 subunits [(14^) and references therein]. The mechanism of this hydrolytic enzyme is similar in principle to that of alkaline phosphatase. It requires the intermediate formation of two different enzyme-substrate complexes (126). E + S;:±ESi->ES2-^E4-P2 Pi

P2 is the galactose part of the substrate. It is not known whether ES2 is a covalent intermediate. A remarkable feature of this Michaelian enzyme is its absolute requirement for Na+ or {93, 95, 121, 138, 139). Very interesting studies have been recently carried out in Wallenfels' laboratory. Van Loan {136) and Wallenfels analyzed the binding of 3-nitro-4-aminophenylethylthio-jff-D-galactoside, a competitive inhibitor of )S-galactosidase, under various conditions of Na+, K+, and Mg^^ con­ centrations and at different pH's. As expected, there are four binding sites for the competitive inhibitor. In the absence of Na*^ or K% the com­ petitive inhibitor shows a sigmoidal binding isotherm characteristic of positive cooperativity. Positive cooperativity progressively disappears as the Na+ concentration is increased, and gives way to negative coopera­ tivity from Na+ concentrations as low as 10 μΜ. Mathematical analysis of the data indicates that there are two identical tight and two identical loose substrate-binding sites. This conclusion remains valid for a wide range of Na"- concentration including, for example, the conditions for optimal activity: pH 7.0, 40 mM NaCl, and 1 mM MgCU. Both positive and negative cooperativity show that the subunits are not independent. The interesting point is that medium conditions ade­ quate for negative cooperativity are absolute requirements for the mani­ festation of catalytic activity. It is tempting to postulate in this case a flip-flop mechanism analogous to that of alkaline phosphatase (Fig. 8). The functional unit in jff-galactosidase appears to be the dimeric structure. Intrasubunit cooperativity would couple ( * E ) the binding of the first molecule of )S-galactoside and the chemical transformation of E S i into ES2. Intersubunit cooperativity in the functional dimer would couple the binding of a second molecule of j3-galactoside to the second subunit and the activation ( * * E ) of the first subunit in view of the decomposition of ES2. E— E

*E—Si —

E

E—S2 + S •*E—S2 Pi

—

I

S

,

E— i *E—Si

P2

with reciprocation

FLIP-FLOP

MECHANISMS

AND HALF-SITE

ENZYMES

293

The data of Van Loan and Wallenfels give no evidence of an inter­ dependence of the two protomers (or functional dimers) in the tetrameric enzyme. D. Eschenchia coli Methionyl-tRNA Synthetase Escherichia coli methionyl-tRNA synthetase is a tetrameric enzyme having apparently identical subunits of molecular weight 43,000 (82,16), This enzyme, like other aminoacyl-tRNA synthetases, catalyzes a three-substrate reaction. Catalysis takes place in two steps. (i)

(ii)

irMethionine + ATP ^ Lrmethionine adenylate + PPi

L-Methionine adenylate + tRNA

i/-methionyl-tRNA -f AMP

As is generally the case for aminoacyl-tRNA synthetases, MichaelisMenten behavior is observed for all substrates (19), The interactions of methionyl-tRNA synthetase with the different ligands involved in the methionine activation reaction (methionine and analogs, ATP, AMP, P P i , methionyl adenylate, and methioninyl adenylate) were examined in great detail by equilibrium dialysis, absorbance difference spectroscopy, and fluorescence measurements at equilibrium and in a stopped-flow apparatus (10). By equilibrium dialysis four binding sites for ATP were found, but only two for methionine. The number of binding sites and the associa­ tion constants of these ligands for the enzyme were not affected by the presence of Mg^^ or EDTA. From ligand-induced fluorescence and absorbance variations, only two binding sites were measured for methionyl adenylate, a structural analog of the intermediate methionyl adenylate. By analogy, it is believed that two molecules of methionyl adenylate associate with the enzyme under the same conditions. From fluorescence data. Waller and his group (10) have inferred that in saturating con­ centrations of L-methionine and ATP the enzyme probably forms and retains two molecules of methionyl adenylate and may, in addition, bind two more molecule of ATP and possibly two of L-methionine. Finally, only 2 moles of tRNA were found to be bound per mole of tetrameric methionyl-tRNA synthetase (11), Since there is at present no evidence for any chemical difference between the enzyme subunits, all these data taken together constitute a convincing demonstration of the half-of-thesites reactivity of E. coli methionyl-tRNA synthetase. Independent information also obtained in Waller's laboratory shows that the enzyme is a double dimer (20, I40). Native methionyl-tRNA synthetase is highly susceptible to proteolytic cleavage. Incubation with trypsin results in the release of about 25% of the original protein, with the consequent dissociation of the tetramer into modified dimers, each now composed of two apparently identical subunits of molecular weight

294

MICHEL LAZDUNSKI

32,000 (as compared to 43,000 for the native enzyme). In spite of its drastically altered molecular structure, the trypsin modified enzyme is characterized by an unimpaired specificity and affinity toward normal substrates and by a practically unchanged catalytic efficiency (80% as active as the native enzyme). Modified dimeric methionine-tRNA syn­ thetase is also a half-site enzyme. It binds two moles of ATP but only 1 mole of methionine and 1 mole of tRNA per mole of enzyme (11), The tetrameric methionyl-tRNA synthetase then appears to be made up of two independent functional dimers each having a molecular weight of 64,000, and of four globules of 11,000 molecular weight which are im­ plicated in the covalent association of the dimers into the tetrameric structure (W, Ij^O), A start has been made on determining the tertiary structure of the dimeric trypsin-modified methionyl-tRNA synthetase by X-ray crystallographic analysis (Ij^), A flip-flop type mechanism may well account for the functional asym­ metry of the tetrameric (or dimeric) methionyl-tRNA synthetase (10). It is the opinion of the author of this review that the absolute negative cooperativity observed in the binding of both methionine and methionyl adenylate may be related to the coupling in the functional dimer (by intersubunit cooperativity) of the two fundamental reactions of the catalysis (i and ii). In such a model the formation and exclusion of methionyl-tRNA on one subunit (reaction ii) would be dependent upon the concomitant formation of methionyl adenylate (reaction i) on the other subunit. Such a mechanism would be perfectly consistent with the Michaelian behavior of the enzyme. More detailed studies concerning the mechanism of methionyl-tRNA synthetase are being carried out by Waller and his associates. It is of great interest that two functionally unrelated tetrameric enzymes, GAPDH and methionyl-tRNA synthetase, which both catalyze the transformation of three substrates using two successive reactions have a similar mode of operation. They are both double dimers and display a very strong anticooperativity for one of the products of the first reaction. Absolute negative cooperativity has been found for the covalent binding of 3-phosphoglycerate to GAPDH; abso­ lute negative cooperativity has also been found for the association of methionyl adenylate with methionyl-tRNA synthetase. It is well known that aminoacyl-tRNA synthetases.generally display Michaelian kinetics, it is also well known that all these enzymes are not tetramers. E, coli phenylalanyl-tRNA synthetase, which is a tetramer may well be a flip-flop enzyme (66), However, there are also dimers such as E, coK prolyl- and seryl-tRNA synthetases {64y 81) or monomers such as £ . coli leucyl-tRNA synthetase {107). One would like to believe, at least as a working hypothesis, that the mechanism involved in the

FLIP-FLOP

MECHANISMS AND HALF-SITE ENZYMES

295

catalytic activity will be common to most aminoacyl-tRNA synthetases. A polymeric structure is needed for half-of-the-sites reactivity and for a flip-flop mechanism. For this reason the latest results obtained with leucyl-tRNA synthetase are of great interest (107), It has been shown that this monomeric enzyme (MW 110,000) is, ih fact, made up of two compact and apparently identical protein fragments (MW 55,000 each) associated covalently through a small peptide of 3000 molecular weight. Leucyl-tRNA synthetase has only one site for ATP, one for leucine and one for tRNA {106). E. Acetoacetate Decarboxylase {96,123) and Transaldolase {Ί2α, 58 and References Therein) Acetoacetate decarboxylase from Clostridium acetobutylicum consists of twelve apparently identical subunits of molecular weight about 29,000. The enzyme dissociates reversibly into inactive dimers at pH 8 in 4 Jlf urea solutions. Acetoacetate reacts with the c-amino group of a specific lysine residue of the active site to form a Schiff base that serves as an essential inter­ mediate in the decarboxylation. Acetopyruvate is a covalent inhibitor of the reaction. It reacts with the same essential lysine side chain to form an enamine. The active site can also be selectively acetylated with acetic anhydride or with 2,4-dinitrophenyl acetate. The acetylated enzyme is completely inactive when only one acetyl group is introduced per dimeric unit. These results definitely show the polydimeric character and half-ofthe-sites reactivity of acetoacetate decarboxylase. These two properties are likely to be correlated with a fiip-flop type mechanism. Transaldolase also forms a Schiff base with its substrates. The enzyme catalyses the following reaction. Fructose 6-phosphate + erythrose 4-phosphate ;=± sedoheptulose 7-phosphate + glyceraldehyde 3-phosphate

In the absence of the acceptor, erythrose 4-phosphate, a stable enzymatically active intermediate accumulates which has been identified as a Schiff base containing dihydroxyacetone (DHA) linked to the c-amino group of a lysine residue at the active site of the enzyme. Transaldolase is a dimeric protein which appears to be a typical half-site enzyme. Horecker and his group have shown that only one equivalent of dihydroxyacetone is bound in the transaldolase dihydroxyacetone complex. Dihydroxyacetone is slowly released from the complex when the enzyme is incubated with the substrate, fructose 6-phosphate (F-6-P).

296

MICHEL LAZDUNSKI

The data may be interpreted as follows (personal communication from Dr. Horecker). E - F6P *E—S ^

E—

G3P / E—DHA

**E—DHA

E—

DHA / E— ^E—S

E—S with reciprocation

Intrasubunit cooperativity would couple (*E) the binding of the first F6P molecule and the Schiff base formation with DHA. Intersubunit cooperativity in the functional dimer would couple the binding of the second F6P molecule to the second subunit and the activation (**E) of the first subunit in view of the release of DHA. It was also shown that the enzyme contains two histidines (one per subunit) and that loss of activity is associated with loss of only one histidine residue. IV. Haif-of-the-Sites Reactivity and Flip-Flop Mechanisms for Allosteric Enzymes A . Main Features of Allosteric Enzymes with Flip-Flop Mechanism It has been emphasized up to now that Michaelis-Menten kinetics do not necessarily mean a functional independence of the subunits in a polymeric enzyme. Half-of-the-sites reactivity and its corollary, the flip-flop mechanism, provide a functional meaning for multisubunit structures. While the characteristic feature of allosteric kinetics is positive co­ operativity this does not exclude a flip-flop mechanism as a basic cata­ lytic process for allosteric enzymes. For the sake of simplicity, our model of an allosteric enzyme will be the tetramer of Monod, Wyman, and Changeux (94), which undergoes a concerted transition from the Τ to the R state. Only substrate binding will be discussed. If the four homologous catalytic sites are intrinsically independent, the saturation function will be (94). L(l + cay+{l

+

ay

where L = T/R (in the absence of substrate), a = S/ÜCR and c = Κ^/Κτ. ÜLR and KT are the microscopic constants for the dissociation of the substrate from its specific binding site in the R and Τ state, respectively.

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This equation for an equilibrium situation is also valuable for steadystate conditions where dissociation constants and Κτ are replaced by corresponding values {30, 74)· Such a model with four inde­ pendent catalytic sites will not be of the flip-flop type. The maximal value of the Hill coeflicient obtained from ν versus [S] profiles will be near η = 4 for c = 0 (or very near 0) and for high values of L (over 10*) (22). Let us suppose now that this tetramer is a double dimer. The func­ tional unit in the new model is the dimeric structure; dimers work catalytically as identical and independent flip-flop units and each func­ tional dimer displays half-of-the-sites reactivity for the substrate. In this case absolute negative cooperativity will characterize the functional dimer whereas positive cooperativity will characterize inter-dimer subunit interactions. The saturation function will be -y - _ Lcajl + Co) + a{l + ca) L{l + ca)'+{l + aY Preferential binding of the substrate to the R state will obviously give a sigmoidal shape for the ν versus [S] profile. Binding and catalysis exclusively by the R state and a very large L value will result in a Hill coefficient of 2 or nearly 2 instead of nearly 4 as in the previous model. The "half-site allosteric enzyme" will bind a maximum number of two substrate molecules on its four subunits (anticooperativity) and the binding isotherm will indicate a maximum value of 2 for the Hill number, that is, half the number of subunits. Half-of-the-sites reactivity in an allosteric enzyme similar to our model will be manifested not only by the number of substrate binding sites (two instead of four, for example), but also by the low value of the Hill coefficients. A number of allosteric enzymes have been found to have Hill co­ efficients practically equal to the number of subunits. For example. Hill numbers of 1.8-2.0 have been found for the saturation of the dimeric Phosphorylase b by the effector AMP (17). Therefore, while several interpretations may be given to Hill coefficients lower than the maximal value (22, 69, 71, 94) (and references therein), this author feels that Hill coefficients (obtained from substrate binding or catalysis) and corre­ sponding to half or less than half the maximal value (i.e., the number of identical subunits) could be indications for a flip-fiop type mechanism. A double dimer with two substrates may well have different Hill coefficients for each of its substrates. Independent binding of substrate Sl to each one of the four subunits, and negative cooperativity for sub­ strate S2 (only two of the four active sites can be saturated) may give a

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flip-flop mechanism. If binding and catalysis are carried out exclusively by the R state (L very high), the maximal values of the Hill coefficients will be 4 for substrate S i and only 2 for substrate S2. The allosteric systems that were just discussed are systems" (94) as defined by Monod et al A fiip-fiop mechanism with an allosteric dimer will give a "F system" (94)- The dimer will present negative coopera­ tivity for the substrate and positive cooperativity only for the other effectors (activators or inhibitors not structurally related to the sub­ strate). Langmuir-type profiles will always be observed for saturation by the substrate, and sigmoidal profiles will be obtained with effectors. For example, if L is very high, exclusive binding of the substrate to the R state, which works catalytically as a fiip-fiop unit (half-of-the-sites reactivity), will produce a Michaelis-Menten profile for substrate satu­ ration. Exclusive binding of an inhibitor to both independent inhibitor sites in the Τ state will give an hyperbolic saturation function in the absence of substrate, and a sigmoidal saturation function in saturating concentrations of the substrate. Under these last conditions, the Hill coefficient will be close to 2. To illustrate the possibility of having a fiip-fiop mechanism with an allosteric enzyme, two examples will now be treated: cytidine triphos­ phate synthetase and aspartate transcarbamylase. B. Cytidine Triphosphate Synthetase CTP-synthetase catalyzes the formation of CTP from NH3, UTP, and ATP. UTP -h N H , 4- ATP -> CTP + ADP + Pi

The enzyme can also utilize glutamine instead of NH3 as a nitrogen donor when GTP is present as an allosteric effector (89) GTP

U T P + Glu—NH2 + ATP — • CTP + G l u - O H + ADP + Pi

The mechanism of action of CTP synthetase from E. coli was re­ cently elucidated; it proceeds through the following steps (86). GTP

Enzyme + glutamine — • glutamyl-enzyme + NHa (bound) Glutamyl-enzyme + H2O —• enzyme + glutamate N H s + U T P ±^ H 2 N - U T P (tetrahedral adduct) H 2 N - U T P + A T P ^ H2N

\ t p ' .(phosphorylated tetrahedral adduct) + A D P H2N ^UTP

CTP + Pi

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In the absence of any ligand, CTP-synthetase exists as a dimer (88) of molecular weight 105,000 comprised of apparently identical subunits. Addition of glutamine or GTP does not change the polymerization state. However, when the substrates ATP and UTP are added at saturating concentrations, the dimer polymerizes to the active tetramer of molecular weight 210,000 (86,86). The enzyme has been shown to have positive homotropic effects for the two tetramer-stabilizing substrates ATP and UTP, and negative homotropic effects for glutamine (87). Half-of-the-sites reactivity was demonstrated with an aflSnity label, 6-diazo-5-oxonorleucine (DON), which mimicks the glutamine structure. Covalent binding of DON abolishes the glutamine activity of OTP synthetase. Binding of the other substrates ATP and UTP, and of the effector GTP are unaffected in the DON-labeled OTP synthetase. The aflSnity label reacts with only one of the two potential glutamine-binding sites. The reaction of DON remains asymmetric in the presence of GTP, but the rate of the covalent modi­ fication is increased by approximately 8-fold. The role of GTP is not to change the anticooperative behavior, but only to "activate" the glutamine site. In saturating concentrations of ATP, UTP, and GTP, CTP-synthetase is transformed into the tetrameric form. DON reacts with the enzyme so that only two of the four sites become covalently blocked. The interested reader is referred to the paper of Levitzski and his associates (87) for a discussion of the different conformational states of CTP-synthetase. A simple mechanism appears to be possible for CTP-synthetase activity in spite of the apparent complexity which is due to the great number of substrates, the great number of steps, and to the simultaneous functioning of positive and negative cooperativity. The tetrameric CTP-synthetase is obviously a double dimer. Each functional dimer behaves as a half-site enzyme and is likely to have a flip-flop mechanism at least for the two first steps of the catalysis. Glutamylation of one subunit of the functional dimer may be associated through a rate-limiting structural change (intersubunit cooperativity) with the transfer of the bound NH3 to UTP in the other subunit. Absolute negative cooperativity for only one substrate suflSces to impose catalytic asym­ metry. Appearance of activity requires a "double dimer" structure be­ cause of the preferential binding of UTP and ATP to the tetrameric form. Positive cooperativity will then be observed for both nucleotides. There are four equivalent and independent binding sites for UTP in the tetramer (87). Therefore it is not surprising that a maximal value of 3.4 for the Hill coeflScient has been obtained with this substrate (89). Similarly, values near 4 (n = 3.8) where obtained with ATP (89).

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C. Aspartate Transcarbamylase The regulatory properties of aspartate transcarbamylase (ATCase) of E. coll have been extensively studied, and the interested reader is referred to recent reviews by Gerhart (43), Kirschner (61), Hammes and Wu (50) and the references therein. The enzyme catalyzes the carbamylation of aspartate with carbamylphosphate, the first step in pyrimidine nucleotide biosynthesis. Carbamyl phosphate + aspartate

carbamyl aspartate + Pi

ATCase behaves as a "if system" as defined by Monod, Wyman, and Changeux (94), The saturation of ATCase by its substrates aspartate and carbamyl phosphate is sigmoidal. The same positive cooperativity is obtained with substrate analogs such as succinate and maleate. CTP, the end product of the pyrimidine biosynthetic pathway, and its analog 5'-bromo CTP are strong inhibitors of the enzyme. The binding sites of ATCase for substrates (and analogs) and inhibitor (and analogs) are located on different subunits. ATCase presents an hexameric structure symbolized by (RC)e (MW 310,000). It contains six apparently identical catalytic chains (MW 35,000) and six identical regulatory chains (MW 17,000) (141)^ Present chemical and crystallographic evidence strongly suggests that ATCase belongs to the Da class of dihedral symmetry. It appears to have three 2-fold axes at right angles to one 3-fold rotational axis. A plausible model proposed by Rosenbusch and Weber (104) appears in Fig. 11. Native hexameric ATCase binds 6 moles of CTP per mole of enzyme. Several investigators (18, 21, 44) 144) have shown that these CTP sites belong to two different classes of three sites: the high aflSnity sites (K^ = 9.3 X 10-^ M) and the low aflSnity sites (iCd = 4 X 10'^ M) (144)^ This very strong anticooperativity also exists in the separated regulatory dimer (43). Moreover half-of-the sites reactivity appears to hold for the substrates. Although there are six potential substrate binding sites in the hexameric ATCase (49, 51), only about three of them could be easily saturated with succinate (21,22), All these results taken together suggest that the hexameric ATCase is composed of three flip-fiop units. Each flip-flop unit would consist of two regulatory and two catalytic monomers. A flip-flop unit would then be a functional dimer, each "subunit" in this dimer being formed by the association of one regulatory and one catalytic chain. The functional dimer would present a 2-fold axis of symmetry and would bind asym­ metrically both CTP and the substrate analog succinate. Such an as­ sembly of three flip-flop units in the allosteric structure would evidently give maximal values of η = 3 for the Hill coeflScient relative to substrate

FLIP-FLOP

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301

FIG. 11. Proposal for the arrangement of the polypeptide chains in ATCase (104), The larger units (light shading) represent the six catalytic chains grouped in two trimers around a 3-fold symmetry (axis) (vertical line). The two trimers are arranged around three 2-fold symmetry axes (one of which is indicated by a horizontal line). The six regulatory chains (dark shading) are intercalated as dimers between the two catalytic trimers. Placing the subunits in register along the 3-fold axis is used for the purpose of better visualization only. Other related models have been proposed by CJerhart (43). From Rosenbusch and Weber (104) (by permission of the American Society of Biological Chemists).

binding and not η = 6 which would be expected for six independent catalytic subunits. It is of a great interest that the Hill coefficients determined for carbamylphosphate and for aspartate and succinate are, respectively, 3.0 and 1.6 (P, 21, 22, 48) very far from the theoretical maximal value of η = 6. A model of ATCase made up of three flip-fiop units would have the following properties: 1. Intrasubunit cooperativity. Binding of the substrates is an ordered process. Carbamyl phosphate binds first to permit the subsequent as­ sociation of aspartate (27). 2. Intersubunit cooperativity (2-fold axis of symmetry) to explain the anticooperative behavior of CTP and succinate. This is of primary importance for the catalytic mechanism. 3. Interprotomer (or inter-flip-flop) cooperativity (3-fold axis of sym­ metry) which is of primary importance for the manifestation of the positive cooperativity. Analysis of half-of-the-sites reactivity has been restricted in this review to substrate binding. Therefore, systems that show strong negative

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cooperativity for effectors [like that of ribonucleoside diphosphate re­ ductase (15)], although they are interesting have not been treated. V. The Functional and Evolutionaiy Advantages of Half-Site Enzymes and Flip-Hop Mechanisms In previous sections it has been shown that half-of-the sites reactivity appears to be a fairly general property among polymeric enzymes. A flip-flop type mechanism definitely provides a satisfactory explanation for the coexistence within a given enzyme of both negative cooperativity for its substrates and Michaelian kinetics. The application of flip-flop mechanisms is not limited to "classical" enzymes with Michaelis-Menten kinetics; alternating operation of the two subunits within a functional dimer may also be the basic mode of action of allosteric enzymes. In a flip-flop mechanism, conformational changes are not mainly used for a modulation of the catalytic activity [as for the allosteric transition (94) ]; they are an integral part of the catalytic mechanism itself. The importance of structural changes for the catalytic activity of enzymes was first formulated by Koshland in the form of the "induced-fit" theory (67, 68). While a fiip-fiop catalysis may have special advantages for a number of enzymes, such as to provide a lack of specificity for the alkaline phos­ phatase, this type of mechanism appears to have more important general properties. 1. The exchange of conformations between the two subunits of a functional dimer (flip-flop transition) ensures the thermodynamic cou­ pling between successive steps of the catalytic mechanism. In alkaline phosphatase, intrasubunit cooperativity couples substrate binding and phosphorylation, intersubunit cooperativity couples substrate binding (a second molecule) and dephosphorylation with product release. 2. Although thermodynamic coupling is undoubtedly of great impor­ tance for the manifestation of catalytic activity, it is not, in the opinion of this author, the main advantage of a flip-flop type mechanism. The main interest appears to be of an evolutionary nature. Intracellular enzymes are part of metabolic pathways. Their Km and Vm values cannot be chance parameters. The Km (or S0.5 for an allosteric enzyme) and Vm values of a given enzyme will obviously be adjusted to the Km and Vm values of the enzyme that precedes and of the enzyme that follows. These two fundamental kinetic parameters Km and Vm must be dis­ cussed independently; they are related to different events of the catalytic process. Km values characterize substrate recognition while Vm values characterize catalytic efficiency. In a metabolic pathway the product of

FLIP-FLOP

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a given enzyme will be the substrate of the following one. This implies analogies in active-site geometries of successive enzymes. Therefore, adjustments of Km values through mutations does not seem to present great problems, at least when the KJs represent true dissociation constants of enzyme-substrate complexes. The situation is quite different for Vm values. Successive transformations in a metabolic pathway involve very different types of chemical reactions. Consider for example four successive steps in glycolysis: transphosphorylation catalyzed by phosphofructokinase, aldolization by aldolase, isomerization by triose phos­ phate isomerase, and oxidation by glyceraldehyde-3-phosphate dehydro­ genase, fccat (Vm = fcoat [Eo]) is the rate constant of the slowest step under steady-state conditions. If this slowest step corresponds to the rate of the chemical transformation of the substrate, adjustment of Vm values of consecutive enzymes will obviously be a considerable challenge with a low probability of achievement. There is no mechanistic relation­ ship between an aldolization and an oxidation reaction. Conversely, the problem of mutual adjustment of Vm values through mutations would be easily solved if slowest steps were always related to structural changes affecting the enzyme substrate complexes. Flip-flop catalysis offers such a possibility. Intersubunit conformational change is an essential step in the mechanism. It means, for example, that binding of one substrate molecule to one subunit of the functional dimer creates a correct geometry of the active site in the other subunit so that catalysis can take place (see Figs. 8 and 9). Catalysis is dependent upon intersubunit rearrange­ ment. It is believed that in many cases, the structural reorganization is much slower, by several orders of magnitude, than the catalytic process which follows. A rate-limiting conformational change of the enzyme offers an easy modulation of Vm values. Any mutation of one residue of the monomeric polypeptide chain appears twice in a symmetrical dimer. The catalytic effect of the single mutation will then obviously be considerably amplified if this particular residue is implicated in the intersubunit conformational change of the functional flip-flop dimer. As already forecast by Monod and co-workers (94), "symmetrical oligomers should constitute particularly sensitive targets for molecular evolution, allowing much stronger pressures to operate in the random pursuit of functionally adequate structures." Rate-limiting conformational changes in enzyme catalysis do not necessitate polymeric structures, but the amplification system of mutational events is lost in a monomeric enzyme. In fact, most enzymes involved in metabolic pathways and requiring a mutual adjustment of Km and Vm parameters are polymeric; monomeric enzymes are usually found among the secretory proteins (trypsin, chymotrypsin, subtilisin.

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carboxypeptidases, RNases, DNases, etc.). The latter enzymes do not have to react in a given order. The existence of intracistronic complementation (29) may be an interesting indication of the existence both of flip-flop mechanisms and of rate-limiting steps controlled by conformational changes. Intra­ cistronic complementation has been observed for a number of enzymes, but for the sake of simplicity, only the work concerning E, coli alkaline phosphatase will be mentioned (37, 113), Hybrid dimers of this enzyme were formed in vitro with subunits from each of various mutants. In vitro complementation of inactive or practically inactive mutant mono­ mers was shown to produce active dimers having specific activities vary­ ing depending on the combination. These observations suggest that hybridization restored the possibility of intersubunit cooperativity, that is, the alternating operation of the two subunits necessary for a flip-fiop mechanism. Different specific activities may be considered to reflect different rate constants for the rate-limiting conformational changes. 3. It is now clear that enzymes are not randomly distributed in the cell but rather are organized into functionally significant assemblies. A number of multienzyme complexes have now been isolated. The pyruvate dehydrogenase complex (102) and acetoacetyl carboxylase (4β, 135) will be taken here as examples of the possible integration of fiip-fiop type mechanisms within such multienzyme structures. The pyruvate dehydrogenase complex is made up of three different types of enzymes: pyruvate dehydrogenase itself, dehydrolipoyl transacetylase, and dehydrolipoyl dehydrogenase. The lipoyl moiety of the complex undergoes a cycle of transformation, i.e., reductive acylation, acyl transfer, and electron transfer. These transformations involve the interaction of the lipoyl part of transacetylase with thiamine pyrophosphate bound to pyruvate de­ hydrogenase and with the prosthetic group of dehydrolipoyl dehydrogen­ ase. Oscillation of the transacetylase-bound lipoyl moiety between pyruvate dehydrogenase and dehydrolipoyl dehydrogenase is a necessary requirement for catalysis. A flip-flop type mechanism would be a satis­ factory interpretation of this reciprocating motion. Acetyl-CoA carboxylase is composed of three essential protein com­ ponents: a carboxyl-carrier protein which contains the covalently bound biotin prosthetic group; biotin carboxylase which catalyzes the ATPdependent carboxylation of the carrier bound biotin, and a carboxyl transferase which has a role in carboxyl transfer from the carboxylated prosthetic group to acetyl-CoA. The catalytic mechanism of acetyl-CoA carboxylase requires the oscillation of the protein-carrier-bound biotin prosthetic group from biotin carboxylase to carboxyl transferase and

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back. Interenzyme translocation of the biotin prosthetic group is likely again to be controlled by a flip-flop type mechanism. 4. Finally, although various types of inhibitions, especially inhibitions by excess substrate, have not been treated in this review, it should be mentioned that flip-flop type mechanisms provide an easy explanation for them. These inhibitions are frequently difficult to explain with classical theories for ordered mechanisms (54). They might be of importance for the autonomous control of enzymatic activity at each step of a metabolic pathway. ACKNOWLEDGMENTS The idea of the flip-flop mechanism for the alkahne phosphatase was initially developed in collaboration with Dr. Claude Lazdunski and Dr. Claude Petitclerc. Further developments were carried out in collaboration with D . Chappelet, Dr. M. Fosset, and C. Cache. I am glad to thank all these associates as well as other members of my laboratory for their enthusiastic and stimulating participation. Careful reading of the manuscript by Dr. D . Grossman is gratefully acknowledged. I thank Dr. Luisi and Dr. Wallenfels for communication of their results before publication and Dr. Bernhard and Dr. Weber for permission to reproduce their data in Figures 10 and 11. REFERENCES 1. Allen, L., Vanecek, J., and Wolfe, R. G., Arch. Biochem. Biophys. 143, 166 (1971). 2. Anderson, S. R., and Weber, G., Biochemistry 4, 1948 (1965). 3. Antonini, E., and Brunori, M., Annu. Rev. Biochem. 39, 977 (1970). 4. Applebury, M. L., and Coleman, J. E., Biol. Chem. 244, 709 (1969). 6. Applebury, M. L., and Coleman, J. E., J. Biol. Chem. 245, 4969 (1970). 6. Atkinson, D . E., in "the Enzymes'' (P. D . Boyer, ed.), 3rd ed., Vol. 1, p. 461. Academic Press, New York, 1970. 7. Banaszak, L. J., Tsernoglou, D., and Wade, M., in "Probes of the Structure and Function of Macromolecules and Membranes," Vol. II, p. 71. Academic Press, New York, 1971. 8. Bernhard, S. Α., Dunn, M. F., Luisi, P. L., and Schack, P., Biochemistry 9, 185 (1970). 9. Bethell, Μ. R., Smith, K. E., White, J. S., and Jones, M. E., Proc. Nat. Acad. Sei. U. S. 60, 1442 (1968). 10. Blanquet, S., Fayat, G., Waller, J. P., and Iwatsubo, M., Eur. J. Biochem. 24, 461 (1972). 11. Blanquet, S., and Waller, J. P., personal communication. 12. Boers, W., Oosthuizen, C , and Slater, E . C , Biochim. Biophys. Acta 250, 35 (1971). 12a. Brand, K., Tsolas, 0., and Horecker, B. L., Arch. Biochem. Biophys. 130, 521 (1969). 13. Branden, C. I., Zeppezauer, E., Boiwe, T., Söderlund, G., Söderberg, Β. Ο., and Nordström, Β., in "Pyridine Nucleotide-Dependent Dehydrogenases" (Η. Sund, ed.), p. 129. Springer-Verlag, Berlin and New York, 1970.

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FLIP-FLOP MECHANISMS AND HALF-SITE ENZYMES

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64. Harada, K., and Wolfe, R. G., J. Biol. Chem. 243, 4131 (1968). 66. Harris, J. I., Meriwether, B. P., and Park, J. H., Nature (London) 198, 154 (1963). 66. Harris, J. I., and Perham, R. N., J. Mol. Biol. 13, 876 (1965). 67. Heppel, L. Α., Harkness, D . R., and Hilmoe, R. J., J. Biol. Chem. 237, 841 (1962). 68. Horecker, B. L., Cheng, T., and Pontremoli, S., / . Biol. Chem. 238, 3428 (1963). 69. Jomvall, H., Eur. J. Biochem. 1β, 25 (1970). 60. Keleti, T., in "Pyridine Nucleotide-Dependent Dehydrogenases" (H. Sund, ed.), p. 104. Springer Veriag, Berlin and New York, 1970. 61. Kirschner, K., this series 4,167 (1971). βΒ. Kirschner, K., J. Mol Biol 58, 51 (1971). 63. Klotz, I. M., Annu. Rev. Biochem. 39, 25 (1970). 64. Knowles, J. R., Katze, J. R., Königsberg, W., and Söll, D., / . Biol Chem. 245, 1407 (1970). 66. Ko, S. H. D., and Kezdy, F. J., J. Amer. Chem. Soc. 89, 7139 (1967). 66. Kosakowski, H. M., and Bock, Α., Eur. J. Biochem. 24, 190 (1971). 67. Koshland, D . E., Jr., Proc. Nat. Acad. Sei. U.S. 44,9$ (1958). 68. Koshland, D . E., Jr., J. Cell Comp. Physiol 54, 235 (1959). 69. Koshland, D . E., Jr., this series 1, 1 (1969). 70. Koshland, D . E., Jr., and Neet, K. E., Annu. Rev. Biochem. 37, 359 (1968). 71. Koshland, D . E., Jr., Nemethy, G., and Filmer, D., Biochemistry 5, 365 (1966). 72. Lardy, H. Α., Paetkou, V., and Walter, P., Proc. Nat. Acad. Sei. U. S. 53, 1410 (1965). 73. Lazdunski, C , Chappelet, D., Petitclerc, C , Leterrier, F., Douzou, P., and Lazdunski, M., Eur. J. Biochem. 17, 239 (1970). 74' Lazdunski, M., and Delaage, M., in preparation. 76. Lazdunski, C , and Lazdunski, M., Biochim. Biophys. Acta 113, 551 (1966). 76. Lazdunski, C , and Lazdunski, M., Eur. J. Biochem. 7, 294 (1969). 77. Lazdunski, C , Petitclerc, C , Chappelet, D., and Lazdunski, M., Biochem. Biophys. Res. Commun. 37, 744 (1969). 78. Lazdunski, C , Petitclerc, C , Chappelet, D., Leterrier, F., Douzou, P., and Lazdunski, M., Biochem. Biophys. Res. Commun. 40, 589 (1970). 79. Lazdunski, C , Petitclerc, C , and Lazdunski, M., Eur. J. Biochem. 8, 510 (1969). 80. Lazdunski, M., Petitclerc, C , Chappelet, D., and Lazdunski, C , Eur. J. Biochem. 20, 124 (1971). 81. Lee, M. L., and Muench, K. H., / . Biol Chem. 244, 223 (1969). 82. Lemoine, F., Waller, J. P., and Van Rapenbusch, R., Eur. J. Biochem. 4, 213 (1968). 83. Leuzinger, W., Biochem. J. 123, 139 (1971). 84. Levine, D., Reid, T. W., and Wilson, I. B., Biochemistry 8, 2374 (1969). 86. Levitzki, Α., and Koshland, D . E., Jr., Biochim. Biophys. Acta 206, 473 (1970). 86. Levitzki, Α., and Koshland, D . E., Jr., Biochemistry 10, 3365 (1971). 87. Levitzki, Α., Stallcup, W. Β., and Koshland, D . E., Jr., Biochemistry 10, 3371 (1971). 88. Long, C. W., Levitzki, Α., and Koshland, D . E., Jr., / . Biol Chem. 245, 80 (1970). 89. Long, C. W., and Pardee, A. B., J. Biol Chem. 242, 4715 (1962). 90. Luisi, P. L., and Favilla, R., Biochemistry 11, 2303 (1972). 91. McQuarrie, R. Α., and Bernhard, S. Α., J. Mol Biol 55,181 (1971).

308

MICHEL LAZDUNSKI

91a. Malamy, M. H., and Horecker, B. L., Biochemistry 3, 1889 (1964). 92. Malhotra, 0 . P., and Bernhard, S. Α., / . Biol. Chem. 243, 1243 (1968). 93. Monod, J., Cohen-Bazire, G., and Cohn, Μ., Biochim. Biophys. Acta 7, 585 (1951). 94. Monod, J., Wyman, J., and Changeux, J.-P., J. Mol. Biol. 12, 88 (1965). 95. Neville, M. C , and Ling, G. N., Arch. Biochem. Biophys. 118, 596 (1967). 96. O'Leary, M. H., and Westheimer, F. H., Biochemistry 7, 913 (1968). 97. Perutz, M. F., Nature {London) 228, 726 (1970). 98. Petitclerc, C , Lazdunski, C , Chappelet, D., Moulin, Α., and Lazdunski, M., Eur. J. Biochem. 14, 301 (1970). 99. Pfleiderer, G., and Auricchio, F., Biochem. Biophys. Res. Commun. 16, 53 (1964). 100. Flocke, D . J., Levinthal, C , and Vallee, B. L., Biochemistry 1 , 373 (1962). 101. Raval, D . N., and Wolfe, R. G., Biochemistry 1 , 1118 (1962). 102. Reed, L. J., this series 1 , 233 (1969). 103. Reynolds, J. Α., and Schlesinger, Μ. J., Biochemistry 8, 4278 (1969). m. Rosenbusch, J. P., and Weber, K., Biol. Chem. 246,1644 (1971). 105. Rothman, F., and Byrne, R., J. Mol. Biol. 6, 330 (1963). 106. Rouget, P., and Chapeville, F., Eur. J. Biochem. 23, 433 (1971). 107. Rouget, P., and Chapevüle, F., Eur. J. Biochem. 23, 459 (1971). 108. Sandler, N., and McKay, R. H., Biochem. Biophys. Res. Commun. 35, 151 (1969). 109. Sanwal, B. D., BacteHol. Rev. 34, 20 (1970). 110. Schlesinger, Μ. J., / . Biol. Chem. 240, 4293 (1965). 111. Schlesinger, Μ. J., / . Biol. Chem. 242,1604 (1967). 112. Schlesinger, Μ. J. Brookhaven Symp. Biol. 17, 66 (1964). 113. Schlesinger, Μ. J., and Levinthal, C , / . Mol. Biol. 7, 1 (1963). 114. Schlesinger, Μ. J., and Olsen, R., J. BacteHol. 96, 1601 (1968). 115. Schlesinger, Μ. J., Reynolds, J. Α., and Schlesinger, S,, Ann. N. Y. Acad. Sei. 166, 368 (1969). 116. Schwartz, J. H., Proc, Nat. Acad. Sei. U. S. 49, 871 (1963). 117. Simpson, R. T., and Vallee, B. L., Biochemistry 7, 4343 (1968). 118. Simpson, R. T., and Vallee, B. L., Ann. N. Y. Acad. Sei. 166, 670 (1969). 119. Simpson, R. T., and Vallee, B. L., Biochemistry 9, 953 (1970). 120. Stadtman, E. R., in "The Enzymes" (P. D . Boyer, ed.), 3rd ed.. Vol. 1, pp. 398-459. Academic Press, New York, 1970. 121. Strom, R., Attardi, D . G., Forsen, S., Turini, P., Celada, F., and Antonini, E., Eur. J. Biochem. 23, 118 (1971). 122. Sund, Η., and Theorell, H., in "The Enzymes" (P. D . Boyer, H. Lardy, and K. Myrbäck, eds.), 2nd ed. Vol. 7, p. 25. Academic Press, New York, 1963. 123. Tagaki, W., and Westheimer, F. H., Biochemistry 7, 891 (1968). 124. Tait, G. H., and Vallee, B. L., Proc. Nat. Acad. Sei. U. S. 56, 1247 (1966). 125. Tate, S. S., and Meister, Α., Proc. Nat. Acad. Set. U. S. 68, 781 (1971). 126. Tenu, J. P., Viratelle, 0 . M., Garnier, J., and Yon, J., Eur. J. Biochem. 20, 363 (1970). 127. Theorell, H., in "Molecular Associations in Biology" (B. Pullman, ed.). Aca­ demic Press, New York, 1968. 128. Theorell, H., and Chance, B., Acta Chem. Scand. 5, 1127 (1951). 129. Thome, C , and Kaplan, N . 0., / . Biol. Chem. 238, 1861 (1963). 130. Torriani, A. M., Biochim. Biophys. Acta 38, 460 (1960).

FLIP-FLOP

131. 132. 133. 134. 135. 136. 137. 138. 139. 140. 141. 142. 143. 144. 146'

MECHANISMS AND HALF-SITE ENZYMES

309

Torriani, A. M., / . Bacteriol 96, 1200 (1968). Trentham, D . R., Biochem. J, 109, 603 (1968). Trentham, D . R., and Gutfreund, Η., Biochem. J. 106, 455 (1968). Tsernoglou, D., Hill, E., and Banaszak, L. J., Cold Spnng Harbor Monographs 36, 171 (1971). Vagelos, P. R., this series 4, 119 (1971). Van Loan, V., Ph.D. Thesis, University of Freiburg, Germany, 1971. Velick, S. F., and Furfine, C , in "The Enzymes" (P. D . Boyer, H. Lardy and K. Myrbäck, eds.). Vol. 7, p. 243. Academic Press, New York, 1963. Wallenfels, Κ., and Malhotra, 0 . P., Advan. Carbohyd. Chem. 16, 239 (1961). Wallenfels, Κ., Malhotra, 0 . P., and Dabich, D., Biochem. Z. 333, 377 (1960). Waller, J. P., Risler, J. L., Monteühet, C , and Zelwer, C , FEBS Lett. 16, 186 (1971). Weber, K., J. Biol Chem. 243, 543 (1968). Wilson, I. B., and Hogness, D . S., / . Biol Chem. 239, 2469 (1964). Wilson, I. B., and Hogness, D . S., / . Biol Chem. 244, 2132 (1969). Winlund, C , and Chamberlin, M., Biochem. Biophys. Res. Commun. 40, 43 (1970). Zabin, I., and Fowler, Α., in "The Lactose Operon" (I. R. Beckwith and D. Zipser, eds.), p. 27. Cold Spring Harbor Lab. Quant. Biol., Cold Spring Harbor, New York, 1970. NOTE ADDED IN PROOF

A number of papers by Halford and his co-workers [Biochem. J. 126, 127 (1972); 126, 108 (1972); / . Biol Chem. 247, 2095 (1972)] have appeared after this review was sent for publication. These papers contain numerous misquotations and mis­ interpretations of our previous work. Some essential corrections are therefore needed for clarification. The statement by Halford that "Lazdunski et al. (1971) were not able to confirm observations on the liberation of only one mole of product/mole of dimeric enzyme . . . at low pH values" is false as can be seen in the discussion of Section Π,Α in this review. Halford et al also write that we postulated that sub­ strate and inorganic phosphate induced "the same conformational change on binding to the dimer" and that this conclusion is incompatible with their results. Far from drawing such a conclusion from our results, we demonstrated that inorganic phoisphate and substrates behave very differently: the former forms only noncovalent complexes at alkaline pH whereas the others form a phosphorylated derivative with the enzyme under the same conditions. Substrates and inorganic phosphate binding do not induce the same conformational change but they both induce an asymmetry (negative cooperativity or half-site reactivity) in the phosphatase structure. In the last paper Halford et al write that "a further alternative for the catalytic mechanism of alkaline phosphatase, from Lazdunski et al is that dephosphorylation . . . deter­ mines the turnover number at both acidic and alkaline pH values and that (Halford's results) . . . can be used as evidence against any mechanism which fails to include a change in the rate-limiting step between acidic and alkaline conditions." A change in the limiting step does obviously occur between acidic and alkaline conditions as was already discussed in reference 80 and also in Section II,C of this review. The important conclusions from the work of Halford et al are the following: 1. Half-site reactivity for the Zn'^ phosphatase of E. coli is observed.

310

MICHEL LAZDUNSKI

2. Ligand-induced conformational changes occur after substrate binding, and unimolecular isomerizations determine the rate of product formation. 3. Although no mechanism has been given, it was suggested that conformational change for substrate binding at one subunit is linked to another conformational change on the other subunit as orthophosphate dissociates from that site. The results and discussions that appear in these papers confirm previous observa­ tions and interpretations (Sections ΙΙ,Α and C of this review). The present author believes that Halford's data provide complementary evidence obtained by stoppedflow and relaxation techniques that the E. coli phosphatase displays a flip-flop type mechanism.

AUTHOR INDEX Numbers in parentheses are reference numbers and indicate that an author's work is referred to although his name is not cited in the text. Numbers in italics show the page on which the complete reference is listed.

Abd-El-Al, Α., 227(1), 228(1), 229(1), 261 Abraham, J. M., 250(92), 263 Abraham, S., 34(1), 66 Acher, R., 7(50), 13(51), IP Acquarone, M. E., 29(113), 38(2), 66, 60 Adair, G. S., 143,166 Adair, L. B., 227(2), 228(2), 229(2), 261 Adam, Α., 42(3), 66 Adelman, R. C , 162(69), 167 Afolayan, 42(4), 43(4, 99), 45(99), 50 (97), 52(4), 67, 69 Aftergood, L., 176(1), 181(1), 202 Ahrens, Ε. Η., Jr., 178(57, 116), 189(57), 201 206 Aithal, Η. Ν., 178(2), 182(120), 191(3), 192(3), 193(3), 202, 206 Aiyar, Α. S., 192(4), 202 Alberti, Μ., 25(59), 26(59), 68 Alberty, R. Α., 65, 67(4, 5, 57), 128, 129, 137, 149(3, 4), 166 Alema, S., 22(18), 40(18, 19), 67 Alfin-Slater, R. B., 176(1), 181(1), 202 Allan, N. C , 44(95), 50(95), 69 Allen, L., 284(1), 295(1), 306 Altman, C , 65,128 Amaya, T., 228(159), 253(159), 266 Ames, B. N., 111(1), 127 Amorosi, E. L., 44(134), ^0 Anderson, A. D., 236(71), 262 Anderson, C , 236(71), 262 Anderson, P. M., 227(3), 228(4), 236(3), 237(103), 239(103), 243(4), 258(4), 261, 263 Anderson, S. R., 287(2), 305 Anderson, W., 12(13), 18 Anderson, W. B., 28(5), 30(5, 6, 73), 31

Anthony, R. S., 152(5), 166 Antonini, E., 218, 221, 224, 226, 226, 284 (3), 292(121), 305, 308 Appel, S. H., 228(5), 234(5), 254(5), 255 (5), 256(5), 257(5), 258(5), 260(5), 261 Applebury, M. L., 270, 279(52), 280(4), 305, 306 Arese, P., 45(27, 28, 29), 57 Armstrong, D . T., 199, 200(5, 8), 202 Aronson, A. I., 3(34), 6(1, 34), 8(1, 34), 9(34), 10(1, 34), 11(1), 12(1), 15(34), 18, 19 Atkins, I. C , 229(126), 245(126), 247 (174), 254(126), 261 265 Atkinson, D. E., 30(139), 60, 64(la, 2), 68(la), 72(3), 95(2), lOO(la), 127, 128, 156(6, 9), 152(6), 157(7), 158 (66), 159(7), 162(8), 166, 167, 286 (6), 305 Attardi, D. G., 292(121), 308 Aubert, J.-P., 7(52), 8(52, 68), 9(52), 10 (68), 11(52), 12(53), 13(51, 52, 53), 19, 20 Austrian, R., 228(6), 229(6), 235(6), 255 (6), 261 Auricchio, F., 287(99), 308 Avigad, G., 29(8), 30(8), 41, 57 Avila, J., 15(2), 18 Avoy, D. R., 175(9), 176(9), 181(9), 202 Β Bach, M. L., 11(3), 18 Bachelard, Η. S., 156(10), ί^β Back, P., 176(10), 178(10), 183(10), 203 Backkolb, F., 23(9), 57 Bagh, J. F., 203 Bain, J. Α., 229(142), 234(142), 247(142), 254(61), 255(61, 142), 256(142), 262,

(5, 6, 73), 35(5, 6), 67, 69 Angelo, N., 6(1), 8(1), 10(1), 11(1), 12 Baker, F. Α., 227(139), 228(139), 229 (1), 18 311

312 (140, 141), 251(140, 144), 252(139), 264 Baker, G. D., 176(13), 179(12, 13), 203 Balassa, G., 1(67), 20 Balinsky, D., 23(161), 50(10), 67, 61 Banaszak, L. J., 2g4(7), 285(45a, 134), 286(134), 305, 306, 309 Bandi, L., 196(122), 206 Banks, J., 51(115), Banks, R. C., 198(42), 203 Bard, R. C , 23(47), 24(47), 58 Barr, D . P., 181(11), 203 Barrett, A. M., 181, 206 Barrett, K. J., 279(37), 304(37), 306 Bartley, W., 30(136), 60 Barto, E., 36(156), (5i Barry, J. M., 34, 35(68), 58, 59 Barwell, C. J., 163(11), ίοβ Basford, R. Ε., 157(65), 167 Beckwith, J. R., 228(6), 229(6), 235(6), 255(6), 261 Becroft, D . M. 0., 254(7), 255(7), 261 Beher, W. T., 176(13), 179(12, 13), 203 Behrens, 0 . Κ., 38, 57 Bekersky, L, 195(131), 206 Bell, R. Μ., 131,166 Bell, V. L., 176(52), 183(52), 204 Belser, W. L., 228(53), 229(53), 262 Bennett, L. L., 189(98), 190(98), 205 Bennett, N. G., 280(48), 306 Benz, J. 0., 190, 204 Bergstrom, S., 179(14), 195, 203 Bernhard, S. Α., 285(8, 91, 92), 288, 289 (35), 290, 291(91, 92), 305, 306, 307, 308 Bernhardt, P. H., 227(163), 228(163), 230, 232(163), 233(163), 237(163), 238 (163), 258(163), 266 Bernhardt, S. Α., 261 Bernlohr, R. W., 3(4), 8(31), 9(6), 10(5, 6), 11(30, 31), 12(6), 13(6), 17(11), 18, 19 Bernstein, R. E., 50(10), 67 Bethell, M. R., 227(8), 228(8), 229(8), 24m). 261, m(9),S06 Beutler, E., 36(12, 129), 37(12, 110, 163, 164), 38(110), 41(13), 42(13, 14), 44 (13), 57, 59, 60, 61 Beyer, R. E., 191(15), 192(15), 203 Beytia, E., 152(13), 166

AUTHOR INDEX

Bhattathiiy, E. P. M., 178(16), 203 Bhose, Α., 176(110), 181(110), 187, 205 Binotti, I., 221, 225 Bishop, S. H., 243(9), 244(9), 261 Bittman, R., 142(37), 166 Black, D . L., 199, 202 Blair, Α., 37(155), 61 Blanquet, S., 285(10), 293(10, 11), 294 (10, 11), 304(11), 306 Blatchford, K., 227(16), 261 Bledsoe, Τ., 197, 203 Bloomfield, V., 128 Bockrath, R., 128 Böck, Α., 294(66), 307 Boers, W., 290(12), 305 Boissonas, R. Α., 293(19), 306 Boiteux, Α., 63(33), 74,128 Boiwe, T., 289(13), 305 Bonnichsen, R., 158(72), 167 Bonsignore, Α., 22(18, 25, 26, 179), 28 (111), 29(16, 17), 30, 40(18, 19), 42 (25), 45(15, 24), 46(23, 24, 25, 26, 46, 179), 47(179), 49(23, 179), 50(21, 22), 52(22, 24, 26, 179), 53(18, 26, 179), 55(20, 26, 179), 67, 68, 69, 61 Booth, Α. Ν., 194(18), 203 Bortz, W. Μ., 184, 203 Bossü, Μ., 44, 68 Bottomley, R. Η., 243, 261 Bourget, P. Α., 245, 248(12), 249(12), 261 Bowers, Μ. D., 236(13), 261 Bowness, J. Μ., 112(7), 113, 121,128 Boyd, G. S., 176(20), 180(20), 181(20, 114), 196(21, 22), 200, 203 205, 206 Boyer, H. W., 5(7), 8(7), 14(7), 18 Boyer, P. D., 164(14), 166 Bradshaw, R. Α., 285(45a), 306 Brand, K , 30(30, 31), 45(27, 28, 29), 57, 285(12a), 295(12a), 305 Brand, L., 65(32), 128 Branden, C. I., 289,305 Bransome, E. D., Jr., 196(23), 203 Bresnick, E., 227(16), 229(14, 15, 19), 245 (18), 247(17), 251(17, 19), 256(19), 261 Briggs, W. W., 180(27), 181(27), 203 Broad, T. E., 31(32), 57 Broadbent, J. Α., 228(25), 234(25), 261 Brockman, R. N., 274(14), 305 Brodie, J. D., 175, 203

AUTHOR INDEX

Brosnan, J. T., 250(20), 261 Brown, B,, 90, ISO Brown, D . H., 23(65), 26, 28, 29(65), 68 Brown,N. C , 302(15), SÖ^ Brownie, A. C , 196(22), Brozowski, T., 227(73), 262 Brunori, M., 218, 221, 226, 284(3), 306 Bruton, C. J., 293(16), 50^ Buc, H., 297(17), 50β Bucher, N. L. R., 176(25), 177(25), 179 (25), 180(25), 183(25), 185, 187(25), 188(25), 203 Buchowicz, J., 227(104), 251(104), 263 Buckman, T., 300(18), S06 Bunn, H. F., 149(15), iöö Bunyan, J., 192(41), 203 Burgess, E. Α., 250(92), 263 Burgoyne, L., 99(8), 128 Burk, D . J., 134,166 Burt, M. E., 237(158), 239(158), 266 Burton, K., 28(33), 57 Butcher, R. W., 115(63), 116(30, 63), 117 (9, 30), 128, 129 Byers, S. 0., 176(49), 180(27), 181(27), 183(49), 189(50), 190(50), 201(49), 203, 204 Byrne, R., 269(105), 308

CahiU, G. F., Jr., 34(34), 67 Cahn, R . D . , 147(16), iöß Calcott, M. Α., 112(50), 113, 129 CaHssano, P., 40(60), 68 Calva, E., 229(21, 22), 261 Calvo, J. M., 94(11), 128 Cama, H. R., 191(99), 206 Campbell, B. J., 180(56), 204 Campbell, J. W., 236(156), 237(156), 243 (9, 156, 157), 244(9), 261, 264, Cancedda, R., 22(25, 26), 38(2), 42(25), 45(24), 46(23, 24, 25, 26), 47(25), 49 (23), 50(21, 22), 51(22), 52(22, 24, 26), 53(26), 54, 55(20, 26), 66, 67 Cannon, W. B., 64, 128 Cantwell, R., 146(32), 166 Carlsson, B., 236(89), 263 Carlton, B. C , 5(7, 20), 8(7, 20), 12(21), 14(7, 21), 18 CaroUne, D. F., 228(24), 231(23), 253(23), 261

313 Cartesegna, C , 40(60), 55 Case, M., 99(8), 128 Cafisio, D., 293(19, 20), 294(20), 306 Celada, F., 292(121), 308 Celikkol, E., 6(62), 14(62), 15(62, 63), 20 Chft, C.-J. M., 160(18), 166 Cha, S., 160(18), 163(17), 166 Chaikoff, I. L., 34(1), 38(61, 62), 66, 68, 176(153), 177(152), 178(72), 179(15, 153), 180(151), 189(72), 191(151), 204, 206 Chaloupka, J., 9(8, 9, 10, 15), 18 Chamberlin, M., 300(144), 309 Chance, B., 63(28), 67(28), 68(28), 74 (28), 128, 164, 166, 289, 308 Changeux, J.-P., 29(36), 68, 64(47), 89, 91(47), 101(47), 125(47), 129, 209, 214, 226, 267(94), 268(94), 296(94), 297(22, 94), 299(94), 300(21), 301(21, 22), 302(94), 303(94), 306, 308 Chao, Α., 149(15), 166 Chapeville, F., 294(107), 295(106, 107), 308 Chappelet, D., 268(80), 269(24, 73, 77, 78, 80), 270(24, 77, 78, 80), 271(77, 78, 80), 272(80), 273(80), 274(80), 275 (73), 276(80), 278(77), 279(98), 280 (73, 78, 80), 282(24, 73, 77, 80), 283 (23, 40, 80), 284(80), 285(23, 98), 288 (80), 295(23), 306, 307, 308 Charles, H. P., 228(25), 234(25), 261 Charles, R., 249(26), 261 Chen, B., 12(13), 18 Cheng, T., 285(58), 295(58), 307 Chi, Y.-M., 117(39, 51), 128, 129 Chieffi, 0., 45(15), 67 ChiUa, R., 23(50), 26(50), 68 Chishohn, E. M., 38(146), 39(146), 40 (146), 60 Chiu, M., 187(100), 190(100), 206 Chistian, W., 23(176), 61 Chung, A. E., 46(37), 50(37), 52(37), 53 (37), 54, 68 Ciferri, 0., 118, 128 Citri, Ν., 9 ( 5 8 ) , / Ρ Clark, v., 9(6), 10(6), 12(6), 13(6), 17 (11), 18 Clark-Turri, L., 38(125), 60 Cleland, W. W., 67(14, 15, 16, 17), 78 (17), 120(17), 121(17), 124(17), 128,

314 132, 133, 137(20), 152(13), 158, 160 (26), 162(20), 166, 167, 287(25, 26), 306 Cohen, G. N , 62(82, 83), 90(20, 76, 82), 91(81), 94(20, 82), 99, 100(18, 54, 55), 128, 129, 130, 260(27), 261 Cohen, P., 45(38, 39), 46(38, 39), 49 (39, 40), 50(39), 52(38, 39), 53(39), 68 Cohen, P. P., 227(90, 96, 146), 228(90), 229(21, 22, 81, 146), 235(47, 105), 236 (47, 79, 81, 105), 237(90), 243(146), 247(81, 96), 252(90), 261, 262, 263, 264 Cohen-Bazire, G , 64(48), 101(48), 129, 292(93), 308 Cohn, Μ., 152(48), 167, 292(93), 308 Coleman, G , 3(12), ί5 Coleman, J. E., 270, 279(52), 280(4), 306, 306 Coleman, M. S., 227(28), 228(28), 229 (28), 261 Collins, K. D , 301(27), 306 Collins, Z , 42(14), 67 Colowick, S. P., 30(41), 58 Constantopoulus, G., 196(28), 203 Conway, Α., 268(28), 291(28), 306 Cook, B , 200, 203 Copley, M , 154(21), 166 Corash, L. M , 44(134), 60 Cori, 0., 23(42), 68 Costa, E., 116(31), 128 Cosulich, M. E., 22(25), 42(25), 45(24), 46(24, 25), 47(25), 52(24), 67 Cotton, R. G. H., 91(21), 99, 128 Cox, D . J., 99(62), 129 Cox, G. E , 178(146), 189(146), 206 Creange, J. E., 198(30, 123), 203, 206 Creasey, W. Α., 228(29), 234(29), 254 (29), 261 Crick, F. H. C , 304(29), 306 Criss, W. E , 32(43), 33(44), 50(44), 68 Curci, M. R., 245(30), 261 Curry, J. R., 228(110), 252(108, 110), 263

Dabich, D., 292(139), 309 Dalmasso, A. P., 112(50), 113, 129

AUTHOR INDEX

Dalziel, K., 297(30), Damiani, G , 22(26), 46(23, 26), 49(23), 52(26), 53(26), 55(26), 67 Danielsson, H., 179(14), 195(32), 196(31), 203 Datta, P , 90(22), 128 Davenport, D . D., 44(134), 60 Daves, G. D., 193(115), 206 Davignan, J., 178(57), 189(57), 204 Davis, R. H., 110(24), 111(23), 128, 227 (163), 228(162, 163), 229(31, 32, 33, 34, 36, 162, 165), 230, 231(36), 232(34, 163), 233(34, 162, 163), 237(162, 163), 238(162, 163), 244(162), 251(162), 258 (34, 163), 261, 266 Davis, W. W., 197(112), 206 Dayhoff, M. 0., 50(7), 57 Decker, K., 25(56), 68 Deckner, K., 30(30), 67 D e Crombrugghe, B., 12(13), 18 De Eds, F., 194(18), 203 De Flora, 22(18, 25, 26, 179), 28 (111), 29(16, 17), 30(17), 40(18, 19), 42(25), 45(24), 46(23, 24, 25, 26, 46, 179), 47 (25, 179), 49(23, 179), 50(21, 22), 51 (22), 52(22, 24, 26, 179), 53(18, 179), 53(26), 55(20, 26, 45, 179), 67, 68, 69, 61 Delaage, M., 284(74), 297(74), 307 DeLange, R. J., 5(70), D e Matties, F., 176(33), 183(33), 185(33), 203 Demerec, Μ., 228(169), 229(169), 266 DeMoss, R. D., 23(47), 24(47), 68 Denes, G., 104(26), 128 Denton, R. M., 148(22), 149(22), 166 Dern, R. J., 44(48), 68 DeRobichon-Szulmajster, H., 90(76), 129 Deutscher, Μ. P., 1(29), 2(29), 10(14, 29), 11(14), 12(14), 18, 19 Devenyi, T., 284(31), Sol? De Vijlder, J. J. M., 290(32), 306 Dexter, R. N., 197(34, 112), 203, 206 Dietschy, J. M., 178(35), 203 Di Franco, Α., 285(33), Sö^ Din, F. υ . , 9(15), i 5 Dina, D., 46(46), 55(20), 67, 68 Diplock, A. T., 192(41), 203 Dixon, M., 29(49), 68 Doi, R. H., 1(16), 5(32), 6(32, 33, 64),

AUTHOR INDEX

8(33), 12(23), 14(32, 33), 15(33), 16 18,19, 20 Domagk, G, R, 23(50), 25(51, 54, 56, 59), 26(50, 51, 52, 53, 54, 55, 57, 59), 68 Domschke, W., 23(50), 25(51, 54, 56, 59), 26(50, 51, 52, 53, 54, 55, 57, 59), 68 Donachie, W. D., 228(37), 233(37), 238 (37), 245(30), 261 Donovan, J. Α., Jr., 28(144), 60 Dorfman, R. I., 183(74), 198, 199(36, 74, 105, 106), 203, 204, 206, 207 Dorsey, J. K., 152(13), 166 Douglas, H, C , 118, 129 Douzou, P., 269(73, 78), 270(73, 78), 271 (78), 275(73), 280(73, 78), 282(73), 307 Downing, M., 228(60), 262 Doyle, D., 22(153), 40(153), 61, 237(135), 255(135), 264 Draper, H. H., 187(100), 190(100), 205 Drell, W., 229(106), 263 Drosdorssky, M., 199(105), 205 Drucker, W. D., 196(122), 206 Drum, D . E., 287(34), 306 Dubuc, J., 184(39, 40), 199(40), 203 Duckworth, H. W., 94(64), 129 Duffield, P. H., 227(73), 262 Dugan, R. E., 176(37), 203 Dunn, M. F., 285(8, 35), 288(8), 305, 306 Duphil, M., 238(75), 244(75), 262 Durr, I. F., 185, 203 Dvornik, D., 184(39, 40), 199(40), 203

Eagle, H., 236(134), 237(134), 264 Eakin, R. T., 228(38), 252(38), 262 Fames, D . F., 228(154), 252(154), 253 (154), 264 Eder, Η. Α., 179(111), 181(11), 203, 206 Edwin, Ε. Ε., 192(41), 203 Eger-Neufeldt, I„ 41(58), 68 Eichhorn, J., 196(59), 204 Eigen, M., 142, 166 Eik-Nes, K. B., 196(124), 206 Einarsson, K., 195(32), 196(31), 203 Elek, S. R., 176(49), 183(49), 201(49), 204 Ellar, D . J., 2(17), 18 ElUott, W. H., 9(42), 19 Emerson, 0 . H., 194(18), 203

315

Engel, Η. J., 25(51, 54, 59), 26(51, 54, 59), 68 Engelbrecht, Η. L., 6(62), 14(62), 15(62), 20 England, P. J., 148, 149(22), 166 Engle, W., 23(50), 26(50), 68 Engstr0m, L., 269, 270, 306 Ennis, H. L., 229(39), 253(39), 262 Esnouf, M. P., 115(25), 128 Evans, H., 5(70), 20 F Fabiano, R., 154(80), 167 Fabre, L. F., 198(42), 203 Fagan, V. M., 177(136), 178(135, 137), 206 Fallon, H. J., 254(40), 262 Fan, D. P., 279(37), 304(37), 306 Fantoni, Α., 40(60), 68 Farago, Α., 104(26), 128 Farrel, G., 198(42), 203 Favüla, R., 285(90), 288, 289(90), 307 Fayat, G., 285(10), 293(10), 294(10), 305 Feder, J., 5(18), 18 Ferdmandus, J. Α., 229(160), 247(160), 265 Fernley, H. N., 274, 285(38), 306 Field, F. E., 193(155), 207 Fife, W. K., 274, 306 Filippi, G., 42(158), 61 Filmer, D., 268(71), 297(71), 307 Fimognari, G., 176(43), 179(43), 204 Fink, G. R., 94(11), 99(27), 128, 260(41), 262 Fink, R. M., 229(128), 264 Firkin, B. G,, 254(42), 255(42), 262 Fisher, Ε. Α., 39(177), Fishman, L. Μ., 197(34), 203 Fitch, W. Μ., 38(61, 62), 58 Fitz-James, P. C , 2(82), 20 Fletcher, Κ., 180(44), 181(45), 191(44), 204 Foellner, E., 45(145), 60 Folkers, K., 193(115), 206 Forchielli, E., 183(74), 198(154), 199(36, 74, 105, 106), 203, 204, 206, 207 Fornaini, G., 40(60), 44, 45(15), 67, 68 Forsen, S., 292(121), 308 Fosset, M., 283(23, 40), 285(23), 295(23), 306

316

AUTHOR INDEX

Fowler, Α., 292(145), 809 Fowler, D . D., 198(123), Fox, R. M., 229(43), 254(42), 255(42, 43), 257(43), 259(43), 262 Frantz, I. D., Jr., 176(46), 177(47), 179 (48), 180(47), 183(46), 204 Frederick, E., 251(144), 264 Frederickson, D . S., 179(48, 142), 190, 204, 206 Freese, E., 9(19), 18 Freese, P. K., 5(32), 6(32, 33), 8(33), 12 (33), 14(32, 33), 15(33), 19 Friedberg, S. L., 117(39), 128 Friedman, M., 176(49), 180(27), 181(27), 183(49), 189(50), 190(50), 201, 203,

204 Fromm, Η. J., 131(25, 61, 79), 137, 145 (24), 146(23, 79), 149(55, 62), 150 (56), 152(57), 154(21, 25, 51, 62, 80), 155(51, 55, 56), 156(51), 161(52, 63), 164(58), 166, 167 Fukumoto, J., 12(54), 19 Furfine, C , 290(137), 309

Gache, C , 283(23, 40), 285(23), 295(23), 306 Gage, v., 8(60), 12(60), 15(60), 19 Galliers, E., 9(19), 18 Galofre, Α., 229(44), 247(44), 251(44), 262 Gans, Μ., 229(45), 262 Garces, Ε., 160(26), 166 Garces, Ε., 38(125), 60 Garedry, R., 184(39, 40), 199(40), 203 Garen, Α., 275(41), Garfinkel, D., 63(28), 67(28), 68(28), 74, 128, 163(59), 165(27), 166, 167 Garfinkel, L., 63(28), 67(28), 68(28), 74 (28), 128, 165(27), 166 Garnier, J., 292(126), 308 Garre, C., 28(111), 29(112, 113), 38(2), 56, 69, 60 Garren, L. D., 197(112), 206 Gatt, S., 155,166 Gazith, J., 157(64), 167 George, P., 149(54), 167 Gerding, R., 285(42), 306

Gerhart, J. C., 227(46), 228(46), 262, 284 (43), 300(21, 43, 44), 301(21, 43), 306 Geroch, M. E., 23(132), 24(132), 25(132), 36(132), 60 Giardina, B., 221, 226 Gibson, F., 91(21), 94(58), 99(21, 29), 128, 129 Gües, N. H., 99(8), Güfillan, J. L., 179(73), ^i>4 Gilvarg, C., 11(3, 83), 12(75), 19, 20 Ginsberg, Α., 162(29), 166 Giovenco, S., 221, 226 Giuliano, F., 50(21), 67 Givner, M., 184(39, 40), 199(40), 203 Givol, D., 290, 306 Glaser, L., 23(65), 26, 28, 29(65), 68 Glatthaar, B. E., 285(45a), 306 Glazer, A, N., 5(71), 20 Glock, G. E., 34, 38, 68, 158(30), 166 Goldberg, E. B., 30(41), 55 Goldfarb, P. S. G., 156(10), 166 Goldstein, B. N., 65,130 Gooding, R. H., 157(64), 167 Gottesman, M., 12(13), 18 Gould, Ε., 176(25), 177(25), 179(25), 180 (25), 183(25), 187(25), 188(25), 203 Gould, R. G., 175(9), 176(9, 52), 177(51, 54, 55), 179(53), 181(9), 183, 187(54), 188, 192(54), 202, 204 Grabosz, J., 236(89), 263 Grahame-Smith, D . G., 116, 117, 128 Grazi, E., 21(137), 23(137), 50(142), 60 Green, C. D., 34(68), 35, 55 Green, J., 192(41), 203 Green, S. B., 63(28), 67(28), 68(28), 74 (28), 128, 165(27), 166 Greenbaum, A. L., 22(69), 39(69), 68 Greenberg, D . M., 233(78), 234(78), 254 (78), 256(78), 258(78), 260(78), 263 Greengard, P., 116(31), 128 Greer, S., 100, 130 Grenson, M., 228(87), 229(87), 233(87), 235(87), 258(87), 263 Gries, F. Α., 180(56), Griffin, C. C., 65(32), 128 Grinnan, E. L., 38, 57 Grisolia, S., 235(47), 236(13, 47), 262 Grundy, S. M., 178(57, 116), 189(57), 204,

AUTHOR INDEX

317

Gubler, C. J., 27(126), 60 Guchhait, R. B., 304(46), 306 Guder, W., 180(58), m Günther, Τ., 149(31), 166 Guest, M. J., 176(138), 177(138), 187 (139), 206 Gumaa, J. Α., 22(69, 70), 38(128), 39(69), 58, 60 Gunsalus, I. C , 23(47), 24(47), 58 Guroff, G., 250(58), 262 Gutfreund, Η., 146(32), 166, 274, 280(48), 283(133), 285(133), 306, 309 Guthöhrlein, G., 237(48), 262 Η Haber, J. E., 209, 225 Hageman, J. H., 5(20), 8(20), 12(21), 14 (21), 18 Hager, S. E., 227(50), 229(50), 236(49, 50, 55), 237(50), 245(50), 247(50), 248 (50), 250(49, 50), 251(49), 252(49), 259(49), 262 Hagerman, J. S., 177(55), 204 Haggard, Μ. Ε., 254(51), 255(51), 262 Haidane, J. Β. S., 138,166 Halford, S. E., 280(47, 48), 306 Halkerstein, I. D. K., 196(59), 204 Hall, F. F., 5(22), 7(22), 8(22), 18 HaU, L. M., 235(105), 236(105), 263 Hall, P. F., 190(60), 197, 199(61, 62), 204, 205 Halvorson, H. 0., 1(23), 2(23), 11(25), 18,19 Hammes, G. G., 148, 166, 300(49, 51), 306 Hamprecht, B., 176(10, 121), 178(10), 179 (63), 180(121), 183(10, 64), 184(65), 185(121), 186(64), 187(64), 203, 204, 206 Handschumacher, R. Ε., 228(29), 234 (29), 253(121), 254(29), 261, 264 Hanes, C. S., 65(93), 124(93), 130 Hanson, Α. W., 279(52), 306 Hanson, R. S., 1(24), 2(24), 11(25), 18, 19 Harada, K., 286(53), 287(54), 305(54), 306 Harbert, P., 42(182), 61 Hardy, G. H., 225, 225 Harkness, D . R., 275(57), 307

Harris, A. P., 197(101), m Harris, J. I., 290(56), 291(55), 307 Harris, R. Α., 182(66), m Hartley, Β. S., 293(ip), 306 Hartmann, P. E., 111(1), W Haschemeyer, R. J , 237(158), 239(158), 265 Hasselbach, W., 148, 166 Hatfield, D., 234(52), 254(52), 257(52), 262 Hathaway, J. A , 30(139), 60, 72(3), 128 Hauser, S., 195(130, 131), 206 Haynes, R. C , Jr., 198(67), 204 Hayward, W. S., 228(53), 229(53), 262 Heather, J. V., 22(179), 46(179), 47(179), 49(179), 52(179), 53(179), 55(179), 61 Heaton, F. W., 192(68), 204 Hechter, 0., 196(59), 197(143), 204, ^06 Hemphill, R. M., 163(36), 166 Hendrickson, Ε. Μ., 42(83), 49, 53(82), 54, 59 Henning, U., 260(54), 262 Heppel, L. Α., 274(14), 275(57), 305, 307 Hermann, R. L., 229(95), 237(95), 263 Hermoso, J. Μ., 15(2), 18 Hernandez, Η. J., 176(1), 181(1), 202 Herzfeld, Α., 236(55), 262 Hess, B., 63(33), 74, 128, 163(11), 168 Higgins, M., 185(69), 186, 204 Hill, E., 285(134), 286(134), 309 Hill, J. M., 229(56), 262 Hill, R., 38(61), 58 Hilmoe, R. J., 275(57), 307 Hinkleman, B. T., 176(46), 177(47), 179 (48), 180(47), 183(46), 204 Hirsch, Μ. L., 229(57), 262 Hirschberg, Ε., 45(114), 60 Hirschfeld, Τ. J., 191(15), 192(15), 203 Hitchens, Α. D., 15(63), 20 Hitchings, G. H., 247(17), 251(17), 261 Ho, R. J., 116(10), 128 Hoagland, V. D., Jr., 51, 62 Hodes, S., 236(71), 262 Hoffman, D . H., 229(148), 247(148), 251 (148), 264 Hogans, A. F., 250(58), Hogness, D . S., 285(142, 143), 309 Holloway, B. W., 227(63), 228(63), 229 (63), 235(63), 262 Holmes, W. L., 190, 204

318

AUTHOR INDEX

Holt, S. C., 6(1), 8(1), 10(1), 11(1), 12 Iwatsubo, M., 285(10), 293(10), 294(10), (1), 18 305 Holten, D., 38(146), 39(146), 40(146), 60 J Hoogenraad, N. J., 239(59), 245(59, 93), 246(93), 250(93), 256(93), 262, 263 Jacob, F., 64(47), 89, 91(47), 101(47), 125 Hopfield, J. J., 212, 225 (47), 129, 228(6), 229(6), 235(6), 255 Horecker, B. L., 21(72), 23(71), 26(173, (6), 261 174), 59, 61, 274(91a), 285(12a, 58), Jacobasch, G., 45(145), 60 295(12a, 58), 307 Jaffe, E. R., 42(75),59 Home, R. N., 28(6), 30(6, 73), 31(6, 73, James, E., 150, 151, 167 74), 35(6), 37, 57, 59 Jayaraman, J., 190(80, 82), 192(83, 84), Hort{)n, B. J., 184(71), 204205 Horton, J. D., 184(71), 204Jeanrenaud, B., 34(3~), 57 Hotchkiss, R. D., 13(35), 19 Jefconte, C. R., 196(22), 203 Hotta, S., 178(72), 189(72), 204Jensen, R. A., 64(35), 90(35), 94(35), 128 Houlahan, M. B., 229(111), 263 Johansen, G., 13(35), 19 Houston, J. Y., 228(60),262 Johansson, G., 195(32, 181), 203, 206 Huff, J. W., 179(73), 204Johnson, A. B., 45(114), 60 Huguley, C. M., Jr., 229(142), 234(142), Johnson, B. C., 38(76), 59 247(142), 254(61, 1(3), 255(61, 142, Jones, F. T., 194(18), 203 1(3), 256(142), 262, 264Jones, M. E., 227(2, 8, 28, 50, 115, 116, Hulme, E. C., 68(34), lt8 127), 228(2, 8, 28, 68, 115), 229(2, 8, Hunt, V. M., 179(73), 20428, 50, 69, 70, 127), 234 (70, 138), 236 Hurwitz, R., 229(117), 236(117), 242(83), (49, 50, 55, 69, 71), 237(50, 122), 239 263 (70, 137), 240, 241(70, 137), 243(115), Hussey, C., 12(26),19 245(50), 246(8, 116), 247(50, 69), 248 (50, 69, 116), 250(49, 50), 251(49), 252(49, 138), 254(138), 257(138), 258 (69, 116, 137, 138), 259(49), 261, 2~, Ichii, S., 183(74), 199(74), 204263, 264-, 301 (9), 305 Imer, D. F., 209, 226 Jornvall, H., 287(59), 307 Inagaki, A., 229(62), 242(62), 262 Joshi, V. C., 176(95), 177(95), 182(95), Inamdar, A. R., 176(76, 77, 94), 180(77), 187(95), 190(82), 191(3, 99), 192(3, 181, 182(120), 184(75), 187(75, 77), 83, 84, 95), 193(3, 85, 95), 202, 205 190(94), 191(75, 76, 94), 192(75, 76, Julian, G. R., 35, 59 77, 94), 193(75, 76), 204-, 206, 206 Jund, R., 228(72), 229(72), 233, 234, 251 Inano, H., 199(79), 204(72), 253(72), 257(52), 262 Ingraham, J. L., 227(1), 228(1, 114), 229 Jungmann, R. A., 181(86), 205 (1), 261, 263 Ionesco, H., 1(67), 20 K Isaac, J. H., 227(63), 228(63), 229(63), Kagawa, A., 38(78), 59 235(63), 262 Kagawa, Y., 38(78), 59 Island, D. P., 197(17, 101), 203, 206 Kahnt, I. W., 197(87),205 Issaly, A. S., 227(64), 228(64), 262 Kalman, S. M., 227(73),262 Issaly, I. M., 227(64), 228(64), 262 Ito, K., 227(149), 228(150, 151), 229(66), Kandutsch, A. A., 178(88), 183(88),205 236(65, 149, 150), 237(66, 67, 1(9), Kaneshige, Y., 39(167), 61 . 240, 245(67, 149), 247(67, 150), 248 Kaplan, B. H., 179(113), 205 Kaplan, J. G., 227(97), 229(74, 76), 233 (150), 256(67), 262, 264-

319

AUTHOR INDEX

(97, 99, 100), 235(98, 100), 237(97, 100), 238(75, 97, 99, 100), 239(76, 97, 99), 244(75, 97, 98), 258(100), 262, 263 Kaplan, N. 0., 147, 166, 285(129), 308 Kapoor, M., 94(64), 129, 228(77), 252 (77), 258(77), 262 Karp, Α., 180(89), 206 Kasbekar, D . K., 233(78), 234(78), 254 (78), 256(78), 258(78), 260(78), 263 Katze, J. R., 294(64), 507 Kawachi, T., 185(69), 186(69, 90), 204, 206 Keay, L., 5(27, 28), 7(27), 19 Keleti, T., 285(60), 291, 307 Kelln, R. Α., 6(33), 8(33), 12(33), 14(33), 15(33), 19 Kemp, R. G., 25(79), 59 Kennan, A. L., 38(135), 60, 236(79), 263 Kerjan, P., 15(38), 19 Kerr, C. T., 228(109, 110), 252(109, 110), 253(109), 263 Kezdy, F. J., 274, 285(65), 307 Khedouri, E., 263 Kiely, M. E., 26(80), 69 Kim, S., 229(81), 236(81), 247(81), 263 King, E. L., 65, 128 Kirkman, H. N., 23(81), 32, 40(81), 42 (83), 44(84), 49, 50(81), 53(82), 54, 59 Kirschner, Κ., 64(37), 128, 142(37), 166, 268(61), 291(62), 300, 307 Kistiakowsky, G. B., 65, 128 Klainer, L. M., 117(39), 128 Kleczkowski, K., 246, 263 Klofat, W., 9(19), 18 Klotz, I. M., 267(63), 507 Knappe, J., 185, 205, 237(48), 262 Kniefel, H. P., 149,167 Knight, M., 25(94), 59 Knowles, J. R., 294(64), 307 Knox, W. E., 229(173), 236(173), 245 (173), 247(173), 248(173), 265 Knudsen, K. Α., 200(8), 202 Ko, S. H. D., 274, 285(65), 307 Kochavi, D., 149, 166 Königsberg, W., 294(64), 307 Kohen, C , 123, 128 Kohen, E., 123, 128

Koritz, S. B., 190(60), 197, 199(61, 62), 204, 205 Kornberg, Α., 1(29), 2(29), 10(14, 29, 74), 11(14, 73), 12(14), 18, 19, 20, 29(86), 69, 227(94), 263 Kosaka, K., 39(167), 61 Kosakowski, H. M., 294(66), 307 Koshland, D . E., Jr., 31(87), 69, 64(41), 124(41), 128, 131, 143, 166, 209, 226, 226, 268(28, 69, 70, 71), 291(28), 297 (69, 71), 299(85, 86, 87, 88), 302(67, 68), 306, 307 Koskimies, 0., 241, 263 Kosow, D. P., 154(39, 40), 166 Kraml, M., 184(39, 40), 199(40), 203 Krebs, Η. Α., 157, 159, 166, 167, 250(20), 261 Kreckova, P., 9(8, 9, 10), 18 Kretchmer, N., 229(44, 117), 236(117), 239(59), 242(83), 245(59, 93), 246 (93), 247(44), 250(93), 251(44), 256 m), 262, 263 Krishnaiah, K. V., 176(93, 94, 95, 96), 177(95), 178(93), 179(93), 182(95, 96, 120), 187(93, 95), 191(94, 97), 192(93, 94, 95, 97), 193(95), 205, 206 Krisnan, P. S., 37(194), 62 Krooth, R. S., 229(84, 125), 253(84, 125, 168), 255(84, 125), 256(125), 257(84, 125, 168), 259(84, 125), 263, 264, 265 Krum, A. Α., 189, 190(98), 205 Kuby, S. Α., 23(127), 27(88, 126, 190, 191), 28(180), 30(88), 35(191), 59, 60, 61, 62 Kuhn, Τ. S., 124(142), 129 Kunkel, Η. Ο., 5(22), 7(22), 8(22), 18 Kuron, Κ. W., 179(148), 206 Kusama, Κ., 236(85), 263 L

LaCroute, F., 227(86), 228(72, 86, 87), 229(72, 76, 86, 87), 233(86, 87), 234, 235(87), 238(75), 239(76), 244(75), 251(72, 86), 253(72, 86), 255(86), 257 (72), 258(86, 87), 262, 263 Lagerkvist, U., 236(89), 263 Laishley, E. J., 8(31), 11(30, 31), 19 Lakshmanan, M. R., 191(99), 206

320

AUTHOR INDEX

Lan, S. J., 227(90), 228(90), 237(90), 252 (90), 263 Lance, E. M., 197(101), 206 Lanczos, C., 65, 88(43), 129 Landon, M., 5(70), 20 Lane, M. D., 304(46), 306 Langdon, R. G., 46(37), 50(37), 52(37), 53 (37), 54, 68 Lardy, H. A., 149, 167, 284(72), 307 Larrson-Raznkiewicz, M., 149(42), 166 Lartigue, D. J., 161 (76), 167 Lawless, M. B., 110(24), 128 Lazdunski, C., 268(80), 269(24, 73, 77, 78, 79, 80), 270(24, 77, 78, 80), 271 (77, 78, 80), 272(80), 273(80), 274(77, 79, SO), 275(73, 76), 276(75, SO), 278(75, 77), 279(76, SO, 98), 280(73, 78, SO), 282 (24, 73, 77, 80), 283(80), 284(80), 285(77, 98), 288(80), 306, 307 Lazdunski, M., 268(80), 269(24, 73, 77, 78, 79, SO), 270(24, 73, 77, 78, SO), 271(77, 78, SO), 272(80), 273(80), 274 (77, 79, SO), 275 (73, 76), 276(75, SO), 278(77, 75), 279(76), 280(78, SO), 282 (24, 73, 77, SO), 283(23, 40, SO), 284 (74, SO), 285(23, 77, 98), 295(23), 297 (74), 306, 307, 308

Lea, M. A., 39(177),61 Leader, D. P., 34(89), 69 Leboeuf, B., 34(34), 67 LeBras, G., 90(20, 76), 94(20), 100(54, 55), 128, 129 Lee, D. J., 187(100), 190(100), 206 Lee, M. L., 294(81), 307 Leighton, T. J., 1(16), 5(32), 6(33, 64), 8(33), 12(33), 14(32, 33), 15(33), 16 (16), 18, 19, 20 Lemoine, F., 293(19, 82), 306, 307 Leoncini, G., 40(60), 45(15), 67, 68 Lessie, T., 23(90), 69 Leterrier, F., 269(73, 78), 270(78), 271 (78), 275(73), 2SO(73, 78), 282(73), 307

Leuzinger, W., 285 (83), 307 Levenberg, B., 227(91), 236(91), 263 Levin, B., 250 (92), 263 Levine, D., 270(84), 307 Levine, L., 147(16), 166

Levine, R. L., 239(59), 245(59, 93), 246 (93), 250, 256(93), 262, 263 Levine, S. N., 112(44), 121, 129 Levinthal, C, 269(100), 275(41), 279(37, 113), 304(37), 306, 308 Levisohn, S., 3(34), 6(34), 8(34), 9(34), 10(34), 15(34), 19 Levitzki, A., 299(85, 86, 87, 88), 307 Levy, H. R., 23(91, 132), 24(130, 132), 25 (130, 132), 35(92), 36(91, 93, 122, 123), 69, 60 Li, T. K., 287(34), 306 Liddle, G. W., 177(147), 196(147), 197 (17, 34, 101),203, 205, 206 Lieberman, I., 227(94), 263 Lieberman, S., 196(122), 206 Lilly, E. M., 176(52), 179(53), 183(52), S04LinderstrflSm-Lang, K., 13(35), 19 Lindstedt, S., 195(102), 206 Lineweaver, H., 134, 166 Ling, G. N., 292(95), 308 Linn, T. C., 177(103), 180(103), 185(103), 186(103), 206 Lipmann, F., 23(42), 68 Lockhart, L. H., 254(51), 255(51), 262 London, J., 25(94), 59 Long, C. W., 298(89), 299(88, 89), 307 Lorenzoni, I., 22(18, 25), 29(16, 17), 30 (17), 40(18, 19), 42(~5), 45(24), 46 (23, 24, 25~ 46), 47(25), 49(23), 50 (21, 22), 51(22), 52(22, 24), 53(18), 55(20), 67, 68 Losick, R., 11, 12(26), 14, 15(36, 37), 19 Lotz, I. V., 179(53), 204 Lotz, M., 254(40), 262 Lou, M. F., 229(95), 237(95), 263 Loud, A. V., 176(25), 177(25), 179(25, 48), 180(25), 183(25), 187(25), 188 (25), 203, 204

Lovig, C. A., 243, 261 Loviny, T., 100(55), 129 Lowe, J. S., 192(68),204Lowenstein, J. M., 227(96), 229(22), 236, 247(96), 261, 263 Lowry,. O. H., 28(133), 29(133), 60 Lubin, M., 229(39), 253(39), 262 Lue, P. F., 227(97), 233(97, 99, 1(0), 235 98, 100), 237(97, 1(0), 238(97, 99,

321

AUTHOR INDEX 100), 239(97, 99), 244(97, 98), 258 (100), S63

Lueck, J., 250(101), 263 Luisi, P. L., 285(8, 90), 288(8), 289(90), 306, 307

Lund, P., 248(102),263 Lundgren, D. G., 2(17), 18 Luzzatto, L., 42(4), 43(4, 99), 44(95), 45 (99),50(95, 97), 52(4, 96), 54(35), 67, 69 Lynen, F., 176(10, 121), 178(10), 179"(65), 180(121), 183(10, 64), 184(65), 185 (121), 186(64), 187(64), 203, 204, 206, 206

Lyon, M. F., 42, 69

M Maas, W. K., 104(89), 130 McClure, W. R., 149, 161 McConn, J. W., 5, 6(43), 19, 20 McCurdy, P. R., 44(48, 85), 68, 69 Macfarlane, R. G., 112(45), 115(25), 128, 129 McGarrahan, K., 176(25), 177(25), 179 (25), 180(25), 183(25), 187(25), 188 (25), 203

Machintoch, J. E., 182(66), 204McIssac, W. M., 198(42), 203 McKay, R. H., 287(108), 308 McKean, C. M., 188(126), 189(126), 206 McKems, K. W., 32(43, 102, 104, 105), 33(44, 101, 103, 105, 106), 50(44), 68, 69 McLean, P., 22(69, 70), 32(128), 34(107, 108, 109), 38, 39(69), 68, 69, 60, 158 (30), 166 McMurray, C. H., 146(32), 166 McQuarrie, R. A., 285(91), 291(91), 307 McQuate, J. T., 162(45),167 Maia, J. C. C., 15(38),19 Malamy, M. H., 274(91a), 307 Malhotra, O. P., 285(92), 290(92), 291 (92), 292(138, 139), 308, 309 Mandelstam, J., 1(39), 10(40), 12(40), 11) Mandula, B., 37(110, 163), 38(110), 69, 61 Mangiarotti, G., 22, 28(111), 29(112, 113), 59,60

Mangiarotti,. M. A., 22(18), 29(16, 17), 30(17), '40(18, 19), 46(46), 53(18), 67,68 Marco"R., 164, 167 Markland, F. S., 5(70, 71), 20, 151(46), 167 Marks, P. A., 32(116), 44, 45, 49, 51 (115), 60, 61

Marr, J., 152(13), 186 Marshall, M., 235(105), 236(105), 263 Masri, M. S., 194(18), 203 Masson, M., 229(45), 262 Matschinsky, F., 180(56), 204 Matsubara, H., 5(41), 19 Matsuda, T., 40(192), 41(192), 60, 62 Matthews, S. L., 237(103), 239(103), 263 May, B. K., 9(42), 19 Mayer, L. A., 37(119), 60 Mayfield, E. D., Jr., 229(19), 247(19), 251(19), 256(19), 261 Mazus, B., 227(104), 251,(104), 283 Meister, A., 227(3), 228(4), 236(3), 237 (158), 239(158), 243(4), 258(4), 261, 263, 266, 285(125), 308 Melchior, J. B., 149(47), 167 Meng, H. C., 116(10), 128 Menkes, J. H., 188(104), 206 Menon, K. M. J., 198(154), 199(36, 105, 106), 203, 206, tor Menon, T., 117(79), 129 Meriwether, B. P., 291(55), 307 Merker, H.-J., 243, 264Messmer, I., 229(74, 76), 239(76), 262 Metzenberg, R. M., 235(105), 236(105), 263 Metzger, R. P., 37, 60 Meyer, J., 26(53), 68 Michalis, F., 189(50), 190(50), 204Michel, J., 3(44),6(45), 12(45), 19 Michelson, A. M., 229(106), 263 Migicovsky, B.B., 176(125), 179(107, 108, 125), 180(125), 187(125), 206, 206 Mildvan, A. S., 152(48),167 Miller, L. S., 200(8), 202 Miller, R. W., 228(107, 109, 110), 252 (107, 109), 253(109), 263 Miller, W. G., 65, 129 Millet, J., 5(47, 48), 6(45, 48), 7(46, 50, 52), 8(46, 48, 52, 68), 9(48, 52), 10

322

AUTHOR INDEX

(68), 11(52), 12(45, 53), 13(49, 51, 52, 53), 19, 20 Milman, L. S., 30(193), Minamura, N., 12(54), 19 Miraglia, J., 44(134), 60 Mitchell, H. K., 228(38), 229(106, 111), 252(38), 262, 263 Mitropoulos, K. Α., 181(109), 205 Mitton, J. R., 196(21), 203 Mobley, P. W., 37(119), 60 Monod, J., 64(47, 48), 89, 91(47), 101(47, 48), 129, 209, 214, 226, 267(94), 268 (94), 292(93), 296(94), 297(94), 299 (94), 300, 302(94), 303, 308 Monro, R. E., 10(55), 19 Monteilhet, C , 293(140), 295(140), 309 Moore, H. W., 193(115), 205 Morelli, Α., 50(21), 57 Morihara, K., 5(56), 19 Morris, D. R., 110(49), l^P Morris, H. R., 178(137), 206 Morris, M. D., 189(98), 190(98), 205 Morrison, J. F., 150, 151,167 Morrison, M , 36(12, 129), 37(12), 57, 60 Morton, R. Α., 192(68), 204 Mosbach, Ε. Η , 195(130, 131), 206 Moser, P. W., 5(28), 7, Moss, J , 304(46), 50^ Mosse, H , 229(19), 245(18), 247(19), 251 (19), 256(19), 261 Motulsky, A , 42(121), 42(120), 44(184), 60, 61 MouHn, Α., 279(98), 283(40), 285(98), 306, 308 Mourad, N., 160(50), 167 Muench, K. H., 294(81), 307 Mukherjee, S , 176(110), 181(110), 187, 205 Muller-Eberhard, H. J., 112(50), 113, 129 Munkres, K. D., 229(147), 231(147), 264 Murad, F., 117(51), 129 Murrell, W. G., 1(57), 2(57), 19 Musil, J., 30(30), 57 Myant, N. B., 179(111), 180(44), 181(45, 109), im^^), 204, 205 Ν Nagahushanam, Α., 233(78), 234(78), 254 (78), 256(78), 258(78), 260(78), 263 Naiman, J. L., 44(85), 59

Nakanishi, S., 237(67), 245(67), 247(67), 256(67), 262 Nalbandov, A. V., 200(29), 203 Nasser, D. S., 64(35), 90(35), 94(35), 128 Natale, P. J., 248(12), 249(12, 112), 261, 263 Naylor, A. W., 227(119), 228(119), 237 (119), 243(119), 246(119), 256(119), 264 Nazario, M., 100(52), 109(52), 129, 228 (113), 263 Neda, Κ., 154, 61 Neet, Κ. Ε., 64(41), 124(41), 128, 268 (70), 307 Neher, R., 197(87), 205 Neidhardt, F. C., 23(90), 59 Nelson, D. L., 1(29), 2(29), 10(29), 19 Nelson, D . R., 137, 145(24), 166 Nelson, N. M., 118, 129 Nemethy, G., 209, 226, 268(71), 297(71), 307 Nester, Ε. W., 64(35), 90(35), 94(35), 128 Neuhard, J., 227(118), 228(114, 118), 229 (118), 229, 234(118), 251(118), 263, 264 Neumann, J., 227(115, 116), 228(115), 243 (115), 246(116), 258(116), 263 Neumark, R., 9(58), 19 Nevaldine, B. H., 35(122, 123), 36(93, 122, 123), 59, 60 Neville, M. C , 292(95), Ney, R. L , 116(30), 117(30), 128, 197 (34, 112), 203, 205 Nicolini, Α., 22(26), 46(23, 26), 49(23), 52(26), 53(26), 55(26), 57 Nielson, M. H., 32(124), 60 Niemeyer, H., 38(125), 60 Nilsson, U. R., 112(50), 113, 129 Ning, J., 154(51), 155(51), 156(51), 167 Nisbett, D . Α., 161(52), 1(57 Nissley, P., 12(13),i8 Niswender, G, D., 200(29), ^05 Noble, W. M., 191(15), 192(15), 203 Nolte, I., 180(58), 204 Noltmann, E. Α., 23(127), 27(88, 126, 190, 191), 28(191), 30(88), 35(191), 59, 60, 62 Nordlie, R. C., 28(5, 6), 30, 31(5, 6, 74), 35(5, 6), 37, 57, 59, 250(101), 263 Nordman, Y., 229(117), 236(117), 263

AUTHOR INDEX

Nordstrom, B., 289(13), 306 Norton, H. W., 200(29), 203 Novello, F., 38(i28), 60 Nüssler, C , 179(63, 65), 183(64), 184 (65), 186(64), 187(64), Nyc, J. F., 229(111), 263 Nygaard, A. P., 158(72), ίβ7

O'Donovan, G. Α., 227(118), 228(118), 229(118), 229, 504(118), 251(118), 264 Ogawa, S., 212, 226 Ogilvie, J. W., 179(113), ^05 Ogunmola, G. B., 54(35), 67 Ohno, S., 36(129), 00 Oka, T., 5(56), 19 O'Leary, M. H., 285(96), 295(96), 308 OHve, G., 23(132), 24(130, 132), 25(130), 36(132), 60 Oliver, I., 242(83), 263 OUver, M. F., 181(114), 206 Olsen, R„ 275, 308 Olsen, R. Κ , 193(115), ^05 O'Neal, Τ. D., 227(119), 228(119), 237 (119), 243(119), 246(119), 256(119), 264 Ong, B. L., 227(120), 229(120), 237(120), 243(120), 246(120), 264 Ono, T., 38(138), 60 Oosthuizen, C., 290(12), 306 Orgel, L. E., 304(29), 300 O'SulUvan, W. J., 229(43), 254(42), 255 (42, 43), 257(43), 259(43), 262 Ott, W. H., 179(148), ^00 Ottesen, M., 5, 19 Overath, P., 185, 203 Overholzer, V. G., 250(92), 263

Paetkou, v., 284(72), 307 Pardee, A. B., 64(95), 89, 101(95), 110 (49), 111(95), 129, 130, 227(170), 228 (6, 171, 172), 229(6, 172), 235(6), 255 (6, 172), 261, 266, 298(89), 299(89), 307 Park, J. H., 291(55), 307 Parks, R. E , Jr., 160(18, 50), 166, 167 Parsons, W. W., 193(115), 206 Passonneau, J. V., 28(133), 29, 60 Pastan, I., 12(13), 18

323 Pasternak, C. Α., 253(121), 264 Patte, J.-C., 100(54, 55), 129 Patterson, R. B., 254(133), 255(133), 264 Patton, D., 178(146), 189(146), 206 Payne, H. W., 36(129), 60 Peck, E. J., 148,149(53), i07 Peller, L., 67(4, 5, 57), 128, 129 Peng, L., 237(122), 264 Penny, D . G., 176(13), 179(13), 203 Peraino, C., 38(135), 60 Perham, R. N., 290(56), 307 Perlman, R., 12(13), 18 Perutz, M. F., 268(97), 305 Peterson, J. Α., 1(24), 2(24), 11(24), 18 Peterson, N . Α., 188(126), 189(126), 206 Peticlero, C., 268(80), 269(24, 73, 77, 78, 79, 80), 270(24, 77, 78, 80), 271(77, 78, 80), 272(80), 273(80), 274(77, 79, 80), 275(73), 276(80), 278(77), 279 (80, 98), 280(73, 78, 80), 282(24, 73, 77, 80), 283(80), 284(80), 285(77, 98), 306, 307, 308 Pfleiderer, G., 287(99), 308 PhilUps, L. I., 254(7), 255(7), 261 Phillips, R. C., 149(54), 167 Pickard, B. M., 44(84), 50 Pierard, Α., 227(124), 228(87, 123, 124), 229(87, 124), 233(87), 235(87), 258 (87, 124), 263, 264 Pinsky, L., 229(125), 253(125), 255(125), 256(125), 257(125), 259(125), 264 Piomelli, S., 44(134), 60 Piszkiewics, D., 37(119), 60 Pitot, H. C,, 38(135), 60 Pittard, J., 94(58), 99(29), 128, 129 Plocke, D . J., 269(100), 308 Pogell, B. M., 41(166), 07 Polakis, E. S., 30(136), 00 Polley, M.J., 112(50), 113,129 Pontremoli, S., 21(137), 23(137), 26(143), 50(142), 60, 285(58), 295(58), 307 Poore, G. Α., 229(148), 247(148), 264 Port, L. Α., 110(24), 128 Porter, F. S., 254(133), 255(133), 264 Porter, J. W., 175(24), 176(37), 166, 203 Porter, R. W., 300(49), 300 Potter, V. R., 38(138), 00 Prager, Μ. D., 229(126), 245(126), 247 (174), 254(126), ^04, ^05

324

AUTHOR INDEX

Pratt, I., 11 (76), 20 Prescott, J. M., 5(22), 7(22), 8(22), 18 Prescott,·L. M., 227(127), 229(127), 264Prestidge, L., 8(60), 12(60), 15(60), 19 Pries, N., 38(135), 60 Pring, M., 63(28), 67(28), 68(28), 74(28), 128, 165(27),166 Purich, D. L., 149(55), 150(56), 152(57), 154(51), 155(51, 55, 56), 156(51), 164 (58), 167 Pynadath, T. I., 229(128), 264-

Q Quackenbush, F. W., 182(66), 204Queener, S. F., 229(160), 247(160), 266 Quintas, E., 178(116), 206

R Racker, E., 155, 156(73), 166, 167 Radford, A., 229(129, 130), 231 (129, 130), 264Raineri, R. R., 36(93), 69 RaIl, T. W., 117(39, 51, 59, 79), 128, 129, 198(67), 204-

Ramaiah, A., 30(139), 60 Ramakrishna Kurup, C. K., 190(82), 206 Ramasarma, T., 176(76, 77, 94, 95, 96, 144), 177(95), 179(144), 180(77), 181, 182(95, 96, 120), 183(144), 184(144), 187(77, 95, 119, 144), 189(118), 190 (119),191(3,76,94,97, 99),192(3, 76, 77, SO, 82, 83, 84, 94, 95, 97), 193(3, 76, 85, 129), 194(117, 118), 202, 204-, 206, 206

Randle, P. J., 148(22), 149(22), 166 Ranganathan, S., 189(118), 194(117, 118), 206

Ransil, B. J., 149(15), 166 Rapoport, S., 45(145), 60 Rasore-Quartino, A., 44(149), 61 Rattazzi, M. C., 45(140), 49(141), 60 Raval, D. N., 67(60), 129, 285(101), 308 Ray, W. J., Jr., 148, 149(53), 167 Reddy, S., 45(98), 69 Reed, L. J., 99(61, 62), 129, 304(102), 308 Rege, D. V., 192(4), 202 Regen, D., 176(121), 180(121), 185(121), 206

Reichard, H., 236(131), 264

Reichard, P., 227(132), 236(89), 263, 264, 302(15), 306

Reid, T. W., 270(84), 307 Reissig, J. L., 227(64), 228(64, 113), 262, 263 Reithel, F. J., 35(77), 69 Renold, A. E., 34(34), 67 Reynolds, J. A., 276(103), 279(115), 308 Rhoads, D. G., 163(59), 167 Riepertinger, C., 176(121), 180(121), 185 (121), 206

Rihova, L., 9(9), 18 Ringlemann, E., 185(91), 206 Rippa, M., 26(143), 50(142), 60 Risler, J. L., 293(140), 295(140), 309 Rivera, M., 45(27, 28, 29),67 Rivers, S. L., 254(61), 255(61), 262 Robbins, D. J., 194(18),203 Roberts, E., 236(85), 263 Roberts, K. D., 196(122), 206 Roberts, S., 198(30), 198(123) ,203, 206 Robison, G. A., 115(63), 116(63), 129 Rodkey, F. L., 28(144),60 Rodwell, V. W., 176(43, 127, 128), 177 (128), 179(43), 183(127, 128), 184 (128), 185(128), 186(127), !O4, 206 Rogers, L. E., 254(133), 255(133), 264Rogers, S., 284(31), 306 Roigas, H., 45(145), 60 Roscher, R., 179(63), 204Rose, I. A., 25(79), 69, 148, 149(60), 154 (39, 40), 166, 167 Rosemeyer, M. A., 45(38, 39), 46(38, 39), 49(39, 40), 50(39), 52(38, 39), 53 (39), 68 Rosenbusch, J. P., 300, 301(104), 808 Rosenman,. R. H., 180(27), 181 (27), 203 Ross, C., 258(166), 260(166), 266 Rossi, E., 44(172), 61 Rossi Fanelli, A., 224, 226 Roth, H., 37(155), 61 Rothman, F., 269(105), 308 Rouget, P., 294 (107), 295 (106, 107), 308 Rubin, M. M., 297(22), 301(22), 306 Rudack, D., 38, 39(146), 40(146), 60 Rudney, H., 174(157, 158), 175, 176(158), 180(158), 183, 185(69, 158), 186(69, . 90), 187(158), 193(115), 208, 204-, 206,: 206

AUTHOR INDEX

Rudolph, F. B., 131(61), 149(62), 154 (62), 161(63), 167 Russ, E. M , 181(11), ^03 Rutman, R. J., 149(54), 167 Rutter, W. J., 28, 60 Ryter, Α., 1(67), 2(61),

Sabine, J. R., 184(71), 204 Sadoff, H. L., 6(62), 14(62), 15(62, 63), 20 Salas, M., 15(2), 18, 23(148), 30(148), 60 Sallach, H. J., 227(90), 228(90), 237(90), 252(90), 263 Salzman, N. P., 236(134), 237, 264 Samuels, L. T., 196(124), 206 Sandler, N., 287(108), 308 Sandrin, E., 293(19), Sansone, G., 44(149), 61 Santo, L. M., 6(64), 20 Sanwal, B. D., 23(150), 24(150), 61, 94 (64), 268(109), SOS Sassoon, H. F., 38(76), 55 Sato, G. H., 154, 61 Saucier, S. E., 178(88), 183(88), 206 Savageau, M. Α., 64(68), 65(66), 72(66), 74(66, 67), 75(70), 87(66), 88(69), 89 (68, 69), 94(72), 97(72), 101(71), 120 (69), 124(65), 129 Scaife, J. F., 176(125), 179(125), 180(125), 187(125), 206 Schachet, M., 32(151), 33(151), 61 Schachman, H. K., 300(21, 44), 301(21), 306 Schack, P., 285(8), 288(8), 289, 306 Schaeflfer, P., 1(66, 67), 3(65, 66), 8(66, 68),10, ll,;gO Schettini, F., 44(84), 69 Schimke, R. T., 22(152, 153), 40, 61, 237 (135), 255(135), 264 Schimmer, Β. P., 154, 61 Schlegel, Η. G., 23(9), 67 Schlesinger, Μ. J., 269(112), 275, 276 (103), 279(37, 110, 111, 115), 280 (111), 304(37), 306, 308 Schlesinger, S., 279(115), 308 Schneider, H. S., 177(47), 179(48), 180 (47), 204 Scholan, N. Α., 196(21), 203 Schnitze, I. Τ., 157(64), 167

325

Schnitze, Μ., 45(145), 60 Schulz, D. W., 28(133), 29(133), 60 Schwartz, G. P., 157(65), 167 Schwartz, J. Η., 269, 270, 281(116), 308 Schweppe, J. S., 181(86), 206 Scoggins, R. B., 254(61), 255(61), 262 Sebring, E. !>., 236(134), 237(134), 264 Segal, S., 37(155), 61 Segni, P., 45(15), 67 Shah, S. N., 188, 189(126), i^öß Shapiro, B. M., 67(73), 68(73), 95(73), 129 Shapiro, D. J., 176(127, 128), 177(128), 183(127, 128), 184(128), 185(128), 186 (127), 206 Sharma, B. V. S., 193(129), 206 Shaw, C. R., 36(156,157), 61 Shaw, W. H. R., 65,128 Shefer, S., 195(130, IZl), 206 Shen, L. C , 158(66), 167 Shepherd, M. G., 31(32), 67 Sheppard, H., 176(153), 177(152), 179 (153), 206 Shigesada, K., 228(151), 236(136), 245, 247(151), 249, 250(136), 264 Shimazono, N., 38(78), 69 Shoaf, W. T., 234(138), 239(137), 240 (137), 241(137), 252(138), 254(138), 257(138), 258(137, 138), 264 Shorenstein, R. G., 11(36), 14(36), 15 (36), 19 Shulman, R. G., 212, 226 Sia, C. L., 26(174), öi Siegel, Η., 179(148), 206 Signorini, Μ., 26(143), 60 Silengo, L., 29(113), 55(20), 67, 60 Silverstein, E., 164(67), 167 Simpson, E. R., 196(22), 203 Simpson, R. T., 269(118), 270(119), 280 (117), 308 Singhal, R. L., 39(177), W Siniscalco, M., 42(158, 159), 61 Siperstein, M. D., 171, 175, 176(138), 177 (133, 136, 138), 178(16, 35, 134, 135, 137), 181, \^(m),20S,206 Skarda, J., 34(68), 35(68), 55 Slakey, L. L., 176(37), i^OS Slapikoff, S., 7(69), 10(69), 13(69), 20 Slater, E. C , 249(26), 261, 290(12, 32), 306, 306

326 Smith, E. C.,72{d),m Smith, E. L., 5(70), 20 Smith, J. L., 193(115), 206 Smith, K. E., 301(9), 505 Smith, L. H., Jr., 227(139), 228(139), 229 (140, 141, 142), 234(142), 247, 251 (140, 144), 252(139), 254(40, 143), 255 (m),256iU2),262,264 Smymiotis, P. Z., 23(71), 69 Söderberg, Β. Ο., 289(13), 306 Söderlund, G., 289(13), 306 Söll, D., 294(64), 507 Sörensen, N., 23(50), 26(50), 68 Sokolski, W., 304(46), 306 Soldin, S. J., 23(161), 50, 61 Sols, Α., 23(148), 30(148), 60, 164, 167 Sonenshein, Α. L., 11, 12(26), 14(36), 15 (36, 37), 19 Sora, S., 118, 128 Spector, L. B., 152(5, 75), Ιββ SpeziaU, G. A. G., III(73a), 129 Spitzer, J. L., 7(69), 10(69), 13(69), 20 Spizizen, J., 1(72), 3(72), 8(60), 12(60), 15(60), 19, 20 Spolter, P. D., 162(69), 167 Spors, S., 243, 264 Spudich, J. Α., 1(29), 2(29), 10(29, 74), 11(73), 19, 20 Squire, P. G., 32(151, 162), 33(151), 61 Sreenivasan, Α., 192(4), 202 Srere, P. Α., 163,167 Srinivasan, V. R., 11(25), IP Srivastava, S. K., 37(110, 163, 164), 38 (110), 69, 61 Stadtman, E. R., 64(74), 67(73, 94), 68 (73, 94), 90(75), 95(73), 100, 129, 130, 157(71), 167, 193, 206, 268(120), 308 Stallcup, W. B., 299(87), 307 Stamatoyannopoulos, G., 44(184), 61 Stamm, N. B., 39(177), 6/ Stanier, R. Y., 90(20), 94(20), 128 Stark, G. R., 300(49), 301(27), 306 Stein, L. I., 227(146), 229(146), 243(146), 264 Steinberg, D., 179(142), 190, 206 Steinmann, L., 42(182), 61 Stent, G. S., 122(77), 129 Stetten, O., m(S9),206 Stone, D., 197(143), W

AUTHOR INDEX

Strijkert, P. J., 101(78), 108, 129 Strom, R., 292(121), 308 Subba Rao, G., 176(144), 179(144), 183 (144), 184(144), 187(144), 206 Suelter, C. H., 163(36), 166 Sulebele, G. Α., m(i),202 Sulimovici, S., 200, 206 Sullivan, Μ., 229(141), 251(144), 254 (143), 255(143), 264 Sund, Η., 287(122), 308 Sussenbach, J. H., 101(78), 108, 129 Sussmän, A. J., 12(75), 20 Sutherland, Ε. W., 115(63), 116(10, 30, 63), 117(9, 30, 39, 51, 59, 79), 128, 129, 198(67), 204 Sutterlin, N. S., 200(29), 203 Suyama, Y., 229(147), 231(147), 264 Svendsen, I., 5, 19 Sweeney, Μ. J., 229(148), 247(148), 251 (148), 264 Swyryd, Ε. Α., 175(9), 176(9), 177(54), 181(9), 187(54), 188, 192(54), 202,

204 Sykes, Η. Β., 32(162), 61 Szeinberg, Α„ 51(115), βΟ Szulmajster, J., 15(38), 19

Tabita, R., 23(165), 67 Tagaki, W., 295(123), 308 Tager, J. M., 249(26), 261 Tait, G. H., 275(124), 308 Taketa, K., 39(167), 41(166), 61 Tamoki, B., 199(79), 204 Tan, E. L., 26(80), 59 Tanaka, Α., 39(167), 61 Tate, S. S., 285(125), 505 Tatibana, M., 227(149), 228(150, 151), 229(62), 236(65, 136, 149, 150), 237 (67, 149), 240, 242(62), 245(67, 149), 247(67, 150, 151), 248(150), 249, 250 (136), 256(67), 262, 264 Taylor, A. L., 228(152), 229(152), 264 Taylor, C. B., 177(55), 178, 189, 204, 206 Taylor, M. L., 228(153, 154), 252(153, 154), 253(153, 154), 264 Taylor, W. H., 228(153, 154), 252(153, 154), 253(153, m),264 Tchen, T. T., 196(28), 203 Teinzer, Α., 41(58), 68

327

AUTHOR INDEX

Temple, T. E,, 177(147), 196(147), m Tennert, D. M., 179(148), 206 Tenu, J. P., 292(126), 308 Teplitz, R. L., 42, 61 Tepperman, H. M., 38(169, 170), 39(169, 170), 61 Tepperman, J., 38(169, 170), 39(169, 170), 61 Terada, M., 237(67), 245(67), 247(67), 256(67), 262 Tewari, K. K , 37(194), 0^ Thayer, S. Α., 229(155), 264 Theorell, H., 28, 61, 158(72), 167, 275 (124), 287(122), 289, 305 Thibodeau, P. S., 229(155), 264 Thomas, G. B., Jr„ 69(80), 71(80), 129 Thorne, C., 285(129), 305 Thorp, J. M., 176(149), 181(149), 206 Threlfall, D . R., 193(155), 207 Tiboni, 0., 118,128 Tipper, D . J., 11(76), 20 Tönz, 0., 44(172), 61 Tomita, Κ., 228(159), 253(159), 26δ Tomkins, G. Μ., 176(153), 177(152), 179 (151, 153), 180(151), 191(151), 206 Toren, D., 198(154), 207 Torriani, A. M., 274(130), 275, 279(37), 304(37), 300, 305 Tramell, P. R., 236(156), 237(158), 243 (156, 157), 264, 266 Travers, A. Α., 12(77), ^0 Tremblay, G. C., 245, 248(12), 249(12, 112), 261, 263 Trentham, D . R., 274, 280(48), 283(133), 285(133), 291(132), 306, 309 Trotta, P. P., 237(158), 239(158), 266 Truffa-Bachi, P., 62(82, 83), 90(82), 91 (81), 94(82), 99, 100, 129, 130 Tsernoglou, D., 284(7), 285(134), 286 (m), 306, 308, 309 Tsolas, O., 26(173, 174), 61, 285(12a), 295 (12a), 306 Tsuru, D., 6(43), 19 Tsutsui, E. Α., 49, 61 Tsuzuki, H., 5(56), 19 Turini, P., 292(121), 305 U üchino, H., 229(66), 237(66), 248, 262 Udenfriend, S., 250, 262

Ullrich, J., 25(56), 55 Umbarger, H. E., 64(84), 89, 90, 95(84), 101(83, 84), 111(83), i30 Umezu, K., 228(159), :?05 Urba, R. C , 10(78), ^0 Usanga, E. Α., 45(98), 50 Utter, Μ. F., 162(45), 167 Uyeda, Κ., 155, 156(73), 167

Vaccaro, D., 7(69), 10(69), 13(69), 20 Vagelos, P. R., 304(135), 309 Vallee, B. L., 269(100, 118), 270(119), 275 (124), 287(34), 280(117), 306, 308 Vanecek, J., 284(1), 295(1), 305 Van Loan, V., 285(136), 292, 309 Van Rapenbusch, R., 293(82), 307 Van Wijk, R., l l l ( 7 3 a ) , ij?0 Vapnek, D., 100, 130 Veech, R. L., 157, 159,167 VeUck, S. F., 290(137), 300 Veneziano, G., 44(149), 61 Vergara, F. E., 38(125), 00 Vinuela, E., 15(2), 18, 23(148), 30(148), 60 Viratelle, 0 . M., 292(126), 305 Vogel, H. J., 104(87), 130 Voight, B., 142(37), 166 Volkenstein, M. V., 65, 130 von Hinüber, C , 26(52, 57), 55 Vyas, S., 104(89), 130

W Wade, M., 284(7), 305 Wadkins, C. L., 151(46), 107 Waites, W. M., 10(40), 11(79), 12(40), 19, 20 Wald, G., 112(90), 130 Walker, P. G., 274, 285(38), 300 Wallenfels, K., 292(138, 139), 309 Waller, J. P., 285(10), 293(10, 11, 19, 82), 294(10, 11, 20), 295(140), 304(11), 305, 306, 307, 309 Walsh, C. Τ., Jr., 152(75), 167 Walter, P., 284(72), 307 Waltinger, G., 179(63, 65), 184(65), 204 Walton, G. M., 150(9), 166 Warburg, O., 23(176), 61 Warford, L. R., 254(133), 255(133), 264 Waring, W. S., 176(149), 181(149), 206

328 Warmer, I., 177(55), m Warren, J. C , 32(124), 60 Warren, R. A. J., 6(33), 8(33), 12(33), 14 (33), 15(33), 19 Warren, S. C , 2(80), 12(80), 20 Wasson, G., 175(24), ^05 Watson, J. D., 122(91), 130 Waygood, E. R., 228(77), 252(77), 258 (77), 262 Webb, E. C., 29(49), 55 Weber, G., 39(177), 61, 229(160), 247 (160), 265, 287(2), 305 Weber, K., 300(141), 301(104), 308, 309 Weinhouse, S., 162(69), 167 Weise, Μ., 26(53), 55 Weise, P. Α., 122(92), 130 Weiss, L., 41(58), 5 5 Westheimer, F. H., 285(96), 295(96, 123), 308 Whistance, G. R., 193(155), 207 White, J. S., 301(9), 305 White, L. W., 174(157, 158), 175, 176 (156, 158), 180(158), 182, 183, 185 (158), 186, 187(158), 207 Wiame, J. M., 227(124), 228(87, 124), 229 (87, 124), 233(87), 235(87), 258(87, 124), 263, 264 Wick, A. N., 37(118, 119), 60 Wieland, 0., 41(58), 58, 180(56, 58), 204 Wilcox, S. S., 37(118), Wild, J., 229, 259, 265 Williams, L. G., 227(163), 228(162, 163), 229(162), 230, 232(163), 233(162,163), 237(162, 163), 238(162, 163), 244 (162), 251(162), 258(163), 265 WiUiams, L. S., 101(96), 130 Williams, V. R., 161(76), Williamson, D. H., 250(20), 261 Wilson, D. W., 227(167), 228(167), 252 (167), 265 Wilson, I. B,, 270, 285(142, 143), 307, 309 Wilson, T. H., 28(33), 57 Winlund, C., 300(144), 309 Wold, F. J., 179(148), Wolfe, R. G., 35(77), 59, 67(60), 129, 284 (1, 31), 285(42, 101), 286(53), 287 (54), 295(1), 305(54), 305, 306, 308 Wong, J. T.-F., 65(93), 124(93), 130

AUTHOR INDEX Wood, J. D., 179(108), 205 Wood, M. H., 229(43), 255(43), 257(43), 259(43), 262 Wood, T., 26(80), 59 Woodward, V. W., 229(36, 56, 147, 164, 165), 231(36, 147, 164), 261, 262, 264, 265 Wolcott, J. H., 258(166), 260(166), 265 Woolfolk, C. Α., 67(94), 68(94), 130 Wratten, C. C , 137, 158,167 Wrigley, N . G., 22(179), 46(179), 47(179), 49(179), 52(179), 53(179), 55(179), 61 Wu, C. W., m m , 306 Wu, J., 28(180), Wu, R., 227(167), 228(167), 252(167), 265 Wuu, K.-D., 253(168), 257(168), 265 Wyckoff, H. W., 279(52), 306 Wyman, J., 209, 210, 214, 221, 225, 226, 267(94), 268(94), 296(94), 297(94), 299(94), 300, 302(94), 303(94), 308 Wyngaarden, J. B., 234(52), 254(52), 257 (52), 262

Yamamoto, T., 12(54), 19 Yan, Y., 228(169), 229(169), 265 Yasunobu, K. T., 5, 6(43), 19, 20 Yates, R. Α., 64(95), 89, 101(95), 111 (95), 130, 227(170), 228(171, 172), 229 (172), 255(172), 265 Yem, D . W., 101(96), 130 Yip, M. G. M., 229(173), 236(173), 245 (173), 247(173), 248(173), 265 Yogi, N., 178(146), 189(146), 206 Yon, J., 292(126), 5Ö5 Yoshida, Α., 23(181), 42(121, 181, 182, 187, 188), 43(183, 186, 188), 44(184, 186), 49(181), 50(181, 185), 51, 53 (ISl), 58, 60, 61,62 Yoshimoto, Α., 228(159), 253(159), 265 Young, I . E . , 2(82), Young, J. E., 229(126), 245(126), 247 (174), 254(126), 264, 265 Young, P. L., 198(30), 203 Yousten, A. Α., 1(24), 2(24), 11(24), 18 Yue, R. H., 27(190, 191), 28(191), 35 (191), 62

AUTHOR INDEX

Yugari, Y., 11(83), 20, 40(192), 41(192), 60, 62 Yurowitzki, Y. G., 30(193), 62 ^ Zabin, I., 292(145), 309 Zaheer, N., 37, 62

329

Zannetti, M. E., 179(148), 206 Zelwer, C , 293(140), 295(140), 309 Zeppezauer, E., 289(13), 306 Zewe, v . , 131(25, 79), 146(79), 154(25, 80), 166, 167 Zielke, C. L., 163(36), 166 ZwiUing, E., 147(16), 166

SUBJECT INDEX A Acetoacetate decarboxylase, :flip-flop mechanism of, 295-296 ACTH, effects on adrenal steroid hormone biogenesis, 197 Actinomycin, effects on cholesterol biogenesis, 185 Adrenal cortex, glucose-6-phosphate dehydrogenase from, 32-34 Adrenals, steroid hormone biogenesis in 196-199 ' Alcohol dehydrogenase, kinetic mechanisms of, 157-159 Aldolase, modification by serine protease 14-16 ' Aldosterone, control of biogenesis of, 197-198 Alkaline phosphatase of E. coli, flip-flop mechanism of, 268283

comparison with intestinal enzyme

~3

'

Allosteric models, 209-226 effect of second ligand, 218-220 effect of subunit heterogeneity, 220-221 MWC and induced-fit models, 214-218 parent model, 211-214 trout hemoglobin as, 221-224 cAMP, role in steroid hormone biogenesis, 198 Aspartate transcarbamylase, as half-site enzyme, 300-302

B Bacilli, extracellar proteases of, ~ Bacillus cel·eus, protease of, 6-7, 8 Bacillus licheni/ormis, proteases of, 7, 8 Bacillus megaterium, protease of, 7, 8 Bacillus 8ubtilis proteases of, 5-6, 8 intracellular, 12-14 Bacteria proteases in, role in sporulation, 1-20 uridylic acid biosynthesis in, 234-235 Bile acids 330

biogenesis of, 194-196 cholesterol 7a-hydroxylase, 195-196 control sites, 195 effects on cholesterol biogenesis 179 Biochemical systems ' mathematical description of, 65-66 methods of analysis of, 66-74 computer simulation, 67-69 linearization, 69-71 nonlinear approximation, 71-74 Biosynthetic pathways, feedback inhibition of, 79-101 Brewer's yeast, glucose-6-phosphate dehydrogenase from, 26-31

C Candida utilis, glucose-6-phosphate dehydrogenase from, 25-26 Carbamyl phosphate synthetase-aspartate transcarbamylase complex, of fungi cellular activity of, 247-248 metabolic control of, 244-247 structure of, 238-244 Carbamyl phosphate synthetase-aspartate transcarbamylase-dihydroorotate complex, of mammals cellular activity of, 247-248 structure of, 239-243 Chlorophenoxyisobutyrate, effects on cholesterol biogenesis, 181-182 Cholestenone, effects on cholesterol biogenesis, 179 Cholesterol biogenesis, in animals, 171189 actinomycin effects on, 185 altered conditions of, 175-185 list, 176 bile and bile acid effects on, 179 chlorophenoxy isobutyrate effects on, 181-182 cholestenone effects on, 179 circadian rhythm in, 183-184 control points in, 187-189 during cholesterol feeding, 175, 177-179 estrogen effects on, 181

SUBJECT INDEX

331

experimental validity of studies on, in enzymatic action, 119-120 174-175 natural selection of, 89-101 /3-hydroxY-f3-methylglutaryl-CoA Corpora lutea, progesterone biogenesis in, reductase in, 185 regulation, 199-200 /3-hydroxy-p-methylglutaryl-CoA synCreatine kinase, kinetic mechanisms of, thetase in, 186-187 150-153 inhibition of, effects on ubiquinone Cytidine triphosphate synthetase, as halfsynthesis, 190-193 site enzyme, 298-299 norepinephrine effects on, 184 D phenyl acid inhibition of, 188-189 Dihydroorotate dehydrogenase radioactive tracer studies of, 171 isolation and properties of, 251-253 serum cholesterol levels and, 189 metabolite control of, 253 site of control of, 174 starvation and refeeding effects on, 179- Dihydroorotase, isolation and properties of, 250-251 181 thyroxine effects on, 180-181 E Triton-WR 1339 effects on, 183 Enzyme(s) ubiquinone effects on, 182-183, 190 cascaded mechanisms of, 111-122 x-irradiation effects on, 183 of eukaryotic cells, in uridylic acid bioCholesterol metabolism synthesis, 235-260 in ovarian tissue, 200-201 glucose-6-phosphate dehydrogena&e of, pituitary role in, 201 41-56 Cholesterol 7a-hydroxylase, effects on bile aging of erythrocytes and, 45 acid biosynthesis, 195-196 apoenzyme-coenzyme interactions, Cholestyramine, effects on cholesterol 49-52 biogenesis, 179 chemical properties, 49 Computer, simulation of biochemical sysdeficiency of, 43-45 tems using, 67-69 genetic polymorphism of, 41-43 Control systems (biochemical), 63-130 interconversion of forms, 52-56· analysis of, 74-89 M. W. and subunits of, 45-49 concepts and techniques in, 87-89 half-site type, flip-Hop mechanisms of, of uncontrolled biosynthetic pathway, 75-79 267-309 kinetic mechanisms for actual systems, in cascaded enzymatic mechanisms, 111-122 149-162 alcohol dehydrogenase, 157-159 amplification, 113-119 distribution in nature, 112-113 aspartase, 161-162 feedforward inhibition as, 101-111 creatine kinase, 150-153 with other control mechanisms, 104hexokinase, 153-157 108 nucleoside diphosphokinase, 160-161 physiological implications, 108-111 Michaelis constants for, 162-163 power law approach in, regulation of, 131-167 contributions of, 124-126 product effects on enzyme velocity, validity of, 123-124 132-142 feedback inhibition as product effects on substrate binding, analysis, 79-83 142-145 in biosynthetic pathways, 89-101 velocity responses of, substrate effects, branched, 94-98 145-149 unbranched, 91-94 a·bortive complex formation, 145-149 consequences of, 85-87 Escherichia coli

332

SUBJECT INDEX

alkaline phosphatase of, as model for flip-flop mechanisms, 268-283 glucose-6-phosphate dehydrogenase from, 24 methionyl-tRNA synthetase of, 293295 Estrogens, effects on cholesterol bio­ genesis, 181 Eukaryotic cells, uridylic acid biosyn­ thesis in, 227-265

F Feedback inhibition, of biosynthetic pathways, 79-101 Feedforward inhibition, of biosynthetic pathways, 101-111 Flip-flop mechanisms of allosteric enzymes, 296-302 aspartate transcarbamylase, 300-302 cytidine triphosphate synthetase, 29S-299 evolutionary advantages of, 302-305 of half-site enzymes, 267-309 acetoacetate decarboxylase, 295-296 i8-galactosidase, 292-293 glyceraldehyde-3 phosphate dehydro­ genase, 290-291 malate and alcohol dehydrogenase, 284-290 methionyl-tRNA synthetase, 293-295 transaldolase, 295-296 model for (alkaline phosphatase), 268283 Fungi uridylic acid biosynthesis in, genetic control of enzymes, 229-235 comparison with bacteria, 234-235

G ^-Galactosidase, flip-flop mechanism of, 292-293 Gonads, steroidogenesis in, regulation, 198-201 Glucose dehydrogenase, glucose-6-phosphate dehydrogenase and, 30-31 Glucose-6-phosphate dehydrogenase (G6PD) chemical properties of, 26, 49 coenzyme specificity of, 24-25

comparison with glucose-6-phosphate dehydrogenase, 37 dimer-monomer equilibrium of, 53-55 general properties of, 23 glucose dehydrogenase and, 30-31 kinetic properties of, 23, 25, 35-36 from mammalian cells, 31-56 adrenal cortex, 32-34 erythrocytes, 41-56 liver, 36-41 mammary gland, 34-36 ovary, 31-32 from microorganisms, 24-31 Brewer's yeast, 26-31 Candida utilis, 25-26 Eschenchia coli, 24 Leuconostoc mesenteroides, 24-25 physical properties of, 26-28 regulatory properties of, 21-62 ion and metabolite effects on, 28-29 substrate specificity of, 23 tetramer-dimer equilibrium of, 52-53 thermodynamic properties of, 28 Glyceraldehyde-3 phosphate dehydro­ genase, flip-flop mechanisms of, 290291 Η Half-site enzymes evolutionary advantages of, 302-305 flip-flop mechanisms of, 267-309 Hemoglobin, from trout, as allosteric model, 221-224 Hexokinase, kinetic mechanisms of, 153157 Hexose phosphate dehydrogenase, com­ parison with glucose-6-phosphate de­ hydrogenase, 37 Hexose phosphate shunt, role in erythro­ cyte metabolism, 41 /3-Hydroxy-y?-methylglutaryl-CoA reduc­ tase, in cholesterol biogenesis, 185186 ;8-Hydroxy-/8-methylglutaryl-CoA syn­ thetase, in cholesterol biogenesis, 186-187 I Isoprenoid biogenesis, in animals, 169207

333

SUBJECT INDEX

bile acids, 194-196 cholesterol, 171-189 coenzym~ A in, 169-170 hydroxylations in, 170-171 pathways of, 169-171 diagram, 172-173 pyrophosphate in, 170 steroid hormones, 196-201 sterols in, 170 ubiquinone, 190-194

L Lactation, glucose-6-phosphate dehydrogenase in mammary gland during, 34-35

Leuconostoc mesenteroides, glucose-6phosphate dehydrogenase from, 24-25 Liver alcohol dehydrogenase, :ftip.Hop mechanism of, 287-290

M Malate dehydrogenase, Hip-Hop mechanisms of, 284-287 Mammary gland, glucose-6-phosphate dehydrogenase from, 34-36 Methionyl-tRNA synthetase, flip-Hop mechanism of, 293-295 Michaelis constants, in kinetic studies of enzyme regulation, 162-163

N Norepinephrine, effects on cholesterol biogenesis, 184 Nucleoside diphosphokinase, kinetic mechanisms of, 160-161

o Orotidylic enzymes, 253-258 in fungi, 253-254 in mammals, 254-258 in plants, 258 Ovarian tissue, cholesterol metabolism in, 200-201 Ovary, glucose-6-phosphate dehydrogenase from, 31-32

p Penicillium dupontii, glucose-6-phosphate dehydrogenase from, 31

Phenyl acids, as inhibitors of cholesterol biogenesis, 188-189 Pituitary gland, \ role in cholesterol metabolism, 201 Pregnenolone, in steroid hormone biosynthesis, 197 Progesterone, biogenesis in corpora lutea, regulation, 199-200 Protease (s) of Bacillus cereus, 6-7, 8 of Bacillus licheniformis, 7, 8 of Bacillus megaterium, 7, 8 of Bacillus 8ubtilis, ~, 8 in sporulation, 1-20 proteolytic activity in, 4 regulation of, 8-10

R RNA polymerase, modification of, by serine protease, 14-16

5 Saccharomyces carlsbergensis, see Brewer's yeast Serine protease, modification by RNA polymerase and aldolase, 14-16 Sporulation protease role in, 1-20 protein turnover during, 10-14 degradation, 12 synthesis, 11-12 proteolytic activity in, 4 stages in, 2 Steroid hormone biogenesis in adrenals, 196-199 in gonads, 198-201

T Testosterone, biogenesis of, in testis, regulation, 198-199 Thyroxine, effects on cholesterol biogenesis, 180-181 Transaldolase, flip-flop mechanism of, 295-296 Triton-WR 1339, effects on cholesterol biogenesis, 183

u Ubiquinone biogenesis, 190-194 aromatic pathway of, 193-194

334

cholesterol biogenesis and, 182-183, 190-193 inhibition effects, 192 control point in, 193 Uridylic acid biosynthesis in eukaryotic cells, 227-265 enzymes of, 235-260

SUBJECT INDEX

in fungi, genetic control of enzymes for, 229-235 general pathway for, 227-229 ^ X-irradiation, effects on cholesterol biogenesis, 183