Contributors to Volume 3 MAX M. BURGER H. W. DUCKWORTH M. KAPOOR JOSEPH LARNER IRA PASTAN ROBERT L PERLMAN B. D. SANWAL WILHELM SCHONER WERNER SEUBERT CARLOS VILLAR-PALASI CURRENT TOPICS IN Cellular Regulation edited by Bernard L Horecker · Earl R. Stadtman Albert Einstein College of Medicine National Institutes of Health Bronx, New York Bethesda, Maryland Volume 3 7977 ACADEMIC PRESS New York and London COPYRIGHT © 1971, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS. ACADEMIC PRESS, INC. Ill Fifth Avenue, New York, New York 10003 United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. Berkeley Square House, London W1X 6BA LIBRARY OF CONGRESS CATALOG CARD NUMBER: 72-84153 PRINTED IN THE UNITED STATES OF AMERICA List of Contributors Numbers in parentheses indicate the pages on which the authors' contributions begin. MAX M. BURGER (135), Department of Biochemical Sciences, Princeton University, Princeton, New Jersey H. W. DUCKWORTH (1), Department of Medical Cell Biology, University of Toronto, Toronto, Canada M. KAPOOR* (1), Department of Medical Cell Biology, University of Toronto, Toronto, Canada JOSEPH LARNER (195), Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia IRA PASTAN (117), Laboratory of Molecular Biology, National Cancer Insti- tute, National Institutes of Health, Bethesda, Maryland ROBERT L. PERLMAN (117), Department of Physiology, Harvard Medical School, Boston, Massachusetts B. D. SANWAL (1), Department of Medical Cell Biology, University of Toronto, Toronto, Canada WILHELM SCHONER (237), Physiologisch-Chemisches Institut der Universität, Göttingen, Germany WERNER SEUBERT (237), Physiologisch-Chemisches Institut der Universität, Göttingen, Germany CARLOS VILLAR-PALASI (195), Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia * Present address: Department of Biology, University of Calgary, Calgary, Canada. vii 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 ever- increasing 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 IX Contents of Previous Volumes 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-1,4-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 xi xii CONTENTS OF PREVIOUS VOLUMES Pyruvate Carboxylase Merlon F. Utter and Michael C. Scrutton Author Index—Subject Index Volume 2 DPN-Linked Isocitrate Dehydrogenase of Animal Tissues Gerhard W. E. Plant The Regulation of Biosynthesis of Aromatic Amino Acids and Vitamins J. Pittard and F. Gibso?i Regulation of Cholesterol Biosynthesis in Normal and Malignant Tissues Marvin D. Siperstein The Biogenesis of Yeast Mitochondria Anthony W. Linnane and J. M. H aslant 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 Metabolic 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 The Regulation of Branched and Converging Pathways I B. D. SANWAL I M. KAPOOR* H. W. DUCKWORTH I Department of Medical Cell Biology I University of Toronto | Toronto, Canada I. Introduction 1 A. Functional Classification of Pathways and Levels of Their Regulation 3 B. Patterns of Control of Nonlinear Pathways 6 C. Nature of Regulatory Enzymes 12 D. Identity of Repressors (Inducers) in Inducible and Repressible Systems 17 E. Scope of Review 22 II. Regulation of Nucleotide Biosynthesis and Related Pathways 23 A. Control of 5'-Phosphoribosyl Pyrophosphate (PRPP) Synthetase. 23 B. The Common Purine Pathway: Synthesis of IMP 26 C. Synthesis of AMP and GMP, and Their Interconversions 30 D. Repression Effects on the Enzymes of Purine Biosynthesis 40 E. Histidine Biosynthesis 42 F. Other Pathways Branching from Purine Biosynthesis 48 G. Interconversion of Nucleotides 49 III. Regulations of Branched Biosynthetic Pathways for Amino Acids.. . 56 A. Aromatic Amino Acid Pathway 56 B. Cross-Pathway Regulation 93 C. The Aspartate Family Amino Acids in Escherichia coli 97 IV. Conclusions 102 References 103 I. Introduction A. Functional Classification of Pathways and Levels of Their Regulation From a physiological standpoint the various pathways of intermediary metabolism may be classified (108) into three major categories: biosyn- thetic, catabolic, and amphibolic. The former two pathways are uni- directional and unifunctional whereas the amphibolic routes are bidirectional and bifunctional; i.e., the enzyme systems constituting the amphibolic pathways function both in a catabolic and biosynthetic capacity (384), * Present address: Department of Biology, University of Calgary, Calgary, Canada. 1 2 B. D. SANWAL, M. KAPOOR, AND H. DUCKWORTH and they supply intermediates for energy generation (in the form of ATP) as well as for biosynthesis. This functional categorization of pathways serves to focus attention on the means by which regulation of these routes is accomplished. It is now well documented that catabolic routes are induced in the presence of compounds which are destined to be degraded through these routes {428), and the enzymes of a biosynthetic pathway are repressed by the end product of that pathway (94, 154, 299, 467, 477). By contrast, the enzymes of amphibolic routes are generally "constitutive" or, in some cases, regulated by both repression and induction {384). This behavior is in keeping with the nature of amphibolic routes, which serve a biosynthetic as well as a catabolic function. Because a majority of biosyn- thetic routes are repressible, the impression has gained ground that induc- tion as a genetic control mechanism is not of any great consequence in strictly biosynthetic pathways. In a number of cases, however, true induc- tion has been demonstrated. In the leucine pathway of Neurospora crassa (161), leucine represses the synthesis of the first enzyme of the pathway (isopropylmalate synthetase) whereas the product of this enzyme, α-isopropylmalate, induces the formation of the succeeding two enzymes. Similarly, out of the five isoleucine-valine pathway enzymes in Escherichia coli, Aerobacter aerogenes, and Salmonella typhimurium, four are multi- valently repressed (see Section I, B) but the fifth, acetohydroxy acid isomeroreductase, is induced by its substrates, acetohydroxybutyrate and acetolactate (225). In yet another case, the pyrimidine pathway of yeast (253), limiting amounts of uracil in the growth medium derepress the enzymes of the pathway, but this does not occur in a mutant lacking the first enzyme (aspartate transcarbamylase), a finding which suggests that the product of this enzyme may be necessary for the induction of one or more remaining enzymes. Evidence exists (254) that this may be brought about in a sequential manner, such that the product of the first enzyme induces the second enzyme and the product of the latter induces the remaining three enzymes of the pathway. Similarly, in Pseudomonas putida the last two enzymes of the tryptophan biosynthetic pathway, tryptophan synthetase A and B, are inducible by indole glycerol phosphate, the product of the first feedback sensitive enzyme of the pathway, anthranilate synthetase (462). It is well known that in another bacterium, E. coli, these same enzymes are repressible by tryptophan (Section III, A). As opposed to induction in some biosynthetic pathways, true repression, however, has not been found in purely catabolic routes. A process referred to as "multi sensitive repression" has been described (285) in converging catabolic routes (Section I, B). Here, a number of end products of inter- locking converging pathways repress the synthesis of blocks of enzymes which are responsible for the synthesis of the end products. This mechanism, REGULATION OF BRANCHED AND CONVERGING PATHWAYS 3 however, differs from true repression in that the repression here is com- pletely reversible by the inducer(s) of the pathway. In linear (unbranched) biosynthetic pathways repression by the end product, and in catabolic routes induction by the initial substrate, affects all the enzymes of a given pathway. This repression or induction may be coordinate (7) or noncoordinate. Generally, coordinate repression occurs in concatenated gene sequences constituting an operon, but this is not a prerequisite for such repression, as shown, for example, by Taylor et al. (442) for the pyrimidine pathway in E. coli. Many speculations have appeared in the literature regarding the need for opérons in bacteria (293). It is clear that with respect to regulation no advantages can accrue to the organism by having the genes governing the synthesis of enzymes of a linear pathway placed adjacent to one another. It is quite possible, as suggested by Harris (167), that clustering of genes may be required by the sexual mechanism of E. coli where the chromosome is transferred in an orderly sequence. It would be a selective advantage to keep genes of a pathway grouped together because breaking of the chromosome in the middle of such a group might conceivably create a lethal situation. One point of considerable interest in the area of genetic control of path- ways is the identity of the inducing or repressing effector of a given metabolic sequence. To avoid confusion the term effector is used here to denote the small molecular weight metabolite participating in the process of induction or repression which may also include one or more macro- molecular components (298). In the oldest known and by far the most intensively studied system, the lactose operon, Burstein et al. (69) demon- strated that it is not lactose itself, but a product of ß-galactosidase, which serves as the effector. This "product induction" is now known to occur in the glycerol regulon (173) of E. coli and the histidine degradative pathway of Aerobacter aerogenes (393) and Pseudomonas aeruginosa (326). In the former case L-a-glycerophosphate and in the latter pathway urocanate, the immediate products of the first enzymes of these pathways, are the inducing effectors. In the catabolism of hydroaromatic compounds (Section I, B) by pseudomonads, the inducing effector, however, is the product of an enzyme much further removed in the sequence (231, 338). In E. coli, only in a few catabolic opérons has it been rigorously demonstrated that the initial substrate is the inducing effector per se. These are, for example, the galactose and arabinose opérons and the ace (governing the synthesis of the pyruvate dehydrogenase complex) locus. In these sequences, D-galactose (293), L-arabinose (407), and pyruvate (113), respectively, are inducers. As is evident from the examples given above, there seems to be no rule determining whether the initial substrate or the intermediary products of the pathway will serve as effectors. It is instructive to mention also that there is no uniformity with regard to the identity of effectors of a given