[1st Edition] 9781483217079

Table of contents :
Content:
Contributors to Volume 8Page ii
Front MatterPage iii
Copyright pagePage iv
List of ContributorsPages ix-x
PrefacePage xiBERNARD L. HORECKER, EARL R. STADTMAN
Preface to Volume 8Page xiiiBERNARD L. HORECKER, EARL R. STADTMAN
Contents of Previous VolumesPages xv-xx
A Molecular Model for Morphogenesis: The Primary Septum of YeastPages 1-32ENRICO CABIB, RODNEY ULANE, BLAIR BOWERS
Metabolic Regulation by Multifunctional Glucose-6-phosphatasePages 33-117ROBERT C. NORDLIE
Glutamine Synthetase as a Regulator of Enzyme SynthesisPages 119-138BORIS MAGASANIK, MICHAEL J. PRIVAL, JEAN E. BRENCHLEY, BONNIE M. TYLER, ALBERT B. DELEO, STANLEY L. STREICHER, ROBERT A. BENDER, C. GREGORY PARIS
Acetyl Coenzyme A Carboxylase*Pages 139-195M. DANIEL LANE, JOEL MOSS, S. EFTHIMIOS POLARIS
Regulation of Lipogenesis in Animal TissuesPages 197-246SHOSAKU NUMA, SATOSHI YAMASHITA
Deamidation of Glutaminyl and Asparaginyl Residues in Peptides and ProteinsPages 247-295ARTHUR B. ROBINSON, COLETTE J. RUDD
Pasteur Effect and PhosphofructokinasePages 297-345ABBURI RAMAIAH
Subject IndexPages 347-350

Citation preview

Contributors to Volume 8 ROBERT A. BENDER BLAIR BOWERS JEAN E. BRENCHLEY ENRICO CABIB ALBERT B. DELEO M. DANIEL LANE BORIS MAGASANIK JOEL MOSS ROBERT C. NORDLIE SHOSAKU NUMA C. GREGORY PARIS S. EFTHIMIOS POLAKIS MICHAEL J. PRIVAL ABBURI RAMAIAH ARTHUR B. ROBINSON COLETTE J. RUDD STANLEY L. STREICHER BONNIE M. TYLER RODNEY ULANE SATOSHI YAMASHITA

CURRENT TOPICS IN

Cell ular Regulation edited by Bernard L. Horecker · Earl R. Stadtman Roche Institute of Molecular Biology Nutley, New Jersey

National Institutes of Health Bethesda, Maryland

Volume 8 1974

ACADEMIC PRESS New York and London A Subsidiary of Harcourt Brace Jovanovich, Publishers

COPYRIGHT © 1974, 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, INC. Ill Fifth Avenue, New York, New York 10003

United Kingdom Edition published by A C A D E M I C PRESS, INC. ( L O N D O N ) LTD. 24/28 Oval Road, London NW1

LIBRARY OF CONGRESS CATALOG CARD N U M B E R : 72-84153

ISBN 0 - 1 2 - 1 5 2 8 0 8 - 1 P R I N T E D IN T H E U N I T E D STATES O F AMERICA

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

A. BENDER (119), Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts BLAIR BOWERS (1), National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland JEAN E. BRENCHLEY* (119), Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts ENRICO CABIB (1), National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland ALBERT B. DELEO (119), Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts M. DANIEL LANE (139), Department of Physiological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland BORIS MAGASANIK (119), Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts JOEL MOSS (139), Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland ROBERT C. NORDLIE (33), Department of Biochemistry, University of North Dakota School of Medicine, Grand Forks, North Dakota SHOSAKU NUMA (197), Department of Medical Chemistry, Kyoto University Faculty of Medicine, Kyoto, Japan C. GREGORY PARIS (119), Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts S. EFTHIMIOS POLARIS (139), Department of Physiological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland MICHAEL J. PRivALf (119), Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts ABBURI RAMAIAH (297), Biochemistry Department, All-India Institute of Medical Sciences, New Delhi, India ARTHUR B. ROBINSON (247), Institute of Orthomolecular Medicine, Menlo Park, California

ROBERT

* Present address: Department of Microbiology, Pennsylvania State University, University Park, Pennsylvania. t Present address: Office of Toxic Substances, U.S. Environmental Protection Agency, Washington, D.C. IX

X

LIST OF

CONTRIBUTORS

J. RUDD (247), Molecular Biology Institute, University of California, Los Angeles, California STANLEY L. STREICHER (119), Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts BONNIE M. TYLER (119), Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts RODNEY ULANE (1), National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland SATOSHI YAMASHITA (197), Department of Medical Chemistry, Kyoto University Faculty of Medicine, Kyoto, Japan

COLETTE

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 number of isolated enzyme systems, together with results derived from physiological 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. However, 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 intend 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 developments 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 conformity 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.

XI

STADTMAN

Preface to Volume 8 This volume summarizes recent developments in several widely different areas of cellular regulation. The first article discusses the roles of multiple protein factors in the conversion of chitin synthetase zymogen to its catalytically active form, which is subject to allosteric activation by iV-acetylglucosamine and divalent cations. Activation of chitin synthetase is regarded as the first step in septum formation in the budding cycle of yeast and it is proposed that this process may serve as a molecular model for studies in morphogenesis. The mechanisms that underlie differential metabolic control of the diverse functions of glucose-6-phosphatase are discussed in the second article. The available evidence relative to the physiological significance of each function in metabolism is summarized and a possible role of glucose-6-phosphatase in glucose transport is considered. The third article is a highly provocative presentation, which summarizes the results of recent studies in Klebsiella aerogenes, showing that in addition to its role in the biosynthesis of glutamine, glutamine synthetase in this organism serves as a co-inducer or a co-repressor of the synthesis of other enzymes in nitrogen metabolism, and is an autoregulator of its own synthesis. The article on acetyl-CoA carboxylase compares a huge body of information on the physical-chemical properties of the enzymes from animals and bacteria. It is noted that different modes of regulating this enzyme in the two kinds of organisms reflect differences in the physiological role of the carboxylase reaction. In animals, the carboxylase reaction is the first committed step in the major energy storage pathway and is regulated through feedforward activation by citrate; whereas in Escherichia coli the carboxylase reaction is the initial step in the biosynthesis of membrane components and its activity is regulated by the growth modulator guanosine tetraphosphate. Other articles are concerned with the mechanisms of regulating lipogenesis in animal tissues, the deamidation of glutamyl and asparaginyl residues in peptides and proteins, and a detailed analysis of the regulation of phosphofructokinase, especially as it pertains to the Pasteur effect. BERNARD L. HORECKER EARL R. STADTMAN

xiii

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-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 XV

XVI

CONTENTS OF PREVIOUS VOLUMES

Pyruvate Dehydrogenase Complex Lester J. Reed 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 Metabolic Regulation A. Sols and R. Marco

CONTENTS OF PREVIOUS VOLUMES

XVÜ

A Discussion of the Regulatory Properties of Aspartate Transcarbamylase from Escherichia coli J. C. Gerhart Author Index—Subject Index

Volume 3

The Regulation of Branched and Converging Pathways B. D. Sanwal, M. Καφοοτ, and H. Duckworth The Role of Cyclic AMP in Bacteria Robert L. Penman and Ira Pastan Cell Surfaces in Neoplastic Transformation Max M. Burger Glycogen Synthase and Its Control Joseph Lamer 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 Exclusion Mechanisms J. M. Wiame

cerevisiae:

XVÜi

CONTENTS OF PREVIOUS VOLUMES

The Lac Repressor Suzanne Bourgeois L-Glutamate Dehydrogenases Barry R. Goldin and Carl Frieden Regulation of Fatty Acid Biosynthesis P. Roy Vagelos Kinetic Analysis of Allosteric Enzymes Kasper Kirschner Phosphorylase and the Control of Glycogen Degradation Edmond H. Fischer, Ludwig M. 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

CONTENTS OF PREVIOUS VOLUMES

XIX

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 Regulation of Histidine Biosynthesis in Salmonella

typhimurium

Robert F. Goldberger and John S. Kovach Author Index—Subject Index

Volume 6

Role of Proteases in Sporulation Roy H. Doi Regulatory Properties of Glucose-6-Phosphate Dehydrogenase A. Bonsignore and A. De Flora The Behavior of Intact Biochemical Control Systems Michael A. Savageau A Possible Role for Kinetic Reaction Mechanism Dependent Substrate and Product Effects in Enzyme Regulation Daniel L. Punch and Herbert J. Fromm Control of Biogenesis of Isoprenoid Compounds in Animals T. Ramasarma

XX

CONTENTS OF PREVIOUS VOLUMES

On Allosteric Models Jeffnes

Wyman

Regulation of Uridylic Acid Biosynthesis in Eukaryotic Cells Mary Ellen Jones Flip-Flop Mechanisms and Half-Site Enzymes Michel

Lazdunski

Author Index—Subject Index

Volume 7

Ribulose 1,5-Diphosphate Carboxylase: A Regulatory Enzyme in the Photosynthetic Assimilation of Carbon Dioxide Bob B. Buchanan and Peter Schürmann Glycolate Biosynthesis N. E. Tolbert Molecular Mechanisms in Blood Coagulation Earl W. Davie and Edward P. Kirby Enzymatic ADP-Ribosylation of Proteins and Regulation of Cellular Activity Tasuku Honjo and Osamu Hayaishi Selected Topics on the Biochemistry of Spermatogenesis Irving B. Fritz Enzyme Degradation and Its Regulation by Group-Specific Proteases in Various Organs of Rats Nobuhiko

Katunuma

Author Index—Subject Index

A Molecular Model for Morphogenesis: The Primary Septum of Yeast E N R I C O CABIB RODNEY ULANE BLAIR

BOWERS

National tabolism, and the Institute National Bethesda,

Institute of Arthntis, Meand Digestive Diseases National Heart and Lung Institutes of Maryland

Health

I. T h e Biological Problem I I . T h e Model A. Budding Cycle and Septum Formation in Yeast . . . B . T h e Bud Scar: Its Biogenesis, Architecture, and Chemical Composition C. T h e Synthesis of Chitin in Vivo D . T h e Effect of Polyoxin D on the Yeast Cell . . . . E . Septum Formation as a Model for Morphogenesis . . . I I I . T h e Enzymatic Mechanism of Chitin Synthesis . . . . A. Properties of Chitin Synthetase B . T h e Zymogen-Activating F a c t o r - I n h i b i t o r System. . IV. Intracellular Distribution of t h e Components of the Chitin Synthetase System A. T h e Zymogen B . T h e Activating Factor . C. T h e Inhibitor V. A Working Hypothesis for Septum Initiation . . . . VI. Some Final Comments References " . . . And, again, Often it matters vastly with what others, In what positions the primordial atoms Are bound together, and what motions, too, They give and get among themselves; for these Same atoms do p u t together sky, sea, lands, Rivers, and sun, grains, trees, and breathing things, But yet commixed they are in divers modes With divers things, forever as they move. Lucretius: De Rerum Natura (English translation by William Ellery Leonard) 1

2 2 2 6 7 10 13 13 13 16 23 23 24 27 27 28 30

2

ENRICO CABIB, RODNEY U L A N E , AND BLAIR BOWERS

I. The Biological Problem The generation of the myriad of structures which confer shape upon living organisms constitutes a major biological problem. Although an extensive body of knowledge has accumulated about the descriptive aspects of morphogenesis, little is known of the underlying molecular mechanisms. Reciprocally, the extensive biochemical studies undertaken during the present century have elucidated a great number of metabolic pathways, including many which lead to the biosynthesis of structural components. Nevertheless, the products have usually been obtained as amorphorus chemical compounds, not as components of an organized structure. Progress in this field would undoubtedly be greatly stimulated if adequate structural models for the study of the basic mechanisms of morphogenesis became available. What properties should we ask from such a model? Obviously, we would like it to be as simple as possible, so as to facilitate its study. However, it should not be as simple as a self-assembling structure, lest we be deprived of a glimpse of the cellular construction machinery in action. It would certainly be desirable that the structure to be studied consist of a minimal number of chemical entities, yet, it should possess a definite shape. In our hypothetical structural model two other features would be most helpful; these could be described as localization in space and localization in time. By the first we mean that the object under study should occupy only a specific portion of the cell, by the second that its formation should occur only at a specific time in the cell cycle. If both requirements were met, it would be easier to dissociate the synthesis of the model structure and its regulation from the overall process of cell growth and division. Finally, it is useful to distinguish two stages in a morphogenetic event. The first stage consists in the activation of certain genes and the consequent manufacture of the corresponding proteins through the processes of transcription and translation. The next stage consists in the organization of the synthesized proteins in order to construct a certain structure, utilizing the building blocks available in the cell. It is with this second stage that we shall be concerned here. II. The Model A. Budding Cycle and Septum Formation in Yeast During vegetative growth of Saccharomyces cerevisiae, as well as of many other yeasts (20), cell division occurs by budding. At a specific time in the cell cycle a protuberance, the bud, appears on the surface of the cell (-47). The bud progressively enlarges, while numerous orga-

A MOLECULAR MODEL FOR

3

MORPHOGENESIS

a

'%,

FIG. 1. See page 5 for legend.

4

ENRICO CABIB, RODNEY U L A N E , AND BLAIR BOWERS

FIG. 1 (continued).

See facing page for legend.

A MOLECULAR MODEL FOR

MORPHOGENESIS

5

FIG. 1. Sequence of septum formation in Saccharomyces cerevisiae X2180: (a) State preceding septum emergence; (b) primary septum grows inward; (c) primary septum is completed; (d) secondary septa are laid down on primary septum: the latter can still be seen at arrow; (e) secondary septa are completed; cells are ready to separate; (f) bud scar remaining on mother cell after separation; (g) stage similar to that of (e) above in S. cerevisiae 316 (a temperature-sensitive mutant of A364A, kindly furnished by L. H. Hartwell). The primary septum (PS) and electron-lucent lateral areas (LA) are clearly seen. The material in the lateral areas is laid down before the septum crosses the channel between cells. In succeeding figures, the bar represents 1 μΐη except where otherwise indicated.

nelles migrate into it from the mother cell: small vesicles (53), mitochondria, vacuoles (69), then part of the nucleus which proceeds to divide into two nuclei, one for each of the two cells. Finally, a septum is formed between the two cells and the plasma membranes are pinched off on both sides. A detailed view of the septal region during this process is shown in Fig. 1 and, in schematic form, in Fig. 2. Electron-transparent material can often be seen accumulating around the constriction between mother and daughter cell (46, 62). Later, the electron-lucent substance extends centripetally, giving rise to a thin disk, the primary septum (Fig. 1, a-c). After the channel between mother and daughter cell has been closed, secondary septa are laid down on the primary septum from both sides (Fig. 1, d and e). The texture of these septa is similar to that of the cell wall.*"* Finally, the two cells separate, but in an asymmetric fashion. * In the micrographs of Fig. 1 the cell wall shows relatively poor contrast. Thus, the accumulation of electron-lucent material before the septum crosses the constriction between the two cells is not apparent. The intensity of staining of the cell wall is rather unpredictable and seems to depend both on the yeast strain and on the particular preparation. An electron micrograph in which the primary septum is more clearly distinguishable from secondary septa is shown in Fig. lg, and others may be found in Marchant and Smith (4#).

6

ENRICO CABIB, RODNEY ULANE, AND BLAIR BOWERS

FIG. 2. Scheme of septum formation in budding yeast. The dotted lines represent the plasma lemma and the cross-hatched areas indicate the cell walls and secondary septa. The primary septum is drawn dark for clarity. PS, primary septum; SS, secondary septa; BiSc, birth scar; BuSc, bud scar. Reprinted, with permission, from Cabib et al. (11).

On the surface of the daughter cell only a somewhat flattened region, the birth scar (5), can be seen. A more conspicuous structure, the bud scar, remains on the surface of the mother cell (Fig. If). The bud scar resembles a shallow crater with a raised rim and a convex center. In Saccharomyces and similar budding yeasts new buds never emerge at the site of an old scar. Each scar, about 1 /mi in diameter, covers only a small fraction of the total cell surface. As many as 45 buds have been seen to emerge successively from the same cell (54), whereby a large portion of the surface is covered with bud scars. Strains with elongated shapes tend to bud preferentially in the polar regions. B. The Bud Scar: Its Biogenesis, Architecture, and Chemical Composition The simplest explanation of the asymmetry which results from cell separation is that the mother cell retains both the primary and secondary septa which go to form the bud scar; the daughter cell retains only a secondary septum, the birth scar (Fig. 2). The same interpretation was suggested by Shannon and Rothman (62), who studied the process of cell division in Candida. Indeed, in many electron micrographs, the electron-lucent material which forms the primary septum can still be seen in the bud scar after cell separation (9, 32, 62). It appears therefore that the chemical composition of the primary septum may be inferred from that of the central core of the bud scar. The presence of small amounts of chitin in the yeast cell wall, along with the major carbohydrates, glucans and mannan, was reported many

A MOLECULAR MODEL FOR

MORPHOGENESIS

7

years ago (31). Chitin is a linear polysaccharide, composed of ß-1,4" linked iV-acetylglucosamine residues. More recently Bacon et al. (1) detected a high concentration of hexosamine, presumably chitin, in isolated and partially degraded bud scars from Saccharomyces. This observation has been reinvestigated in our laboratory with the use of specific enzymes (9). The appearance of bud scar residues, after treatment with alkali and acetic acid as described by Bacon et al. (1) is shown in Fig. 3a. This material contains about 50% glucan and 15% chitin. After treatment with chitinase (Fig. 3b), the ridge of the bud scar is eliminated and the remaining wall appears to the electron-lucent lateral areas of Fig. lg. It may be concluded that a chitin disk, which still retains the circular ridge. The ridge corresponds to the electron lucent lateral areas of Fig. lg. It may be concluded that a chitin disk with an annular ridge is sandwiched between glucan layers in the bud scars. This is the expected location of the primary septum (see Fig. 2). It follows that the latter would be composed of chitin, whereas glucan would be a component of the secondary septum. Recent findings by Bauer et al. (4) and by Bush and Horisberger (7) indicate that the original bud scar also contains mannan. This polysaccharide is easily extracted by alkali and was therefore absent from our preparation. Since glucan and mannan are the major components of the cell wall, their presence in the bud scar suggests that the secondary septa are similar in composition, as they are in appearance, to the remainder of the encircling wall. Finally, it should be mentioned that upon staining with certain dyes, such as primulin (65) or Brightener (29), bud scars show a striking fluorescence, especially in the annular region. This fluorescence has been attributed to the interaction of the dye with specially oriented glucan fibrils (5) or with chitin (29). Further work is needed to clarify this point. C. The Synthesis of Chitin in Vivo If indeed chitin constitutes the primary septum, it should be synthesized during a specific period of the cell cycle, i.e., prior to cell division. In order to determine whether this was the case, a synchronous culture of Saccharomyces carlsbergensis was allowed to grow in a medium containing tritiated glucose, and the incorporation into chitin was monitored (10). Both the polysaccharide label and the number of cells increased in stepwise fashion whereas the total radioactivity in the cells increased exponentially (Fig. 4). As expected, the period of chitin synthesis preceded cell division. Since the synthesis of cell wall material proceeds throughout the cell cycle (18a) this is an additional, strong indication

8

ENRICO CABIB, RODNEY U L A N E , AND BLAIR BOWERS

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FIG. 4. Incorporation of tritium from 3 H-labeled glucose into total cell material and into chitin in a synchronous culture of Saccharomyces carlsbergensis. Arrows indicate first appearance of buds, γ , time of addition of glucose- 3 H. Reprinted from Cabib and Farkas (10), with permission of the publisher.

that the location of chitin is limited to the septal region, rather than being diffused over the whole cell wall, as once suggested by Bacon et al. (2). The correlation between the number of bud scars and the chitin content per cell reported by Beran et al. (6) also supports our conclusion, although these authors detected a small amount of chitin in unbudded scar-free cells. Our results are in conflict with those of Hayashibe and Katohda (29), who found synthesis of hexosamine from the very beginning of the budding process. However, they measured total hexosamine after hydrolysis in strong acid, whereas we used degradation with a purified chitinase followed by paper chromatography of the liberated diacetylchitobiose. FIG. 3. Effect of chitinase and glucanase treatment on bud scars of Saccharomyces carlsbergensis. (a) Bud scars after treatment of yeast with alkali and acetic acid according to Bacon et al. (1); (b) material from (a) after incubation with chitinase; (c) material from (a) after incubation with glucanase. From Cabib and Bowers (9), reprinted with permission of the publisher.

10

ENRICO CABIB, RODNEY U L A N E , AND BLAIR BOWERS

In experiments with labeled glucosamine in the growth medium we found (18a) that 65-75% of the amino sugar was incorporated into nonchitinous material, presumably mannan-protein (60, 61). With the method of hydrolysis used by Hayashibe and Katohda, this hexosamine would be assessed as chitin. The same authors also observe "Brightener" fluorescence in early budding, but the significance of this phenomenon, as indicated above, is still in doubt. D. The Effect of Polyoxin D on the Yeast Cell Polyoxins are a group of peptidyl-pyrimidine nucleoside antibiotics (36, 67) from Streptomyces cacaoi which are active against a variety of fungi. It was suggested by Sasaki et al. that their site of action was related to the biosynthesis of cell wall chitin (58). This hypothesis was confirmed in the same laboratory (15, 16, 55) when it was found that Polyoxins inhibit both the incorporation of chitin in the fungal cell wall in vivo and the activity of chitin synthetase in vitro. Applied to the fungus Cochliobolus miyabeanus, Polyoxin D caused the formation of protoplast-like structures (15). In Mucor rouxii, the growing tip of hyphae was found to burst, and both growth and spore germination were inhibited (3). In the toadstool Coprinus cinereus inhibition of elongation and development of the fruiting bodies was detected by Gooday (23). In contrast to their activity on many fungi, Polyoxins appeared to have no effect on several yeasts (36), despite their behavior as powerful inhibitors of yeast chitin synthetase [(40); see below]. Polyoxin A was also without effect on the excretion of chitin by S. carlsbergensis protoplasts (40). On the assumption that the lack of activity was due to poor penetration of the antibiotic, we submitted yeast to several treatments, such as partial drying or the addition of detergents (59) or amphotericin B (51) in order to increase permeability. Despite many such efforts no effect of the antibiotic was detected. In this series of experiments, growth media containing peptone were used. Finally, the report of Mitani and Inoue (52) on the inhibition of Polyoxin action by peptides came to our attention. With a peptide-free minimal medium for the growth of S. cerevisiae and very high concentrations of Polyoxin D, the appearance of abnormallooking cells was detected (6a). Two types of abnormality were observed. In one, a mother-daughter pair of cells suddenly lyses, spewing out the cellular contents through an orifice at the junction between the two cells (Fig. 5a). In the other, the pair becomes intensely refractile when observed by phase contrast microscope (Fig. 5b) and the connecting bridge between the two cells appears to be narrower and often much longer than normal. In some cases the refringent pair separates into two cells abutting in pointed ends, with little or no lysis (Fig. 5c). About half of the cells

A MOLECULAR MODEL FOR MORPHOGENESIS

11

FIG. 5. Phase-contrast photographs of abnormal cells in a culture of Saccharomyces cerevisiae X2180 growing in minimal medium (2% glucose, 0.7% Bacto yeast nitrogen base), in the presence of Polyoxin D (1 m g / m l ) . (a) Pair of lysed cells. Note cell material ejected at cell junction, (b) Pair of refractile cells; three normal cells are shown for comparison in the lower part of the figure, (c) A pair of refractile cells which have just separated. Note pointed ends where separation took place.

show abnormalities after growth for 2 to 3 generations in the presence of Polyoxin D at a concentration of 1 mg/ml. After growth in a medium containing tritiated glucose, it was found that the incorporation of label into chitin was about 90% inhibited in the abnormal cells (6a). The abnormal cells were also examined in the electron microscope (Figs. 6 and 7). Lysed pairs (Fig. 6a) show an orifice in the cell wall, through which the intracellular material escapes, and an abnormal septum (compare Fig. le) in which fibers appear to run parallel to the channel between mother and daughter cells; no primary septum is visible. A normal and an abnormal bud scar which happened to be close to each other are shown in Fig. 6b for comparison. The thin junction between mother and daughter cell, typical of refringent pairs under phase contrast, can be seen in Fig. 7a, whereas two refringent cells which have just separated, like those of Fig. 5c, appear in Fig. 7b. In no case is a primary septum observed. The abnormalities seem to be confined to the septal region and the remainder of the cell wall has a normal appearance. Our interpretation of the results is that Polyoxin

12

ENRICO CABIB, RODNEY U L A N E , AND BLAIR BOWERS

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MORPHOGENESIS

13

D prevents the formation of the chitinous primary septum, thus throwing the process of a cell separation into a state of disarray. Lacking the transversal primary septum, the secondary septum material, if formed at all, would be laid down in parallel fashion to the existing cell wall (see Fig. 6a). In other cells the secondary septa may be absent (Fig. 7a) or only partially formed (Fig. 7b). The cause of the lysis, which may be more or less severe (see Fig. 6a and 7a, respectively) is not clear. One explanation is that in the normal course of cell division some septal material is actually attacked by autolysins in order to facilitate separation of mother and daughter cells. The anomalous pattern of events set in motion by Polyoxin D would result in an abnormal lysis, often with disastrous consequences for the cell. E. Septum Formation as a Model for Morphogenesis From the evidence discussed in the previous sections it may be concluded that the yeast primary septum has a well-defined geometrical shape; it is a disk with a thicker annular zone. Its chemical nature is quite simple, since it consists of chitin, a linear homopolysaccharide. It is localized at a precise site in the cell, the junction between mother cell and bud, and it is formed during a specific period of the cell cycle. Thus, the primary septum appears to meet all the fundamental conditions required in a suitable system for the study of morphogenesis at the molecular level. It is pertinent now to ask how septum formation is triggered at a certain time and site by the cell. In order to seek an answer to this question it is essential to know the enzymatic components necessary for chitin synthesis and to determine their intracellular distribution. III. The Enzymatic Mechanism of Chitin Synthesis A. Properties of Chitin Synthetase The biosynthesis of chitin has the distinction of being the first case in which substantial evidence was presented for the formation of a polysaccharide from a sugar nucleotide. The original report, by Glaser and Brown (22), was concerned with the enzyme from Neurospora crassa. Later, the systems of the fungi Allomyces macrogynus (57), Mucor rouxii (49, 50), and Blastocladiella emersonii (56) were described in some detail FIG. 6. Electron micrographs of abnormal cells of Saccharomyces cerevisiae X2180 formed in the presence of Polyoxin D. (a) Pair of lysed cells, similar to that of Fig. 5a. Note irregular septum, with fibers running parallel to the constriction between mother and daughter cells, (b) A normal and an abnormal bud scar. In the normal bud scar at left the primary septum can still be seen near the surface and the clear lateral areas are also visible. The abnormal bud scar at right shows a fibrous structure similar to that seen in the lysed pair above.

FIG. 7. Electron micrographs of the junction between refractile pairs, (a) Two cells showing incipient lysis, (b) Two refractile cells after separation, similar to those of Fig. 5c. Some fibrous secondary septa seem to have been formed in these cells.

A MOLECULAR MODEL FOR MORPHOGENESIS

15

and found to have similar properties. All these enzymes are particulate, and all are activated by free iV-acetylglucosamine. In several cases activation by chitodextrins and stimulation by divalent cations (49, 56) were observed. The reaction product was chitin and in some cases also diacetylchitobiose (49, 56). We obtained a particulate preparation of chitin synthetase from both S. carlsbergensis (40) and S. cerevisiae (10) by lysis of yeast protoplasts and centrifugation of the lysate. The stoichiometry of the reaction is represented (40) by Eq. (1). nUDP-iV-acetylglucosamine + particle —> n U D P + (iV-acetylglucosamine) n — particle

(1)

The term "particle" is used to indicate an unknown insoluble acceptor. It is important to notice that the transfer of iV-acetylglucosamine from UDP-iV-acetylglucosamine to chitin may take place in more than one step, and therefore chitin synthetase may consist of a discrete number of proteins. The product was characterized as chitin from its solubility properties and from the release of diacetylchitobiose upon treatment with purified chitinase. The synthesized chitin appears to remain attached to the particle, as indicated in Eq. (1). The kinetic properties of these preparations were in general similar to those reported in the literature for other chitin synthetases (40). Thus, free iV-acetylglucosamine caused a marked stimulation, and a divalent cation (Mg2+, Mn2+, or Co2+) was required. The Km for JV-acetylglucosamine was 4.7 milf, and the amino sugar could be partially substituted by much higher concentrations of cellobiose, mannose, glucose, or glycerol. The effects were not additive. No stimulation by chitodextrins was observed with the yeast enzyme The mechanism of action of iV-acetylglucosamine and other activators, and their significance in vivo are not clear. Concentrations of free JV-acetylglucosamine at the level found effective in vitro are extremely unlikely to occur in the cell. McMurrough and Bartnicki-Garcia (49) have detected a very small incorporation of labeled iV-acetylglucosamine into chitin in the presence of cold UDP-iV-acetylglucosamine, and we have confirmed these results with the yeast enzyme (40a). It is conceivable that JV-acetylglucosamine serves as a primer, but this appears unlikely because chitodextrins are ineffective in the yeast system. Another possibility is that iV-acetylglucosamine is first transferred from UDP-JV-acetylglucosamine to an unknown acceptor, X, forming an intermediate, iV-acetylglucosamine-X. In a further step the amino sugar would be transferred to a growing chitin chain. The function of free iV-acetylglu-

16

ENRICO CABIB, RODNEY U L A N E , AND BLAIR BOWERS

cosamine might then be to prevent hydrolysis of the intermediate by a mass action effect, and the incorporation of the free amino sugar in the presence of UDP-iV-acetylglucosamine would be due to an exchange with the intermediate. In vivo the intermediate may be protected by a more hydrophobic environment, and the stabilizing effect of the free amino sugar would not be needed. Lipid intermediates have indeed been detected in many glycosyl transfer reactions (41), but there is no evidence yet that such an intermediate participates in chitin synthesis (40, 49), although the enzyme from Mucor was stimulated by a crude lipid extract (49). As mentioned in Section II, D, the Polyoxins, a group of pyrimidine nucleoside antibiotics (35), act as strong inhibitors of fungal chitin synthetases (3, 15, 16, 40, 55). The inhibition is of the competitive type, in agreement with the similarity between the structure of the antibiotics and that of UDP-iV-acetylglucosamine, as remarked by Isono et al. (35). The Ki for Polyoxin A, with the enzymes from either S. carlsbergensis (40) or S. cerevisiae (8a), is between 5 and 7 X 10~7 M, about 1000-fold smaller than the Km for UDP-iV-acetylglucosamine. The Kh values for Polyoxin D and L are similar to those for Polyoxin A (8a). B. The Zymogen-Activating Factor-Inhibitor System 1. GENERAL

During the study of yeast chitin synthetase, it was found that the supernatant fluid from a protoplast lysate contained an inhibitor of chitin synthesis (see Fig. 8). This inhibitor was found to be heat-stable and nondialyzable, and it was destroyed by incubation with trypsin. On the basis of these results it was provisionally assumed, and later confirmed, that the inhibitor was a heat-stable protein (12). Certain variations of sensitivity of chitin synthetase to the inhibitor, depending on the age of the enzyme, on the phase of growth at which yeast was harvested for enzyme preparations, and on previous treatment of chitin synthetase with small amounts of trypsin, led us to the erroneous belief that the inhibitor acted on chitin synthetase by an allosteric mechanism (12). An entirely different light was shed on these results by studying the effect of sonic treatment on the particulate chitin synthetase (10). After a short exposure of the particles to very mild sonic oscillation, the pellet which was reisolated by centrifugation (Fig. 8) was practically inactive. Incubation with the corresponding supernatant fluid restored the activity. This supernatant fluid could be replaced by crystalline trypsin, therefore all the necessary components for chitin synthesis were in the residue which remained insoluble after sonic treatment. These results were inter-

17

A MOLECULAR MODEL FOR MORPHOGENESIS Yeast cells

I

Protoplasts lysis; centrifugation

f

Supernate (Inhibitor)

1

Pellet (Zymogen + activating factor) sonic oscillation; centrifugation

f

Supernate (Activating factor)

)

Pellet (Zymogen)

FIG. 8. Scheme for the separation of chitin synthetase zymogen, activating factor, and inhibitor.

preted in the following way: the crude enzymatic preparation would contain both an inactive form of chitin synthetase, or zymogen, and an activating factor, presumably an enzyme. The sonic treatment would solubilize the activating factor, leaving the zymogen in the particulate fraction (see Fig. 8). This interpretation raised the possibility that the heat-stable inhibitor might act either by preventing activation of the zymogen or by inhibiting active chitin synthetase. The availability of zymogen and activating factor in separate fractions permitted investigation of these alternatives. It was quickly found that the inhibitor was devoid of action on previously activated chitin synthetase, but blocked completely the activation step (10). The relationship between the different components of the chitin synthetase system is shown in Fig. 9. Chitin synthetase zymogen

Activating factor

y> /N

►

Activating factor inhibitor

7

Active chitin synthetase f

n UDP-/V-acetylglucosamine + particle

11

► n UDP+ (/V-acetylglucosamine),,-particle

FIG. 9. Relationship between the components of the chitin synthetase From Cabib et al. (11), reprinted with permission of the publisher.

system.

18

ENRICO CABIB, RODNEY U L A N E , AND BLAIR BOWERS

2. T H E ZYMOGEN

When appropriate precautions are taken to prevent action by the activating factor, chitin synthetase can be isolated in an almost completely inactive form (8, 14) · The zymogen appears to be very stable. No loss of latent activity was detected when the particles were stored for 1 year at —70° in a suspension containing 33% glycerol. Even after treatment with cholate, most of the zymogen could be converted into active enzyme (11) with either activating factor or trypsin. Knowledge about the structure of the zymogen and its precise relationship to the active enzyme must await its solubilization and purification. The zymogen content of the cell appeared to be practically constant, regardless of the growth phase or of the medium used (10). It was also constant throughout the cell cycle in a synchronous yeast culture (18a). 3. ACTIVE CHITIN SYNTHETASE

The properties of active chitin synthetase have been discussed in Section III, A. However, it should be kept in mind that most kinetic data were obtained before it was realized that the system under study consisted of an inactive form of chitin synthetase plus an activating factor, and that activation proceeded during the assay. Thus, some of the data may reflect in part the activation process rather than the behavior of active chitin synthetase. Nevertheless, later experiments showed that divalent cations and iV-acetylglucosamine stimulated only the activated chitin synthetase, not the activating factor. The Km and K{ values obtained for UDP-iV-acetylglucosamine and for Polyoxins, respectively, using previously activated enzymes were identical to those obtained with earlier preparations. 4. T H E ACTIVATING FACTOR

The properties of the activating factor are those expected from an enzyme. Thus, as the result of sonic treatment of the crude particulate fraction, the activating factor becomes soluble; it can be precipitated by ammonium sulfate and dialyzed (8, 10). It catalyzes a time-dependent and concentration-dependent activation of zymogen (Fig. 10) and is inactivated by heating at 100°C (see Fig. 10). The nature of its action was assumed to be proteolytic, when it was found that trypsin could mimic its effect. The availability of an inhibitor of the activating factor helped to prove this point. It was found that the neutral proteolytic activity present in preparations of activating factor could be completely blocked by adding purified inhibitor (13). Furthermore, when the proteolytic activity and the activating effect on chitin synthetase were titrated in the

A MOLECULAR MODEL FOR

0I

0

1

10

19

MORPHOGENESIS

1

20

T

30 TIME (min.)

1

40

1

50

Π_ 60

FIG. 10. Effect of activating factor on zymogen. After incubation of zymogen and activating factor at 30°C for the times indicated, the activation was stopped by adding excess inhibitor and the amount of active chitin synthetase was measured. A A , No activating factor added; Δ Δ , 0.4 ng of inhibitor added from the beginning, no activating factor added; # # , 3.4 /^g, as protein, of activating factor added; O O, 8.4 ßg of activating factor added; □ , activating factor heated for 1 minute at 100° C before addition.

same preparation with inhibitor, essentially the same titration curve was obtained (Fig. 11). Such a result would be extremely unlikely if different enzymes were responsible for the two activities. The specificity of the activating factor is not well known, but it does differ from that of trypsin. When amounts of the two enzymes which give the same activity on the commercial substrate Azocoll were allowed to act on benzoyl arginine ethyl ester, the preparation of activating factor hydrolyzed only one-twentieth of the ester, as compared to trypsin. The activating factor is inhibited by phenylmethylsulfonyl fluoride and by p-chloromercuribenzoate (13). The amount of activating factor in the cell is greater in the stationary phase of growth than in the logarithmic phase. Its concentration is higher in cells grown in minimal medium than in those from enriched medium 5. T H E INHIBITOR

As already mentioned, the inhibitor behaves as a heat-stable protein. This property was used to advantage by extracting the inhibitor from yeast in boiling water, whereby a large initial purification was obtained. Subsequent steps included passage through a DEAE-cellulose column,

20

ENRICO CABIB, RODNEY ULANE, AND BLAIR BOWERS

100

80

o


A

Hi

•

w

"^^

»

* *

^w

m **

* #

·

·

·

A Ä

*

% m

w

% «

/

* %

· φ

#

♦ lOpfS

\

FIG. 12. A preparation of yeast vacuoles as seen under phase contrast (a) and in the electron microscope (b).

A MOLECULAR MODEL FOR

MORPHOGENESIS

27

C. The Inhibitor Extraction of protoplasts yielded even more inhibitor than did extraction of intact yeast. This result shows that the inhibitor is located intracellularly rather than in the periplasmic space (11). After lysis of protoplasts most of the inhibitor was found in the soluble fraction. A small amount present in the sediment after centrifugation may have been trapped in resealed ghosts of the protoplasts. We conclude that most if not all of the inhibitor is free in the cytosol. V. A Working Hypothesis for Septum Initiation The information gathered about the chemical composition of the primary septum, the timing and the morphological aspects of its emergence, and finally the nature and localization of the enzymatic systems involved in its synthesis, appears to be sufficient to formulate a working hypothesis for the mechanism of septum initiation. Hopefully, this hypothesis will suggest further lines of experimentation and invite comparison with other morphogenetic systems. A scheme that summarizes the proposed mechanism of primary septum initiation was presented in 1971 {10) and is shown in Fig. 13. For the reasons enumerated in Section IV, the zymogen is depicted as bound to the plasma membrane, the activating factor inside vesicles (vacuoles) and the inhibitor free in the cytoplasm. At the time of septum initiation, • Chitin synthetase zymogen ° Active chitin synthetase D> Activating factor

FIG. 13. A tentative scheme for the initiation of the primary septum. In (C) activating factor and inhibitor are shown combined in a complex. From Cabib and Farkas (10) reprinted with permission of the publisher.

28

ENRICO CABIB, RODNEY U L A N E , AND BLAIR BOWERS

FIG. 14. A possible way of obtaining simultaneous zymogen activation around the perimeter of the junction between mother and daughter cell. Same symbols as in Fig. 13.

the vesicles containing the activating factor are directed to the junction between mother cell and bud and coalesce, perhaps in a transitory way, with the plasma membrane. Zymogen and activating factor thus come in contact, activation takes place, and chitin synthesis begins. It is essential for this hypothesis that the activating factor be located inside an organelle in order to circumscribe the activation of zymogen to a precise area. It remains to be explained how the vesicles are directed to the desired spot and how the activation of zymogen takes place simultaneously in a circular area, on the surface of the channel connecting the two cells. One suggestion for the latter problem is indicated in Fig. 14. If the vesicle which carries the activating factor is larger than the channel (about 1 μΐη in diameter) and has to squeeze through in order to cross it, simultaneous contact and therefore zymogen activation around the girth of the channel would be ensured. It is not clear what role the activating factor inhibitor could have in this scheme. It is tentatively assumed that it would act as a safety mechanism to trap any activating factor released into the cytoplasm (Fig. 13) and thus prevent an undesirable initiation of chitin synthesis at other locations of the cell surface. VI. Some Final Comments It is well known to the biochemist that Nature uses a relatively small number of molecular mechanisms to obtain a great variety of effects.

A MOLECULAR MODEL FOR MORPHOGENESIS

29

Consequently, if the hypothesis we put forward is basically correct, one would expect some of its features to reappear in related systems. In a general way, activation of zymogens by proteolytic action is a well known process in biological systems, as found in gastric and pancreatic enzymes, in the proinsulin-insulin transformation and in blood clotting. The novelty in our formulation is the use of this principle to ensure localization of enzymatic action in space. Turning to organisms akin to Saccharomyces, it has already been pointed out above that the process of septum formation in Candida appears to be very similar (62). This is also the case for Metchnikowia krissii (68). In Schizosaccharomyces pombe, a fission yeast, the mechanism of cell division is apparently different. However, here too an electron-lucent primary septum is observed (37). It would be of interest to know whether this septum contains chitin, as is suggested by the appearance of a strongly fluorescent ring at the appropriate site, in the presence of primulin (66). The absence of chitin in the cell wall of this yeast could be explained by the disappearance of the primary septum in the last stage of cell division (37). An interesting observation was made by Katz and Rosenberger (39), who found that a mutant of Aspergillus nidulans, blocked in amino sugar synthesis, incorporated labeled iV-acetylglucosamine only at the growing tip of hyphae. However, if the organism was submitted to osmotic shock or if cycloheximide was added to the culture medium, incorporation occurred at all points along the hyphal wall. This result immediately suggests that chitin synthetase might have been present as a zymogen on the cell surface and that the disturbance introduced by the experimental conditions resulted in a generalized rather than local activation of the enzyme. Another system which would warrant reinvestigation is that of the water mold Blastocladiella enter sonii. In this organism the transition from zoospore to "round cell" is accompanied by rapid formation of a chitinous cell wall, without apparent need for protein synthesis (44, 56, 63). The possibility that the mechanism of septum formation in bacteria might be somewhat related to that of budding yeast has been discussed by Glaser (21) in a recent review. In this respect Fan et al. (18) found that cell wall ends of Bacillus subtilis, which initially were septa, are more resistant to the action of autolysins than the wall sides and therefore appear to be chemically different. There is, however, some indication (17) that this difference may be due to a modification of the septa after they are formed. Whether the formation of yeast primary septum represents a general-

30

ENRICO CABIB, RODNEY U L A N E , AND BLAIR BOWERS

ized mechanism for morphogenesis remains an open question. Also, it is quite possible that important parts of our working hypothesis are in error. Even in that case, however, the work described above may fulfill two different, and perhaps more important purposes: first, to show that, given a suitable model, the present state of the art permits an attack on the problems of morphogenesis at the molecular level; and second, to draw attention to the fact that the attack must be carried out with different simultaneous approaches—cellular, ultrastructural, chemical, enzymological, genetic—if a solution of the biological problem is to be achieved. REFERENCES 1. Bacon, J. S. D., Davidson, E. D., Jones, D., and Taylor, I., Biochem. J. 101, 36C-38C (1966). 2. Bacon, J. S. D., Farmer, V. C , Jones, D., and Taylor, I. F., Biochem. J. 114, 557-567 (1969). 3. Bartnicki-Garcia, S., and Lippman, E., J. Gen. Microbiol. 7 1 , 301-309 (1972). 4- Bauer, H., Horisberger, M., Bush, D. A., and Sigarlakie, E., Arch. Mikrobiol. 85, 202-208 (1972). 5. Beran, K., Advan. Microbiol Physiol. 2, 143-171 (1968). 6. Beran, K , Holan, Z., and Baldrian, J., Folia Microbiol. (Prague) 17, 322-330 (1972). 6a. Bowers, B., Levin, G. and Cabib, E., unpublished results. 7. Bush, D. A , and Horisberger, M., J. Biol. Chem. 248, 1318-1320 (1973). 8. Cabib, E., in "Methods in Enzymology" vol. 28, pp. 572-580. Academic Press, New York, 1972. 8a. Cabib, E., unpublished. 9. Cabib, E., and Bowers, B., J. Biol. Chem. 246, 152-159 (1971). 10. Cabib, E., and Farkas, V., Proc. Nat. Acad. Sei. U.S. 68, 2052-2056 (1971). 11. Cabib, E., Farkas, V., Ulane, R., and Bowers, B., in "Yeast, Mould and Plant Protoplasts" (J. R. Villanueva, I. Garcia-Acha, S. Gascon, and F. Uruburu, eds.), pp. 105-116. Academic Press, New York and London, 1973. 12. Cabib, E., and Keller, F. A., J. Biol. Chem. 246, 167-173 (1971). 13. Cabib, E., and Ulane, R., Biochem. Biophys. Res. Commun. 50, 186-191 (1973). 14· Cabib, E , Ulane, R., and Bowers, B., / . Biol. Chem. 248, 1451-1458 (1973). 14a. Cabib, E., Ulane, R., and Bowers, B., unpublished experiments. 15. Endo, A., Kakiki, K., and Misato, T., J. Bacteriol. 104, 189-196 (1970). 16. Endo, A., and Misato, T., Biochem. Biophys. Res. Commun. 37, 718-722 (1969). 17. Fan, D. P., and Beckman, B. E., / . Bacteriol. 114, 790-797 (1973). 18. Fan, D. P., Pelvit, M. C , and Cunningham, W. P., / . Bacteriol. 109, 1266-1272 (1972). 18a. Farkas, V., and Cabib, E., unpublished results. 19. Ferguson, A. R., Katsunuma, T., Betz, H., and Hölzer, H., Eur. J. Biochem. 32, 444-450 (1973). 20. Fowell, R. R., in "The Yeasts" (A. H. Rose and J. S. Harrison, eds.), Vol. 1, pp. 461-471. Academic Press, New York, 1969. 21. Glaser, L., Annu. Rev. Biochem. 42, 91-112 (1973). 22. Glaser, L., and Brown, D. H., J. Biol. Chem. 228, 729-742 (1957). 23. Gooday, G. W., Biochem. J. 129, 17P-18P (1972). 24· Hasilik, A., and Holzer, H., Biochem. Biophys. Res. Commun. 53, 552-559 (1973).

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MORPHOGENESIS

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25. Hata. T., Hayashi, R., and Doi, E., Agr. Biol. Chem. 31, 150-159 (1967). 26. Hayashi, R., Aibara, S., and Hata, T., Biochim. Biophys. Acta 212, 359-361 (1970). 27. Hayashi, R., and Hata, T., Agr. Biol. Chem. 36, 630-638 (1972). 28. Hayashi, R., Oka, Y., Doi, E., and Hata, T., Agr. Biol. Chem. 31, 1102-1104 (1967). 29. Hayashibe, M., and Katohda, S., J. Gen. Appl. Microbiol. 19, 23-29 (1973). 30. Hedrick, J. L., and Smith, A. J., Arch. Biochem. Biophys. 126, 155-164 (1968). 30a. Holzer, H., personal communication. 31. Houwink, A. L., and Kreger, D. R., Antonie van Leeuwenhoek; J. Microbiol. Serol. 19, 1-24 (1953). 32. Illingworth, R. F., Rose, A. H., and Beckett, A., J. BacterioL 113, 373-386 (1973). 33. Indge, K. J., J. Gen. Microbiol. 51, 433-440 (1968). 31 Indge, K. J , J. Gen. Microbiol. 51, 441-446 (1968). 35. Isono, K , Asahi, K., and Suzuki, S., J. Amer. Chem. Soc. 91, 7490-7505 (1969). 36. Isono, K., Nagatsu, J., Kawashima, Y., and Suzuki, S., Agr. Biol. Chem. 29, 848-854 (1965). 37. Johnson, B. F., Yoo, B. Y., and Calleja, G. B., J. BacterioL 115, 358-366 (1973). 38. Katsunuma, T., Schott, E., Elsässer, S., and Hölzer, H., Eur. J. Biochem. 27, 520-526 (1972). 39. Katz, D., and Rosenberger, R. F., J. BacterioL 108, 184-190 (1971). 40. Keller, F. A., and Cabib, E., J. Biol. Chem. 246, 160-166 (1971). 40a. Keller, F. A., and Cabib, E., unpublished results. 41. Lennarz, W. J., and Scher, M. G., Biochim. Biophys. Acta 265, 417-441 (1972). 42. Lenney, J. F., J. Biol. Chem. 221, 919-930 (1956). 43. Lenney, J. F., and Dalbec, J. M., Arch. Biochem. Biophys. 129, 407-409 (1969). 44. Lovett, J. S., and Cantino, E. C , Amer. J. Bot. 47, 550-560 (1960). 45. Manney, T. R., J. BacterioL 96, 403-408 (1968). 46. Marchant, R., and Smith, D. G., J. Gen. Microbiol. 53, 163-169 (1968). 47. Matile, P., in "The Yeasts" (A. H. Rose and J. S. Harrison, eds.), Vol. 1, pp. 219-302. Academic Press, New York, 1969. 48. Matile, P., and Wiemken, A., Arch. Mikrobiol. 56, 148-155 (1967). 49. McMurrough, I., and Bartnicki-Garcia, S., / . Biol. Chem. 246, 4008-4016 (1971). 50. Murrough, I., Flores-Carreon, A., and Bartnicki-Garcia, S., J. Biol. Chem. 246, 3999-4007 (1971). 51. Medoff, G., Kobayashi, G. S., Kwan, C. N., Schlessinger, D., and Venkov, P., Proc. Nat. Acad. Sei. U.S. 69, 196-199 (1972). 52. Mitani, M., and Inoue, Y., J. Antibiot. 21, 492-496 (1968). 53. Moor, H., Arch. Mikrobiol. 57, 135-146 (1967). 54. Müller, L, Arch. Mikrobiol. 77, 20-25 (1971). 55. Ohta, N., Kakiki, K., and Misato, T., Agr. Biol. Chem. 34, 1224-1234 (1970). 56. Plessman Camargo, E., Dietrich, C. P., Sonneborn, D., and Strominger, J. L., / . Biol. Chem. 242, 3121-3128 (1967). 57. Porter, C. A., and Jaworski, E. G., Biochemistry 5, 1149-1154 (1966). 58. Sasaki, S., Ohta, N., Yamaguchi, L, Kuroda, S., and Misato, T., Nippon Nogei Kagaku Kaishi 42, 633-638 (1968). 59. Scholz, K , and Jaenicke, L., Eur. J. Biochem. 4, 448-457 (1968). 60. Sentandreu, R., and Northcote, D. H., Biochem. J. 109, 419-432 (1968). 61. Sentandreu, R., and Northcote, D. H., Carbohyd. Res. 10, 584-585 (1969).

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62. Shannon, J. L., and Rothman, A. H., J. Bacteriol. 106, 1026-1028 (1971). 63. Soil, D. R., and Sonneborn, D. R., Proc. Nat. Acad. Sei. U.S. 68, 459-463 (1971). 64. Steinman, C. R., and Jakoby, W. B., J. Biol. Chem. 242, 5019-5023 (1967). 95. Streiblova, E., and Beran, K., Exp. Cell Res. 30, 603-605 (1963). 36. Streiblova, E., Malek, I., and Beran, K., / . Bacteriol. 91, 428-435 (1966). 67. Suhadolnik, R. J., "Nucleoside Antibiotics." Wiley, New York, 1970. 68. Talens, L. T., Miranda, M., and Miller, M. W., J. Bacteriol. 114, 413-423 (1973). 68a. Ulane, R., and Cabib, E., / . Biol. Chem., in press. 69. Wiemken, A., Matile, P., and Moor, H., Arch. Mikrobiol. 70, 89-103 (1970). 70. Yu, P.-H., Kula, M.-R., and Tsai, H., Eur. J. Biochem. 32, 129-135 (1973).

Metabolie Regulation by Multifunctional Glucose-6-phosphatase ROBERT C. NORDLIE Department of Biochemistry University of North Dakota of Medicine Grand Forks, North Dakota I. Introduction I I . Multifunctionality and Other Characteristics of the Enzyme . A. Multiplicity of Functions B. Some Relevant Properties of the Multifunctional Enzyme . I I I . Is Phosphotransferase Activity of Glucose-6-phosphatase of Physiological Significance? A. Specific Problems and Responses B. Conclusions IV. Control of Hydrolytic and Synthetic Activities of Glucose-6phosphatase-Phosphotransferase A. Glucose-6-phosphatase as a Site for Metabolic Control . . B. General Mechanisms for Control of Glucose-6-phosphatasePhosphotransferase C. Detailed, Systematic Consideration of Individual Mechanisms for Control V. Some Proposed Physiological Regulatory Roles for Synthetic and Hydrolytic Activities of Glucose-6-phosphatase-Phosphotransferase A. Proposed Roles for Phosphohydrolase and Phosphotransferase Activities in the Control of Blood Glucose Concentrations at the Hepatic Level B. Possible Involvement of Glucose-6-phosphatase-Phosphotransferase in Regulated Glucose Transport . . . . VI. A Concluding Statement Appendix References .

School

33 34 34 37 44 45 47 48 48 52 52

82

82 100 105 105 Ill

I. Introduction Glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase; EC 3.1.3.9), once believed to be a rather specific hepatic phosphatase, is now known to exhibit quite broad specificity, distribution, and multiplicity of functions. It is intrinsically much the most potent hepatic glucose phosphorylating enzymatic activity yet discovered. It is our intention in this chapter to (a) discuss briefly the multifunctional nature of this important biological catalyst, (b) consider in some 33

34

ROBERT C. NORDLIE

detail important catalytic, molecular, and biological characteristics of this complex enzyme, (c) describe a variety of new mechanisms for control of activities of the enzyme which are inherent in its multifunctional nature, and (d) consider, finally, regulation by activities of this enzyme of levels of a prime source of biological energy—blood glucose. The discussions to follow are based on experimental observations (the majority of them from our own laboratory) made primarily over the past 12 years. On the basis of these observations and other information available in the literature we will propose and discuss mechanisms for its bioregulation and also the possible physiological roles which this multifunctional catalyst may play in controlling overall body carbohydrate metabolism. It is the author's firm conviction that the biological roles for this key enzyme are much more complex than previously thought (i.e., catalysis simply of the common, terminal, hydrolytic step in hepatic and renal gluconeogenic and glycogenolytic processes). He intends to present here a synthesis of his current concepts—and experimental support for these concepts—relating to additional roles and regulatory mechanisms affecting or involving this enzyme. II. Multifunctionality and Other Characteristics of the Enzyme Experimental evidence for the multifunctional nature of glucose-6phosphatase, as well as various catalytic and biological characteristics of the enzyme, have been covered in other reviews {125-127, 134), and the interested reader is directed to these sources as supplements to the present chapter. Here, we will place emphasis on control of activities of this enzyme, and on metabolic control by these activities as they affect the relative availability of glucose within various compartments of the body. With these prime objectives in mind we will first summarize briefly the variety of reactions catalyzed by the multifunctional enzyme and those characteristics that are directly relevant to cellular regulation of and by such enzymatic activities. A. Multiplicity of Functions Glucose-6-phosphatase is now known to catalyze the hydrolysis of a variety of phosphate esters and phosphoanhydrides. In addition, the enzyme also effectively catalyzes the transfer of a phosphoryl group from such compounds to D-glucose, or with a lesser degree of efficiency, to various other sugars and polyols as well.

MULTIFUNCTIONAL

GLUCOSE-6-PHOSPHATASE

35

A representative group of reactions includes the following: * Glucose-6-P + H 2 0 -> glucose + Pi Glucose-6-P + glucose- 14 C «=* glucose- 14 C-6-P + glucose Carbamyl-P + glucose - * glucose-6-P + N H 3 + C 0 2 Carbamyl-P + H 2 0 -> Pi + N H 3 + C 0 2 PPi + glucose -> glucose-6-P -f PPj PPi + H 2 0 -> 2 Pi Mannose-6-P + glucose +± glucose-6-P -f- mannose Mannose-6-P + H 2 0 —> mannose -+- Pi A T P (or other nucleoside triphosphate) + glucose —> glucose-6-P + A D P (or other nucleoside diphosphate) PhosphoenoZpyruvate + glucose —> glucose-6-P + pyruvate A D P (or other nucleoside diphosphate) + glucose —► glucose-6-P + A M P (or other nucleoside monophosphate) Nucleoside triphosphate or nucleoside diphosphate + H 2 0 —> nucleoside diphosphate or nucleoside monophosphate + Pi

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12)

Hass and Byrne (79) and Segal {165) first demonstrated catalysis of glucose-6-P:glucose exchange [Reaction (2)] by the enzyme in 1959 and 1960. Hydrolysis of PPi [Reaction (6)] and PPi-glucose phosphotransferase activity [Reaction (5) ] were demonstrated in rat liver "particles" (mitochondria) by Rafter in 1960 (158); Nordlie and his group (131, 132, 137) and Stetten and co-workers (177, 178, 186) thereafter demonstrated the identity of these, and subsequently other similar (5, 76, 108, 133) activities with classical microsomal glucose-6-phosphatase. Most recently, potent carbamyl-P: glucose phosphotransferase which is manifest even at and above pH 7 has been demonstrated in our laboratory (24, 83, 85, 107-109). Indeed, carbamyl-P is the most effective substrate (including glucose-6-P) yet demonstrated for the enzyme. It is apparent that phosphotransferase activities of this enzyme are not simply experimental curiosities—as may well be the case for synthetic activities of nonspecific acid and alkaline phosphatases which maximally constitute but a fraction of a percentage of their hydrolytic capacity, and then only at multimolar levels of phosphoryl acceptor other than water (2, 123). For example, at pH 7, F m a x for carbamyl-P: glucose phosphotransferase [Reaction (3) ] is more than 150% of the 7 m a x for glucose-6-P hydrolysis * Abbreviations: Glucose-6-P, glucose 6-phosphate; carbamyl-P, carbamyl phosp h a t e ; PP A inorganic pyrophosphate; mannose-6-P, mannose 6-phosphate; P t orthophosphate; ACTH, adrenocorticotropic hormone; H E P E S , 2-hydroxyethylpiperazine-iV^-ethanesulfonic acid. For simplicity, the following additional abbreviations are used in the rate equations and for designation of kinetic parameters: glc, D-gluco.se; glc-6-P, glucose 6-phosphate; and CP, carbamyl phosphate. Parentheses indicate molar concentrations. Primes designate apparent Michaelis constant values (107); min = minimum, and max = maximum. Other abbreviations and symbols are those in general use, or are defined specifically where employed.

36

ROBERT C. NORDLIE

1.60 1.40 o ~

1.20

>

1.00

>

y

0.80

^ 0.60 0.40 0.20 "0

»4.0

J

5.0

I

6.0

"»«ΓΖ-ΓΊ ' - jw-i

7.0

8.0

pH

F I G . 1. Effects of p H on carbamyl-P: glucose phosphotransf erase ()> P P i : glucose phosphotransf erase ( V ) , and glucose-6-P phosphohydrolase ( □ ) activities of h u m a n liver microsomes. Phosphate substrate concentration was 10 m M ; t h a t of D-glucose was 180 m M in phosphotransf erase assays. From Herrman et al. (85) (by permission of North-Holland Publishing Co.).

(107). This remarkably high level of synthetic activity of the enzyme also is dramatically apparent from data in Fig. 1, where levels of glucose-6-P phosphohydrolase, carbamyl-P:glucose phosphotransferase, and PPi-glucose phosphotransf erase activity are compared. Apparent Kmy glucose values as low as 23 mM have been observed with such transferase activities (83). The substrates which we presently believe of greatest potential physiological relevance are utilized in Reactions (1) —(12), presented above. In addition to those compounds specifically indicated, the following also have been noted to function as phosphoryl group donors with this enzyme: fructose-6-P, CTP, CDP, deoxy-CTP, GTP, GDP, ITP, phosphoramide, and deoxy-ATP (see Section II, B, 2, b). More than 40 compounds have been found to serve as phosphoryl group acceptors (179), although only mannose and 2-deoxy-D-glucose exhibit Km values approximating those for glucose [the others are considerably higher (133)]. Glycerol is an interesting acceptor, because glycerol-1-P rather than glycerol-3-P (the product of glycerol kinase) is the product (185). The Km for glycerol is, however, exceedingly high—approximately 3 M (185). Ribitol also acts as a phosphoryl acceptor with PPi; again the Km for polyol is very large—5.6 M (183).

MULTIFUNCTIONAL

GLUCOSE-6-PHOSPHATASE

37

In our discussion of possible physiological relevance of newly characterized phosphotransferase activities of the enzyme, we will limit ourselves to water and glucose as phosphoryl acceptors, and to carbamyl-P, PPi, glucose-6-P—and to a lesser extent ATP, ADP, and phosphoenolpyruvate—as phosphoryl group donors. Subsequent developments may, of course, implicate other substrates in important physiological processes. B. Some Relevant Properties of the Multifunctional Enzyme 1. BIOLOGICAL CHARACTERISTICS

a. Location within the Cell. Once believed to be confined exclusively to endoplasmic reticulum of the cell, glucose-6-phosphatase-phosphotransferase has now been unequivocally established as present also in the outer nuclear membrane {74, 96, 139, 140), where roughly 10-15% of the total hepatic activity resides (74). The presence of the multifunctional enzyme in plasma membrane also appears likely {52, 53), and there is evidence to suggest its presence in the mitochondrial membrane as well {lJffi). b. Distribution in Tissues. The presence of the multifunctional enzyme in liver {5, 76, 108, 132, 133, 178), kidney {148, 174, 175), mucosal epithelial cells of the small intestine {113-115), and pancreas {37, 163) is established. Very recent studies in our laboratory {37, 130) also support the presence of this catalyst, with its multiplicity of activities, in adrenals, testes, and brain, and possibly in lung and spleen as well. c. Distribution among Species. Glucose-6-phosphatase-phosphotransferase appears to be ubiquitously distributed in vertebrates and invertebrates. For example, in a recent study {83), significant levels of both phosphohydrolase and phosphotransferase activities were noted in all of 21 such species studied—man, deer, ox, cat, dog, mouse, rat, sheep, guinea pig, rabbit, pigeon, duck, chicken, hummingbird, turtle, garter snake, grass frog, bullfrog, mudpuppy, catfish, and crayfish (see also Section V, A, 1, below). To date, the specific, multifunctional enzyme has not been detected in a variety of plants, bacteria, yeasts, and flagellate which have been examined {83). A number of comparative biochemical studies {5, 74, 83, 107, 132, 133) indicate a general correspondence in catalytic properties of the enzyme from a variety of sources. 2. CATALYTIC PROPERTIES

A thorough discussion of catalytic properties of the enzyme is complicated by (a) the multiplicity of reactions catalyzed, (b) the apparent partial, activity-discriminating latency of the enzyme as obtained in iso-

38

ROBERT C. NORDLIE

lated microsomal preparations (but not avian liver nuclei—see Section IV, C, 9), (c) pronounced pH-dependence of kinetic parameters pertaining to phosphate substrates, and (d) the rather complex kinetics of the phosphotransferase activities at physiological pH. In general, we will concern ourselves with the characteristics of the enzyme in its fully active form—that is, either with activated preparations obtained from isolated microsomal fraction or with certain intact nuclear preparations as noted. Very recent studies (74, 139, 140) reveal that both synthetic and hydrolytic activities of the enzyme present in the membrane of avian hepatic nuclei may be isolated with membranes intact, and are extensively or totally manifest without further treatment, in contrast with endoplasmic reticulum (which must be fragmented before isolation is possible). And the catalytic properties of the enzyme of such isolated, intact nuclei are identical with those of fully activated microsomal preparations (74, 139, 140). It is the author's current view that such properties reflect the ultimate, intrinsic catalytic capability of the enzyme. The potentially great physiological significance of intimate interrelationships which appear to exist between membrane morphology and catalytic behavior of glucose-6-phosphatase-phosphotransferase, which is a part of such membranes, will be considered in detail in Section IV, C, 9, below. a. Effects of pH. Typical pH-activity profiles for some representative activities of the enzyme, obtained with constant, relatively high levels of substrates, are presented in Fig. 1. The profiles vary dramatically and are functionally related to the nature of the phosphoryl substrate rather than the general type of reaction (phosphohydrolase or phosphotransferase) involved (145). In general, phosphoanhydride- or phosphopyruvateinvolving reactions are characterized by sharp pH optima at acid pH, with comparatively little (PPi) or no (nucleotides, phosphopyruvate) activity manifest at or above neutrality. Profiles for both phosphohydrolase and phosphotransferase reactions involving a common phosphoryl substrate are congruent (145). The rather striking differences in pH optima and complete activity-pH profiles appear related to acid dissociation constants of phosphate substrates in the vicinity of pK.A = 4 to 7 (145). An extensive, detailed mechanistic consideration of such pH effects is given in Section IV, C, 3, where the physiological significance of such substrate-discriminant differential effects of pH on activity is also discussed. b. Effects of Varied Substrate Concentrations. Typical primary kinetic plots representative of the rather complex variations of phosphotransferase activity with alterations in substrate concentrations are presented in Fig. 2, A and B. The specific studies presented were carried out with

MULTIFUNCTIONAL GLUCOSE-6-PHOSPHATASE

39

FIG. 2. Primary double-reciprocal kinetic plots obtained in studies at p H 7 of carbamyl-P: glucose phosphotransferase activity of avian liver microsomal glucose-6phosphatase. From Herrman and Nordlie (83) (by permission of Academic Press, Inc.). Key to symbols: Panel B [CP] (ml)

A •

D Δ O

0 1.0 1.3 2.0 4.0 10.0 00

Panel A K

glc

(ml) 23 29 30 31 33 37 42

[Glc] (ml) 0 20 27 40 80 200 00

KC P

(mil/) 1.5 1.8 1.9 2.0 2.2 2.4 2.6

endoplasmic reticulum preparations from avian liver (83); carbamylP : glucose phosphotransferase was assayed with such preparations of the microsomal enzyme at pH 7. As can be seen from Fig. 2, A and B, apparent Km values for carbamyl-P are directly dependent on the concentration of glucose employed. And

40

ROBERT C. NORDLIE

apparent Km values for glucose are likewise dependent upon carbamyl-P concentration. The latter values range from a minimum of 23 m l , extrapolated for concentrations of carbamyl-P approaching zero, to 42 mM for levels of the phosphate substrate extrapolated to infinity. K'm values obtained with various finite levels of second substrate are depicted in Fig. 2, A and B. The physiological relevance of these complex kinetics, relative to regulation of enzymatic activity, are considered in Section IV, C, 2, b, below. Typical kinetic parameters for a number of additional substrates are compiled in Table I. Kinetic parameters for the carbamyl-P:glucose phosphotransferase reaction with preparations from various other species are further considered in Section IV, C, 2, b. c. Effects of Inhibitors. A variety of compounds, many of them physiologically prominent metabolites, have been found to inhibit hydrolytic and synthetic activities of the enzyme. As with substrates, a detailed quantitative consideration of inhibitor effects is complicated by (a) marked pH-dependence near pH 7, and (b) modifiability of K{ values and extent of inhibition by detergents in the case of the enzyme of isolated, but otherwise untreated, microsomes. And, as with other catalytic properties, inhibitor action with the enzyme of isolated, intact avian liver nuclei resembles closely that of maximally activated microsomal enzyme and may best approximate the intrinsic properties of the enzyme in vivo (74) . TABLE I A P P A R E N T M I C H A E L I S CONSTANT V A L U E S FOR VARIOUS SUBSTRATES«

Phosphoryl donor PPi dCTP Phosphoramide 6 CTP GTP CDP ATP ITP Phosphoeno/pyruvate

K'jLmM) 1.3 2.6 2.8 3.1 3.6 3.8 4.7 6.1 6.9

Phosphoryl acceptor D-Mannose 2-Deoxy-D-glucose D-Galactose Glycerol·

K'jmM) 100 133 571 3000

° K'm values generally were determined at p H 5.5 with high, constant levels of second substrate (i.e., 180 m M D-glucose or 10 m M PPi). Partially purified rat liver microsomal enzyme preparations or rat liver microsomal suspensions activated by detergent supplementation served as enzyme source. D a t a are from the work of Nordlie and co-workers (5, 76, 132, 133), except where otherwise specified. b From Parvin and Smith (154). c From Stetten and Rounbehler (185).

MULTIFUNCTIONAL GLUCOSE-6-PHOSPHATASE

41

Inhibitors affecting the enzyme may conveniently be considered under three categories: (a) those inhibiting competitively with respect to phosphate substrates; (b) those competing with water as alternate phosphoryl acceptors; and (c) those exerting other (or undefined) types of inhibition. i. Compounds inhibiting competitively with respect to glucose-6-P or other phosphate substrate. Included in this group are the alternate phosphoryl substrates PPi (5, 132, 178), carbamyl-P (107, 108), ATP, ADP, and other nucleoside triphosphates and diphosphates {76, 133, 142); Pi (79, 197)] bicarbonate (51); molybdate (113, 132, 158, 190)) saccharin (112); cyclamate (112); certain amino acids (143, 144); citrate, isocitrate, oxalate, cyanide, azide, 1,10-phenanthroline, diethyldithiocarbamate, 8-hydroxyquinoline, and other classical metal-chelating agents (144, 146)· Observations with these latter compounds suggest strongly that glucose-6-phosphatase is a metalloprotein, and that enzyme-bound divalent cation participates in the binding of phosphoryl substrates as well as the inhibitors Pi and bicarbonate (and possibly others) (51, 76, 144y 145, 197). A mutual competition between a variety of the above compounds, and substrates, has been repeatedly observed (51, 76, 144, 145,197). The various phosphoanhydrides—PPi and nucleoside di- and triphosphates—as well as phosphoenolpyruvate, interact with the enzyme in a very complex, pH-dependent fashion, functioning both as substrates and as inhibitors (of glucose-6-P phosphohydrolase, for example) at lower, acid pH values, and as inhibitors only, competitive with glucose-6-P, mannose-6-P, or carbamyl-P, at pH 7 or 7.5 and higher (145). A detailed consideration on a mechanistic basis of these complex interactions of enzyme with such compounds is presented in Section IV, C, 3, below, where regulatory implications are also considered at length. ii. Compounds competing with water as alternate phosphoryl group acceptors. Most notable in this group is D-glucose, which actually is a better phosphoryl group acceptor than water [less than 100 mM glucose competes effectively with approximately 55.5 M water, capturing more than 50% of all phosphoryl groups initially transferred to the enzyme (107)]. A variety of other sugars and polyols also have been shown to function as alternative phosphoryl acceptors, with varying, lesser degrees of effectiveness (133, 179 183, 185), and as inhibitors of PPi-glucose phosphotransferase activity, competitive with respect to glucose (115). Structural and configurational features essential for effective binding of such compounds to the (phosphoryl)-enzyme are portrayed in Fig. 3. Hi. Other inhibitors. Inhibitions of one or both types of activity of the enzyme also have been noted with various fatty acyl coenzyme A esters (126, 142), fatty acids (126), lysolecithin (113), divalent cations

42

ROBERT C. NORDLIE

CH2OH

H O N _ — f1 °" ■3

2

FIG. 3. Structural and configurational features of aldohexose substrates necessary for efficient binding to glucose-6-phosphatase-phosphotransferase. From Lygre and Nordlie (115) (by permission of Elsevier Scientific Publishing Co.).

including Mg2+, Cu2+, Zn2+, and Ca2+ {18, 133), fluoride ion {178), bile acids and salts and other detergents (4, 11, 17, 172), various oral hypoglycemic agents {10, 91, 205), silicic acid {101), borate {214), arsenate {190), iodide ion {17), various amino acids {144), dithiothreitol (143, 144), and others. Reversible, time-dependent inhibition by a variety of classical sulfhydryl inhibitors supports the involvement of enzyme thiol group (s) in phosphate substrate binding (36). The aglycon phlorizin inhibits noncompetitively with respect to both phosphate substrates and glucose (114, 174, 214) · Its effects are highly complex, inhibition of glucose-6-P phosphohydrolase being ameliorated by the detergent cetyltrimethylammonium bromide which concomitantly potentiates inhibition by phlorizin of phosphotransf erase activity (114, 174). Although these effects are not presently understood from the mechanistic standpoint, they do point up the fact that synthetic and hydrolytic activities of the enzyme are amenable to differential, activity-discriminant regulation. A role for glucose-6-phosphatase-phosphohydrolase in glucose transport also has been suggested (127) on the basis of its presence in key tissues and its susceptibility to inhibition by phlorizin, noted both in vitro (114,174) and in vivo (174) · Representative Ki values for some inhibitors of potential physiological importance are compiled in Table II. Control of enzymatic activities through inhibitor action is considered in detail in Section IV, C, below. d. Kinetic Mechanism. Extensive kinetic studies with various activities of the enzyme (5, 79, 107, 165) support the kinetic mechanism schematically depicted in Fig. 4. This mechanism is written in terms of reaction of glucose-6-P or generalized alternate phosphoryl substrate RP with the enzyme E. The mechanism of reaction involves kinetically significant binary enzyme-phosphoryl substrate complexes E-glucose-6-P and E-RP, a common phosphoryl-enzyme intermediate E-P, and a compulsory order of enzyme-substrate interactions. Glucose-6-P or RP first binds to the enzyme (Reactions 4 and 1 of Fig. 4) to produce the relevant binary complex. Dissociation of glucose (Reaction 3, Fig. 4) or R (Reaction

43

MULTIFUNCTIONAL GLUCOSE-6-PHOSPHATASE TABLE II Ki

V A L U E S FOR SOME IMPORTANT METABOLITE

INHIBITORS"

Inhibitory metabolite

pH

Ki(mM)

D-Glucose 6 ATP Pi HC03Citrate

7.0 7.0 7.0 7.0 5.6

115 11 10.5 20 5.2

a Ki values were determined with both glucose-6-P phosphohydrolase and either PPi-glucose phosphotransferase or carbamyl-P: glucose phosphotransferase activities of either partially purified r a t liver microsomal enzyme, or detergent-supplemented microsomes from this same source. D a t a are from the work of Nordlie and co-workers {51, 107, 142, 146, 197). b Glucose-6-P phosphohydrolase, carbamyl phosphatase, or inorganic pyrophosphatase.

2, Fig. 4) produces E-P, from which the phosphoryl group is transferred alternatively to water (Reaction 5, Fig. 4) or to glucose (Reaction 3, Fig. 4) to complete the phosphohydrolase or phosphotransferase reaction, respectively.

FIG. 4. Kinetic mechanism of multifunctional glucose-6-phosphatase-phosphotransferase. R P is generalized phosphoryl substrate; E represents enzyme. Further details are given in the text. Modified from Arion and Nordlie (5) (by permission of American Society of Biological Chemists, Inc.).

44

ROBERT C. NORDLIE

Thus the sequence of Reactions 4 -f 3 -f 5, Fig. 4, depicts glucose-6-P phosphohydrolase, Reactions 1 -f- 2 + 5 describe enzymatic hydrolysis of RP, and Reactions 1 + 2 + 3 + 4 describe R P : glucose phosphotransferase activity of the enzyme. e. Chemical Nature of the Active Site. Although glucose-6-phosphatase has not yet been extensively purified, considerable insight as to the nature of functional groups at the active site involved in substrate-binding and catalysis has been obtained by indirect means. To date, the presence at the active site of protein-bound divalent cation {143, 144), imidazolium group of an enzyme-protein histidine residue {61, 146), and a thiol group (or groups) {36), appears strongly indicated by inhibitor, pH-kinetic, and more direct chemical studies; the possibility of the involvement of an e-amino group of a lysine residue also has been suggested (ref. 116, but see also ref. 60). This subject is considered further in Section IV, C, 3, a. 3. PHYSICAL PROPERTIES OF THE ENZYME

Glucose-6-phosphatase has not been purified extensively, owing chiefly to extensive problems encountered in obtaining a stable, solubilized preparation suitable for fractionation by conventional procedures {39, 127). The enzyme is intimately associated with membrane phospholipids which (a) tend to constrain activities {156, 173), while also (b) stabilizing the enzyme {34), and* (c) being essential (specifically, monounsaturated phosphatidylcholine) for activity {69). The protein itself appears to be very hydrophobic, thus also contributing to the problem of solubilization and purification. The enzyme has been described as ". . . either a part of, or extremely strongly attached to, the lipoprotein membrane of the endoplasmic reticulum" {55). Recent studies indicate the presence of both phosphohydrolase {24y 95, 96) and phosphotransferase {74, 139, 110) in the outer membrane of the nucleus, also, and perhaps in plasma membrane of the cell {52, 53) and in mitochondrial membrane (140). A molecular weight of 325,000 has been reported for the enzyme by one group of workers {176b), but this value is open to question {127). III. Is Phosphotransferase Activity of Glucose-6- phosphatase of Physiological Significance? A variety of problems have, over the past 10 years, been encountered which at the time raised serious questions as to the suitability of these activities of the enzyme for significant function under "physiological" conditions. One-by-one these difficulties have, we believe, been overcome. Some major criticisms which we and others have made, and our answers

MULTIFUNCTIONAL GLUCOSE-6-PHOSPHATASE

45

to these objections obtained through further work in the laboratory, are described below. A. Specific Problems and Responses 1. The pH optima—and, indeed, entire pH-activity profiles—(for activities with initially characterized PPi and nucleotide-involving phosphotransferase reactions) lie too far in the acidic range of pH to be of significance (133). Response: Potent, newly discovered phosphotransferase activity with carbamyl-P as phosphoryl donor is manifest over a broad range of pH ranging from the acid through neutrality and well into the alkaline (108,145).* 2. PPi may be produced in abundance in various biosynthetic processes in the cell, but it is all immediately hydrolyzed by ubiquitous inorganic pyrophosphatase action (133). Response: Carbamyl-P, a wellestablished metabolite involved in the synthesis of urea and pyrimidines, is also an excellent substrate for phosphotransferase activity of glucose6-phosphatase (83, 107-109) .f 3. The requirement (Km) for glucose is too high to permit physiological function (133). Response: Apparent Km, glucose values as low as 23 mM have been determined with carbamyl-P:glucose phosphotransferase at pH 7 (83), with carbamyl-P levels approximating the physiological levels. This value compares favorably with Km values of 10-20 mM for glucose with glucokinase (200), which does not function in the absence of insulin when hyperglycemia generally is encountered. 4. Phosphotransferase activity of glucose-6-phosphatase may be unique to rat liver (164). Response: The wide, generalized distribution of the multifunctional enzyme in various species of vertebrates and inver* Some evidence indicating appropriately acid microanatomic p H in the vicinity of the endoplasmic reticulum also has been presented (33, 150). Acidosis often accompanying the diabetic state may also serve to favor transferase activity with phosphoanhydride* substrates (125). Thus activities with nucleotides, P P i , or phosphoenolpyruvate should not, we feel, be summarily dismissed from consideration with respect to physiological function. f PPi levels as high 0.5 to 1.4 m M in liver have quite recently been reported (65); however, this value has now been adjusted downward to 6 μΜ (66). Although this latter value is quite far removed from observed Km values for P P i , it must be borne in mind that glucose-6-phosphatase-phosphotransferase is located exclusively within biological membranes, and Michaelis kinetics possibly may not be applicable (see ref. 176 for a general consideration of basic concepts involved). Consequently, specific systems for PPi production may be located in juxtaposition to, for example, glucose-6-phosphatase-phosphotransferase such that PPi formed may funnel directly from the former to the latter without exposure to, or action upon by, various other pyrophosphatases within the cell.

46

ROBERT C. NORDLIE

tebrates (83), in various tissues (37), and in several cellular structures (see Section II, B, 1, above) is now well documented. Indeed, such hepatic transferase activity appears, on the basis of presently available evidence, to be much more widely distributed in significant amounts than is glucokinase (83). 5. Phosphotransferase activity of the enzyme is latent and must be unmasked through preliminary activation of u intact microsomes" (9, 133, 172). Response: Very recent work by Gunderson and Nordlie (74) reveals that the enzyme, as it exists in the membrane of the intact, isolated avian hepatic nucleus (in marked contrast with that in recovered fragments of the endoplasmic reticulum—microsomes) is nearly totally (>90%) manifest without the need for preliminary activation of any sort. When the integrity of membranes of such nuclei is disrupted, latency develops, indicating an intimate interrelationship between membrane morphology and enzymatic behavior, and suggesting that partial latency as observed with microsomes may be largely or in part an artifact of manufacture and isolation rather than a reflection of the actual behavior of the enzyme in vivo (74). Further, a consideration of data currently in the literature would appear to predict a discriminant latency of various activities of the enzyme relative to glucose-6-P phosphohydrolase, as assayed in isolated microsomes in vitro, even though the various activities all may be relatively fully manifest within the living cell. This conclusion is based on the following evidence and reasoning: The enzyme is located within the membrane of the endoplasmic reticulum such that, for whatever reason (see ref. 127, for example; also see Section IV, C, 9, below), its various functions are vectorially oriented (181). Glucose-6-P phosphohydrolase, which acts upon intracellular glucose-6-P, is assumed to be oriented intracellularly, while certain other activities (carbamyl-P: glucose phosphotransferase, for example) function selectively toward the canaliculus of the endoplasmic reticulum, where phosphorylation of glucose, under hyperglycemic conditions, is the important physiological function (see Fig. 5). Under such circumstances, both glucose-6-P phosphohydrolase and carbamyl-P :glucose phosphotransferase would be, under appropriate physiological conditions, relatively fully catalytically operative. However, under conditions often employed for assay in the test tube, discriminant latency of such transferase activity would be predicted. When such endoplasmic reticulum is fragmented and microsomes prepared therefrom are isolated, glucose-6-P phosphohydrolase would be oriented vectorially outward (see Fig. 5) and hence relatively accessible to substrates under conditions of assay in vitro. In contrast, carbamyl-

47

MULTIFUNCTIONAL GLUCOSE-6-PHOSPHATASE (H) = Glucose-6-P

© Directed toward (H) Directed toward ENDOPLASMIC

Phosphohydrolase

Canaliculus Cytosol

RETICULUM

DISPERSED PREPARATION h B o t a nd ® © Fully Exposed to External Media

FIG. 5. Schematic illustration depicting hypothesized vectorial orientation of glucose-6-P phosphohydrolase and carbamyl-P: glucose phosphotransferase within the membrane of the endoplasmic reticulum. Because of its suggested inward orientation, such transferase activity of microsomes arising from fragments of these membranes is highly latent; dispersion by detergents or exposure to high p H dispels such latency.

P : glucose phosphotransf erase remains vectorially oriented opposite to such glucose-6-P phosphohydrolase activity (i.e., toward the interior of the microsome); hence it is relatively inaccessible to substrates under usual conditions of assay in vitro. Dispersion of such microsomal preparations with detergent (11, 17, 134, 172), exposure to high pH (180, 181, 184) or A1203 (35), or other similar treatment is essential if both such activities are to be compared under in vitro conditions (Fig. 5). In addition to the above considerations, a rather imposing body of information already exists in the literature (see Section V, A, 5, below for details) substantiating the existence of a "high i£ m " enzyme for glucose phosphorylation in addition to already characterized glucokinase. B. Conclusions We believe that phosphotransferase activities of the enzyme may play physiologically significant roles along with the more traditional glucose6-P phosphohydrolase activity of this multifunctional biological catalyst. Consequently, mechanisms for control of both such activities will be discussed concurrently in the following sections of this chapter, which concludes with two sections dealing with proposed regulatory roles for both

48

ROBERT C. NORDLIE

types of activity in the maintenance of glucose homeostasis in vivo under a variety of conditions of health and disease. IV. Control of Hydrolytic and Synthetic Activities of Glucose-6-phosphatase-Phosphotransferase A. Glucose-6-phosphatase as a Site for Metabolic Control Arguments and observations supporting glucose-6-phosphatase as an important site for the control of carbohydrate metabolism in the cell include the following: 1. STRATEGIC METABOLIC LOCATION

i. Glucose-6-P phosphohydrolase catalyzes the terminal reaction common to glycogenolysis and gluconeogenesis, both of which are extremely important processes in the production of blood glucose under appropriate circumstances (see Fig. 6). ii. In addition, glucose-6-P phosphohydrolase, along with apposing hepatic hexokinase plus glucokinase, constitutes one of the three metabolic sites involving separate enzymes for the catalysis of opposing, unidirectional reactions important in gluconeogenic and glycolytic pathways, respectively {57b, 102, 210). (Fructose diphosphatase in apposition to AMINO ACIDS ETC

V

Pyruvate Kinase

LACTATE

I

j

PHOSPHOENOL-< ■; FRUCTOSE-1,6-P2 PYRUVATE " Fructose I \ Phosphofructodiphospha- \ H \ k i n ae s Pyruvate Carboxytase \t I lase + FRUCT0SE-6-P PEP Carboxykinase Glucokinase Hexokinase

PYRUVATE

//

GLUCONEOGENESIS

GLUCOSE

GLYCOGEN

FIG. 6. Central location of glucose-6-P in carbohydrate metabolism of liver and kidney. I, II, and I I I depict the three sites which may be especially susceptible to discriminant metabolic control, since they involve opposing, unidirectional enzymatic reactions for glycolysis and gluconeogenesis (and glycogenolysis as well, for I I I ) .

MULTIFUNCTIONAL GLUCOSE-6-PHOSPHATASE

49

phosphofructokinase, and pyruvate carboxylase plus phosphopyruvate carboxykinase vs pyruvate kinase constitute the other two such sites (576, JO«, «JO); see Fig. 6.) Highly specific, discriminant regulation of enzymes involved at these sites thus would appear to be particularly appropriate, since the capability exists there for the direction and redirection of metabolic flux either toward glucose production or glucose utilization as the immediate metabolic needs of the organism may dictate {57b, 102, 210). 2. RELATIVE LEVELS OF ENZYMATIC ACTIVITIES

Two comparisons are of particular relevance in this regard: a. Glucose-6-phosphatase Compared with Other Enzymes Common to Glycolysis and Gluconeogenesis. To be of possible significance as a metabolic control point, the ambient levels of a particular enzyme must be relatively low (i.e., rate-limiting) in comparison with other, nonregulatory enzymes involved in a particular metabolic sequence. Such appears to be the case with glucose-6-P phosphohydrolase (164) · b. Glucose-6-P Phosphohydrolase Levels Compared with Those of Hepatic Hexokinase plus Glucokinase. Even more important, perhaps, than the comparison in Section a above, is the evidence indicating that under ideal, "unconstrained" conditions in vitro, observed levels of glucose-6-P phosphohydrolase markedly exceed those of hepatic glucokinase plus hexokinase (see, for example, Table I I I ) . Indeed, even with "physiological" levels of substrates, hydrolase activity exceeds that of kinase by a factor of 10 (209). The unconstrained, combined action of such hepatic kinases, Reaction (13), plus glucose-6-P phosphohydrolase, Reaction (1), thus constitutes an energetically wasteful, "futile cycle," serving simply as an ATPase [Reaction (14)] (176a). ATP + glucose

hexokinase -f glucokinase

Glucose-6-P + H 2 0

giucose-6-P

* glucose-6-P + A D P

» glucose + P ,

phosphohydrolase

ATP + H 2 0 - + A D P + P i : N e t

(13) (1) (14)

Thus, effective mechanisms for the differential control of these apposing synthetic and hydrolytic activities are a metabolic necessity* (176a). * Compartmentation within the cell may be important, to a degree at least, in precluding such "non-sense cycling." Glucose-6-P phosphohydrolase is uniquely located in the membrane of the endoplasmic reticulum and nucleus, while hepatic kinases are present either in the cytosol or mitochondria. Directive metabolic channeling thus is a distinct possibility (see Section IV, C, 8 for further consideration of this concept).

50

ROBERT C. NORDLIE TABLE III COMPARISON OF ACTIVITY L E V E L S OF G L U C O S E - 6 - P CARBAMYL-P: GLUCOSE PHOSPHOTRANSFERASE,

PHOSPHOHYDROLASE,

HEXOKINASE,

AND

G L U C O K I N A S E IN L I V E R S OF VARIOUS S P E C I E S , AS ASSAYED WITH CONSTANT SUBSTRATE CONCENTRATIONS

PRESENT"

Enzymatic activity 6

Species P h y l u m Chordata Class Mammalia Ox

Glucose-6-P phosphohydrolase

CarbamylP : glucose phosphotransferase

Glucokinase

Hexokinase

0.03-0.2 (13, 103)

0.2-0.4 (13, 103)

1.8 (13) 0.4 (13) 0.03 (13) 1.2-3.1 (13, 103, 211) 0.9-1.2 (122, 162, 166, 198) 1.3 (13) 1.3 (13) 0.4 (23)

0 . 5 (13) 0.4 (13) 0 . 1 (13, 201) 0.3-0.7 (13, 103, 211) 0.2-0.7 (13, 103, 162, 211) 0.4 (13) 0 . 3 (13) 0.4 (27,83)

0 . 1 (201)

0.4 (201)

26

31

Deer Dog Cat Sheep

23 20 14 10

31 16 16 15

Mouse

13

11

Rat

14

10

9 8 6

9 8 4

29 20 9 17

25 19 9 8

8 3

8 4

— —

— —

3 1 2

5 3 2

— — —

— — —

6

6

—

—

0.4

0.8

—

—

Guinea pig Rabbit Human0 Class Aves Chicken Pigeon Duck Hummingbird Class Reptilia Garter snake Turtle Class Amphibia Grassfrog Mud puppy Bullfrog Class Osteichthyes Bullhead P h y l u m Arthropoda Crayfish

—

— — —

—

— — —

Values for glucose-6-P phosphohydrolase and carbamyl-P: glucose phosphotransferase activity are from Herrman and Nordlie (83), and were determined at p H 7.0 and μ = 0.1 with 5 milf phosphoryl substrate and 100 m l D-glucose (phosphotransferase, only) present. Liver homogenates were supplemented with deoxycholate, to 0.2%, w / v , prior to assay. Additional details are given in Herrman and Nordlie (83). Glucokinase and hexokinase values are from the indicated literature references. A T P - M g 2 + concentrations were generally 5 m l and D-glucose either 0.5 m l (hexokinase) or 100 m l (glucokinase). Other details are given in the indicated references. Assays were at, or activities adjusted to, 30°. Activity values have been rounded for simplicity of presentation. 6 Enzymatic activity is expressed as micromoles of product formed per minute per gram wet liver. c Autopsy samples were used for assay of h u m a n liver glucose-6-P phosphohydrolase and carbamyl-P: glucose phosphotransferase (85). Activities as presented m a y thus represent minimum values.

MULTIFUNCTIONAL

GLUCOSE-6-PHOSPHATASE

51

3. MULTIPLICITY OF FUNCTIONS

Glucose-6-phosphatase is now known to possess the capacity for catalysis of potent synthesis as well as hydrolysis of glucose-6-P (see Section II, above). If both functions are to be of metabolic consequence under varying conditions (see Section V), then it is essential that they be under activity-discriminating, metabolite-effected control. When glucose-6-P hydrolysis is required, the synthetic function may be contraindicated, and vice versa. 4. EXPERIMENTAL PATTERNS OF RESPONSE TO DIETARY, HORMONAL, AND OTHER MANIPULATIONS

Patterns of response of glucose-6-P phosphohydrolase to experimental dietary and hormonal manipulations noted to date (and described in some detail in Section IV, A, 4 and C, 1, below) would appear to support the hypothesis that this enzyme constitutes an important site for metabolite control. The inherited (40, Iß, 90) or artificially induced (54) absence of this enzyme is accompanied by marked hypoglycemia, pathological accumulations of hepatic glycogen and early death. In general, those factors, hormonal or dietary, stimulating hepatic glucose production also evoke significant elevations in glucose-6-P phosphohydrolase activity, whiie contrasting counterparts of such dietary regimens or hormonal alterations that tend to lower glucose production also effectively bring about lowered levels of hepatic (and renal) glucose-6-P phosphohydrolase. Clear-cut "crossover points" in metabolic flux in isolated, perfused livers very recently have been obtained between glucose-6-P and glucose in response to experimental manipulations involving diabetes/insulin administration (56, 57a), and less pronounced suggestions of "crossovers" at this site also have been noted involving adrenalectomized diabetic and glucocorticoid-treated adrenalectomized diabetic rats {56, 57). That hepatic glycogen pools persist to a degree even in diabetes {68), and are significantly increased above normal in response to glucocorticoid therapy (68), also argue for metabolic control between glucose-6-P and glucose. 5. SUSCEPTIBILITY AND SENSITIVITY TO MODIFICATIONS BY METABOLITES

As indicated in following portions of this chapter (Section IV, C), activities of glucose-6-phosphatase-phosphotransferase are sensitive to modifications—both inhibitory and activational in nature—which may

52

ROBERT C. NORDLIE

be produced by a variety of naturally occurring cellular metabolites. Thus the potential for continuously functional regulation of activities of the multifunctional enzyme by normally present metabolites, and for the instantaneous alterations of effective levels of such activities in response to changes in concentrations of critical metabolites, is established. 6. UBIQUITOUS PRESENCE IN VERTEBRATES AND INVERTEBRATES

Hepatic (and other) glucose-6-phosphatase is widely distributed in higher animals (83). B. General Mechanisms for Control of Glucose-6-phosphatasePhosphotransf erase Although the enzyme has not been extensively purified (127), a variety of interesting mechanisms, of potential physiological significance, for the control of both hydrolytic and synthetic activities of glucose-6-phosphatase are apparent. Included are the following general types of possible control mechanisms: (1) induction and repression of enzyme synthesis through hormonal, nutritional, or other manipulations; (2) variations in intracellular substrate concentrations; (3) variations in intracellular pH; (4) product inhibition; (5) mutual inhibitions among alternate substrates; (6) metabolite inhibition; (7) activity-discriminating effects of divalent cations; (8) compartmentalization and metabolic channeling; and (9) catalytic alterations induced through membrane modifications. C. Detailed, Systematic Consideration of Individual Mechanisms of Control 1. INDUCTION AND REPRESSION OF ENZYME SYNTHESIS

In vivo responses of levels of hepatic (and in some cases renal) glucose6-phosphatase to a variety of artificially and naturally induced conditions are summarized in Table IV. Included are responses to various hormonal and dietary manipulations and drug administration, as well as an indication of variations in activity levels in developing embryos, livers of individuals affected with congenital or acquired type I glycogen storage disease, regenerating livers, tumors, and the like. In general, activity levels have been found to increase under those conditions in which accelerated hepatic glucose production is encountered, and to decrease when hepatic glucose production is diminished or contraindicated. The essentiality of this enzyme in overall metabolism is dramatically demonstrated in von Gierke's disease, where huge, pathological deposits of liver glycogen and concomitant short life expectancy are encountered in the congenital absence of glucose-6-phosphatase (40, 42). Recently,

53

MULTIFUNCTIONAL GLUCOSE-6-PHOSPHATASE T A B L E IV

SOME N O T E D R E S P O N S E S OF HYDROLYTIC AND SYNTHETIC ACTIVITIES OF H E P A T I C GLUCOSE-6-PHOSPHATASE-PHOSPHOTRANSFERASE

TO HORMONAL AND

N U T R I T I O N A L M A N I P U L A T I O N S AND O T H E R FACTORS«

Selected, illustrative literature references Manipulation, treatment, or condition A. Treatments or conditions precipitating increases in activity levels Diabetes mellitus Diabetes, experimental Glucocorticoids c (a) Administered to adrenalectomized animals (b) Administered to normal animals (c) Administered to diabetic animals Growth hormone, administered to normal animals d ACTH Glucagon Thyroxine Acute fasting 6 High-fructose diet High-galactose diet High-fat diet High-protein diet Developing fetus: Appears very early in chick embryo Appears at day 18 in r a t embryo (a) Markedly increased immediately following birth (b) Markedly increased in r a t embryo, in utero, by injection of glucagon, thyroxine, or dibutyryl cyclic A M P Ethanol administration B. Treatments or conditions involving decreases in activity levels Adrenalectomy (a) Normal animals (b) Diabetic animals

Phosphohydrolase

Phosphotransf erase

(12, 31)b (12, 81, 64, 133, 188, 147, 157)

(64, 183, 138, 147, 157)

(7, 12,31, 64, 135, 138, 147) (7, 12, 31, 64, 135, 138, 147) (157)

(7, 64, 185, 138, 147) (7, 64, 135, 188, 147) (157)

(12, 81, 38a) (12, 31) (12, 31) (12, 31) (12, 31, 38a, 64, 136, 151a, 151b) (12, 81, 25) (12, 31) (12, 31) (12, 31)

(6, 88a, 64, 136) (25)

(25)

(25)

(25, 71, 72)

(25)

(25, 71, 72) (

(25)

(71, 72)

(123a)

(12, 31, 64, 135) (12, 31, 64)

(64, 135)

(64) {continued)

54

ROBERT C. NORDLIE

T A B L E IV

(Continued) Selected, illustrative literature references

Manipulation, treatment, or condition Growth hormone, administered to hypophysectomized rats Hypophysectomy High-maltose diet Congential, genetic aberrations: (a) Von Gierke's disease (b) Radiation-induced deletion Hep atom as Induced carcinogenesis Administration of hypoglycemic agents Administration of phenobarbital

Phosphohydrolase

Phosphotransf erase

(12, 31) {12, 31, 38a) (25) (12, (54) (12, (12, (12,

(25)

31, 40, 42)

(90)

31) 31) 31)

(93)

(151b)

"Literature references are intended to be illustrative rather than exhaustive; literally hundreds of references exist documenting responses of glucose-6-P phosphohydrolase in diabetes or to glucocorticoids, for example. 6 A large number of primary references are contained in earlier reviews by Ashmore and Weber (12) and Cahill et al (31). c I t appears t h a t the principal effect of glucocorticoids is an activation of existing enzyme, rather than induced enzyme protein biosynthesis. Responses observed with unsupplemented liver homogenates largely or completely disappear when such preparations are fully activated with detergent prior to assay. Such patterns of response have been observed in the presence of actinomycin D, for example (7), and have been noted with normal, adrenalectomized, and diabetic animals (7, 135, 147, 157). d Both an increase and no change have been reported. e Responses to fasting are complex. Both activities increase exactly parallelly if liver homogenates are first fully activated prior to assay. In the absence of such activation, a significant increase only in phosphohydrolase activity is noted, suggesting t h a t synthetic activity newly induced in response to fasting m a y be masked (6, 136).

Cori and associates (54) also have demonstrated that radiation may induce a mutation in mice, which results in a deficiency or absence from the liver and kidney of glucose-6-phosphatase. This condition results in marked hypoglycemia and death shortly after birth, when the enzyme normally would appear in functional form. In both of these situations, PPi -glucose phosphotransf erase and PPi hydrolase also were absent or decreased proportionately to glucose-6-P phosphohydrolase (54, 90). Although increases in assayable levels of glucose-6-phosphatase have been noted in both glucocorticoid therapy and insulin deprivation (either experimental diabetes or acute fasting) (see Table IV), basic differences in mechanisms of response are indicated by studies from my laboratory (77, 135, 136, 138, 147). Positive responses of both hydrolase and phosphotransferase activities noted in insulim insufficiency are magnified when

MULTIFUNCTIONAL GLTJCOSE-6-PHOSPHATASE

55

F I G . 7. Illustrations of the contrasting modifying effects of detergent supplementation on responses of PPii glucose phosphotransferase activity of rat liver to experimental diabetes and cortisone therapy. Liver microsomal preparations from normal ( Δ ) , diabetic (0)> a n d cortisone-treated rats ( A ) were assayed for activity at the indicated p H value, either without (A) or with supplemental deoxycholate (to 0.2%, w / v ) added (B). From Nordlie et al. (188) (by permission of Elsevier Scientific Publishing Co.).

assays are carried out with detergent-activated microsomal preparations or liver homogenates (136, 138, 147). In marked contrast, highly significant responses to glucocorticoid administration noted with untreated microsomes or homogenates are completely, or nearly totally, abolished when such enzyme preparations are first activated with detergents prior to assay (135, 147) (see Fig. 7). The latter response pattern is demonstrable even in actinomycin D-treated animals (7) ; and it can be superimposed upon diabetic as well as normal subjects (157). It thus appears that while induction of synthesis of new enzyme protein may be involved in the response of this enzyme to insulin insufficiency, glucocorticoid administration may involve principally or exclusively an activation of existing enzyme molecules rather than induced protein synthesis (7). This subject also is discussed further in Section IV, C, 9, below. Because of the sluggishness in response of glucose-6-phosphatase levels to glucocorticoid therapy, or to insulin administration to diabetic animals, compared with the relatively rapid changes in blood glucose levels (see Fig. 8, A and B), it appears that alterations in effective enzyme levels serve for long-term readjustments in metabolism and that other mechanisms for more immediate, highly sensitive regulation of glucose-6phosphatase activity must also be operative. Possible such mechanisms are considered in succeeding sections, below. Greengard (71, 72) has concluded from embryological studies that 3',5'-cyclic AMP (cAMP) acts as a "second messenger'' in "turning on" glucose-6-phosphatase activity in rats shortly after birth, in response to epinephrine or glucagon.

56

ROBERT C. NORDLIE

A. |L ^Blood Glucose Cone. ( a ) \ Glucose-6-P • Λ^ · · ··.../Phosphohydrolase(b)

Gluconeogenic R a t e ( c )

i

Glucose-6-P Pnosphohydrolase(b)

12 24 36 48 6 12 18 24 0 HOURS AFTER HOURS AFTER HYDROCORTISONE INSULIN FIG. 8. Changes with time of blood glucose levels, liver glucose-6-P phosphohydrolase activity, and hepatic gluconeogenic rate after (A) administration of insulin to diabetic rats, or (B) administration of cortisone to adrenalectomized-diabetic rats. Note that the changes in blood glucose concentrations and hepatic gluconeogenic rates precede apparent alterations in levels of the liver enzyme. From Cahill et al. (31) by permission of Dun-Donnelley Publishing Co.

Differences in the need for carbohydrate production in the developing avian embryo as compared with the mammalian embryo are dramatically apparent from comparative developmental studies in which glucose-6phosphatase levels were measured {25, 123b). In the bird embryo, which develops independently of the mother, glucose production in early stages is essential, and glucose-6-phosphatase activity is detectable very early during incubation of domestic chicken eggs {24, 98). In contrast, the rat embryo utilizes glucose from the mother's blood, and does not manifest significant glucose-6-phosphatase activity until immediately postpartum {25, 71, 72). The potential for this activity is present in the embryo, however, and administration of dibutyryl-cAMP to the fetus, in utero, leads to rapid appearance of glucose-6-phosphatase activity {71, 72). In contrast to glucose-6-phosphatase-phosphotransferase which normally appears at birth in rats, glucokinase is not demonstrable until approximately 16 days postpartum {199). The significance of this observation will be considered further in the last section of this chapter. In well developed hepatomas, glucose-6-P phosphohydrolase is undetectable {204) · To this author's knowledge, no attempt has yet been made to measure phosphotransferase activities of this enzyme in tumors. Conceivably, these activities, in direct contrast with phosphohydrolase, may persist consistent with the reversion to a purely glycolytic state as the tumor develops. 2. EFFECTS OF VARIATION IN SUBSTRATE CONCENTRATIONS

a. Glucose-6-P Phosphohydrolase. The Km value for glucose-6-P, generally reported to be between 1 and 2 m l at physiological pH {5, 77,

MULTIFUNCTIONAL

GLUCOSE-6-PHOSPHATASE

57

107, 132), is far removed from the probable hepatic cellular concentration of glucose-6-P, which has been calculated to be between 0.05 and 0.13 raM if it is assumed to be homogeneously distributed in cell water constituting 60% of cell mass (see refs. 142, 197). Thus the ratio, cellular glucose-6-P concentration/Km value for glucose-6-P, is between 0.025 and 0.13, based on available information.* Two very interesting implications with respect to physiological control mechanisms are immediately apparent from this observation. First, enzymatic activity should be highly responsive to variations in substrate concentration in the physiological range (which is much below Km values); and second, glucose-6-P phosphohydrolase should be especially susceptible to competitive inhibition by various metabolites (alternate substrates, Pi, bicarbonate, etc.; see Section IV, C, below). This sensitivity to variations in substrate concentration parallels that of the apposing, phosphorylating glucokinase for its substrate, glucose. And the same is also true of phosphotransferase activities of glucose-6phosphatase (see below). b. Phosphotransferase Activities of Glucose-6-Phosphatase. Three interesting mechanisms for control of phosphotransferase activity associated with variations in substrate levels are suggested by the experimental data. Typical Lineweaver-Burk (104) double-reciprocal plots obtained in the study of the kinetics of carbamyl-P: glucose phosphotransferase activity of chicken liver at pH 7 are indicated in Fig. 2, A and B. In these studies, the effect of varied carbamyl-P concentration was investigated at several constant concentrations of glucose (Fig. 2A), and, similarly, variations in velocity as a function of alterations in glucose concentration were assessed at several constant levels of carbamyl-P (Fig. 2B). Various kinetic parameters thus determined for a variety of species are given in Table V. Of particular relevance is the observation in these studies, carried out at pH 7, that apparent Km values for glucose are considerably smaller than previously (5, 77, 132) determined for PPi-glucose phosphotransferase activity at lower pH. One earlier obstacle to the establishment of the feasibility of physiological phosphorylative functions for phosphotransferase activity of this enzyme thus appears to have been resolved. A detailed, quantitative consideration of this subject is given in Section V, A, below. * Because of the membrane-bound nature of the enzyme, kinetic parameters can only be considered to be apparent. However, values discussed here and below are in general (unless otherwise specified) those determined with maximally activated microsomal preparations, or with untreated nuclear preparations which are insensitive to detergents, etc. Values obtained with these two preparations agree well and may reflect the ultimate intrinsic catalytic properties of the enzyme.

58

ROBERT C. NORDLIE

TABLE V A P P A R E N T M I C H A E L I S CONSTANT V A L U E S FOR C A R B A M Y L - P AND D-GLUCOSE, D E T E R M I N E D FOR A N U M B E R OF S P E C I E S "

"Second substrate" and concentration (ml)

Apparent K m values determined for indicated species ( m l ) Ox

Mouse

Guinea pig

Rabbit

Apparent Km values for glucose Carbamyl-P 0 1.0 1.3 2.0 4.0 10.0 OO

33 37 39 42 48 58 86

31 35 36 38 43 49 60

29 35 37 40 47 60 82

27 33 36 39 46 58 90

Apparent Km values for carbamyl-P D-Glucose 0 20 27 40 80 200 oo

3.0 4.0 4.2 4.8 5.7 7.0 8.4

2.8 3.4 3.6 4.0 4.3 4.9 5.2

3.8 4.8 5.1 5.5 6.4 7.2 8.9

2.4 3.3 3.6 4.1 4.9 6.0 8.1

α D a t a are from the work of Herrman and Nordlie (83). Liver microsomal preparations, supplemented to 0.2%, w / v , with sodium deoxycholate, were employed as enzyme source. Assays were at p H 7.0 and μ = 0.1. Additional details are given in Herrman and Nordlie (83).

The first of the interesting possible control mechanisms apparent from these data (Fig. 2, A and B; Table V) relates, as in the case of glucose-6-P phosphohydrolase activity discussed above, to the apparently great discrepancy between Km or apparent Km values for glucose and physiological blood sugar levels (note that free permeability of glucose to hepatic cells (31) is relevant in this regard). K'm values for glucose range between approximately 23 m l (low carbamyl-P concentrations) and 50 mTIf (infinite carbamyl-P levels), while normal blood glucose levels in man or rat approximate 100 mg/100 ml (slightly in excess of 5 m l ) , postabsorptively. Thus the ratio, glucose concentration: Km(glucose) or apparent Km(glucose), is between approximately 0.1 and 0.2 under normal circumstances, but may be equal to or even exceed a value of 1 in the diabetic animal. Again, this system should be highly sensitive to variations in glucose concentration, a characteristic also manifested by the established hepatic regulatory enzyme glucokinase for which a Km value for glucose of approximately 10-20 m l has been found (47, 199, 200).

MULTIFUNCTIONAL GLUCOSE-6-PHOSPHATASE

59

Second, this activity, and also glucose-6-P phosphohydrolase, should be highly susceptible to the action of inhibitors competitive with glucose; this susceptibility should be manifestly less in the hyperglycemic state. The third possible mechanism for metabolic control, apparent from Fig. 2, A and B, and Table V, relates to the relationship between "second substrate"* and apparent Km values for the first substrate. As glucose concentration is increased, the K'm value for carbamyl-P also increases (see Fig. 2A); likewise, as carbamyl-P concentration is raised, apparent Km for glucose increases. In both cases, apparent maximal reaction velocity (attained with finite concentration of one substrate but extrapolated to infinite concentration of second substrate) rises directly with elevations in the level of first substrate. Thus we have a system in which increases in substrate levels lead to an increase in the velocity term, but at the same time decrease the apparent affinity (i.e., increases Km values). This property would serve to constrain (i.e., dampen) to a degree the extent of response of enzymatic activity to rapid changes in intracellular substrate concentrations and would appear to fit a key homeostatic, regulatory enzymatic activity. 3. EFFECTS OF VARIATIONS IN P H

Activity-pH profiles for a variety of synthetic and hydrolytic activities of glucose-6-phosphatase-phosphotransferase are displayed in Fig. 9. Even from these studies (145) carried out at relatively high substrate * As employed here, "second substrate" and "first substrate" do not relate to the order of enzyme-substrate binding, but simply to the two substrates involved in any given transferase reaction of the enzyme.

LU

> ^ z I

CO

8 2

*

9 6 3 0 ~

5

■ -

6

■

- ■

7 8 9 pH F I G . 9. Comparison of pH-activity profiles of glucose-6-P phosphohydrolase ( O ) , carbamyl-P: glucose phospho transf erase ( f ) , P P i : glucose phospho transf erase ( ♦ ) , and ATP:glucose phosphotransferase ( Δ ) . Phosphate substrates were 5 m l , and glucose concentration was 120 m M in phosphotransferase assays. From Nordlie {127) (by permission of Academic Press, Inc.).

60

ROBERT C. NORDLIE

concentrations, it is clear that variations in intracellular pH may provide a mechanism for discriminating amongst the various activities of the enzyme. For example, reactions involving glucose-6-P, mannose-6-P, and carbamyl-P as substrates (either hydrolytic or synthetic activities) are manifest over relatively wide ranges of pH varying from acid through neutrality and well into the alkaline range. In contrast, activities involving phosphoanhydrides or mixed phosphate anhydrides (PPi, nucleoside tri- and diphosphates, phosphoenolpyruvate) peak near pH 5.5 and thereafter drop extremely rapidly as pH approaches neutrality (145). No activity is discernible with nucleotides or phosphopyruvate at or above pH 7, while PPi -involving activities are significantly diminished between pH 6 and 7.5 and cannot be detected at pH 8 or above. From the physiological point of view, this difference in the effects of pH on activity levels may be significant in that, as diabetes develops, glucokinase disappears (47, 198, 199) and acidosis concomitant with the diabetic state may provide for the increasing importance of possibly compensatory PPi-glucose (or ATP-glucose) phosphotransf erase as a mechanism of hepatic glucose phosphorylation. In direct contrast with their diminished or total inability to function as substrates as pH increases in the vicinity of neutrality, phosphoanhydrides (and phosphopyruvate) remain highly capable of inhibiting, in a manner competitive with respect to phosphate substrates, other activities of the enzyme which are manifest at pH 7 and above (14$) · "Nature [thus] has provided a most interesting and ingenious means of defining precisely discriminant roles of various metabolically prominent phosphate compounds with respect to their several modes of interaction (participation as substrates, inhibitors, or both) and glucose-6phosphatase-phosphotransferase in a highly critical range of pH" (14$) · Mechanistic Considerations. A direct correlation between certain acid dissociation constants for phosphate compounds and their ability to function as substrates is apparent (14$)· A proposed mechanism of enzyme action (Fig. 10) incorporates as functional groups in the active site enzyme-bound divalent cation (143, 144), histidine imidazolium group (60, 67, 146)i and enzyme thiol (36) and is totally consistent with the kinetic mechanism of the enzyme previously formulated by Nordlie and coworkers (5, 107) on the basis of extensive studies of the various activities of this complex system (see Fig. 4). On the basis of pH kinetic studies, Nordlie and Lygre (146) first implicated an imidazolium group of enzyme-bound histidine in the catalytic mechanism of this enzyme. Parvin and Smith (154) and Feldman and Butler (59, 61) later demonstrated that enzyme-bound 32P-labeled histidine [iV-3-phosphorylhistidine (61)] could indeed be formed from 32 P-

MULTIFUNCTIONAL

GLUCOSE-6-PHOSPHATASE

61

FIG. 10. Proposed mechanism of action for multifunctional glucose-6-phosphatasephosphotransferase, based on currently available information. Details are presented in the text, and phosphate-substrate-discriminant effects of p H are rationalized there on the basis of this mechanism which involves, as substrate-binding sites, enzyme-bound divalent cation, thiol group, and imidazolium group of histidine.

labeled glucose-6-P, PPi, or phosphoramidate and microsomal preparations, and produced evidence that this is an intermediate in glucose-6phosphatase action. Feldman and Butler (61) also presented evidence that the free energy of hydrolysis of this phosphoryl-enzyme intermediate

62

ROBERT C. NORDLIE

is low enough so that formation from enzyme and glucose-6-P (as well as "high-energy" phosphate anhydrides) is entirely feasible. The placement of an enzyme-bound divalent cation at the enzyme's active site, and its proposed role in the initial binding of phosphate substrates, is based on a variety of observations by Nordlie and Johns {14%, 144)) who studied extensively the kinetics of inhibition by a variety of metal chelating agents. Thiol group involvement is supported by recent pH-kinetic and sulfhydryl-reagent inhibition studies of Colilla and Nordlie (36). This mechanism, as postulated in Fig. 10, involves initially the binding of the phosphate-substrate to enzyme-bound divalent cation (I - » I I , Fig. 10). This stabilizes the doubly-dissociated organophosphate anion and increases the electronegativity of the bridged oxygen; then through a subsequent series of proton transfers (see II, Fig. 10), the enzyme histidine residue is left as a transient imidazole. This transient nucleophile attacks the electrophilic phosphorus (II, Fig. 10) to form iV-3-phosphohistidine (III, Fig. 10), which as Bruice and Benkovic (28) have pointed out, is, like creatine phosphate, an excellent phosphoryl donor. Such zwitterion compounds as creatine phosphate and iV-3-phosphohistidine are very susceptible to nucleophilic attack by water and alcohols (28), and, indeed, nucleophilic attack by phosphoryl acceptors [R'-OH, which may be either water, glucose, or other hexose or polyol (5, 107, 132) ] is postulated to occur next (IV). Finally, the reaction process is completed through a series of proton transfers (IV) (analogous to the reversal of II -^ III) followed by dissociation of the product Pi, glucose-6-P, or other phosphate ester ( I V - > I ) . Correspondence of the individual reaction steps in Fig. 10 with those in the general kinetic mechanism proposed by Nordlie and co-workers (see Fig. 4) is as follows: For R-P:glucose phosphotransferase (where R-P represents generalized phosphoryl donor), reaction I -> II corresponds with Reaction 1 in Fig. 4, II -» I l i a with Reaction 2; I l l b -> IV with reversal of Reaction 3; and IV -» I with reversal of Reaction 4. For glucon-6-P phosphohydrolase, reaction I ~> II corresponds with Reaction 4 in Fig. 4; II —» I l i a with Reaction 3; and I l l b -» IV -> I with Reaction 5. And for R P hydrolysis, I -> II corresponds with Reaction 1, Fig. 4; II -> I l i a with Reaction 2; and I l l b -» IV -> I with Reaction 5. A key step in the overall mechanism depicted in Fig. 10 involves nucleophilic attack by histidine ring nitrogen on phosphoryl phosphorus (see II, Fig. 10). Since earlier studies (146) have shown that the imidazolium ion—an electrophile, per se—was the active species, it was necessary that any proposed mechanism involve its conversion to a good nucleophile, by abstraction of an imidazolium proton, prior to formation

MULTIFUNCTIONAL

GLUCOSE-6-PHOSPHATASE

63

of iV-3-phosphohistidine. As indicated in II, Fig. 10, this may be achieved by a concerted mechanism using the unpaired electrons of the bridged oxygen of phosphate substrate and the sulfur atom of enzyme-bound thiol. This portion of the proposed mechanism is patterned after the mechanistic concepts suggested by Watts and Rabin (202) regarding creatine kinase. In this latter case, abstraction of an active proton of the guanidinium ion to produce a guanidine group through a concerted sequence of interactions at the active site of the enzyme analogous to those described here involving histidine, is proposed as a requisite step preliminary to phosphoryl transfer to enzyme-bound creatine. Participation of enzyme imidazolium and thiol groups in enzymatic phosphoryl transfer, as outlined in Fig. 10, is similar in many aspects to the recent proposal by Horecker, Tsolas, and Lai (87) with respect to the cleavage of fructose diphosphate by muscle aldolase. A key feature of the mechanism proposed in Fig. 10 thus is the conversion of the imidazolium group of enzyme-bound histidine to a transient imidazole—a good nucleophile—thus permitting attack by histidine N on phosphoryl P, a necessary prerequisite to phosphoryl-enzyme formation. On the basis of this and other concepts incorporated into this scheme, a variety of unique, pH-, and substrate-discriminant modes of interaction of a variety of phosphate compounds with this complex system may be rationalized. It previously has been pointed out (145) that "increased charge density in the vicinity of the terminal phosphoryl group of PPf - , A D P 3 - , or ATP 4 ~, for example, appears in some manner to preclude further reaction requisite to the genesis of the phosphoryl-enzyme intermediate from initial binary complexes . . . ." According to the mechanism outlined in Fig. 10, compounds such as hexose phosphates and carbamyl-P should remain active even when fully ionized, as the maximum of two negative charges accumulating on their phosphoryl groups would be effectively masked through interactions with enzyme-bound divalent cation. In contrast, an increase in negative charge density about the phosphoryl phosphorus atom, which would result as phosphoanhydride compounds further dissociated (i.e., PP?" -» H+ + PPf"; A D P 2 - -> H+ + A D P 3 - ; ATP 3 " -» H+ + ATP 4 "; etc.) between pH 6.5 and 7,* could not be thus masked, and such an increase in negative charge density about phosphoryl phosphorus would preclude the nucleophilic attack by histidine nitrogen on the subject phosphorus atom (see II, Fig. 10) and thus prevent overall activity. These ionic species of phosphate anhydrides would, however, remain effective competitive inhibitors of glucose-6-P, * pK& values for the designated dissociations of PP? - , ADP 2 - , and ATP 3 - are, respectively, 6.9, 6.4, and 6.5 (see refs. 22, 208).

64

ROBERT C. NORDLIE

carbamyl-P, mannose-6-P, etc. hydrolase and phosphotransferase activities, since they are capable of initial binding to the metal ion of the active enzymatic site (I —> II, Fig. 10) although not of reacting further. The presence of a negatively charged dissociated carboxyl group of phosphoenolpyruvate in addition to the doubly dissociated phosphoryl group of this compound would appear to preclude net reactivity at pH ^ 7 in a similar manner. The experimental observation that Pi inhibits, competitively with respect to phosphate substrates, both phosphohydrolase and phosphotransferase reactions of the enzyme (ref. 197; also see Section IV, C, 4, b, below), although 32Pi does not exchange with cold glucose-6-P in the presence of the enzyme (154) nor can incorporation of isotope from labeled 32Pi into enzyme protein-bound JV-3-phosphohistidine be demonstrated (59, 61), may also be resolved on the basis of the above concepts. In this case, R' of generalized compound O

II

R'— O—P— O-

I oin Fig. 10 (between IV and I) would represent H. Reversal of IV —> I is possible, as indicated by observed competitive inhibitions by Pi of various activities of the enzyme. However, when R' in IV is H, further reactivity leading possibly to generation of enzyme-phosphohistidine is precluded; the presence of such a proton attached to the bridge oxygen of this compound prevents necessary interaction of the proton of the enzyme-bound sulfhydryl with this same bridge oxygen atom, thus making impossible further reaction. 4. PRODUCT INHIBITION

Both of the products of glucose-6-P hydrolysis are effective inhibitors of enzymatic activity. And the inhibition by glucose of phosphohydrolase activity (5, 79, 107, 132, 165) and by Pi of both phosphohydrolase (18, 79, 197) and phosphotransferase (197) has been studied in detail. a. Inhibition by Glucose. As indicated in Figs. 4 and 10, glucose functions as an alternate phosphoryl acceptor competitively with respect to water. Thus reduction of net rate of hydrolysis of glucose-6-P is actually a manifestation of glucose-6-P: glucose phosphotransferase activity [Eq. (2) ]. Autoregulation of net release of hepatic glucose based on this mechanism has been suggested (31, 120, 121, 125-127, 133) ; as blood glucose levels rise, inhibition of glucose-6-P hydrolysis increases concomitantly and hyperbolically. The extent of inhibition by various concentrations of glucose-6-P hydrolysis is readily apparent in the illustrations which are presented and discussed in detail in Section V, A, below. It is obvious

MULTIFUNCTIONAL

GLUCOSE-6-PHOSPHATASE

65

from data in these diagrams (see Section V, A) that the system is highly sensitive to variations in glucose concentrations encountered under normal, physiological conditions, as well as in diabetes. b. Inhibition by Pi. Inhibition by Pi of both phosphohydrolase (18, 79, 197) and phosphotransferase reactions (197) of microsomal preparations is highly detergent-sensitive (ref. 197; see Fig. 11) and has the potential to exert highly significant control at physiological levels. For example, assuming physiological levels of glucose-6-P and Pi to be 0.13 m l and 10 m l , respectively, Km for glucose-6-P = 1 . 8 mM, and Κχ for Pi as 10.5 mM (see Appendix for details and references), it may be calculated that 47% inhibition of phosphohydrolase activity will result. It has been suggested (197) that control through Pi may become increasingly important in diabetes, where Pi levels increase approximately 50% (161). Added control of increased absolute levels of glucose-6-phosphatase encountered in diabetes thus is assured (77, 83, 133, 147, 197). The significance of this observation will be considered in more quantitative detail in the last section of this chapter. Certain control features involving Pj along with ATP and ADP are considered in the following section. 5. MUTUALLY COMPETITIVE INHIBITIONS AMONG PHOSPHORYL SUBSTRATES

As depicted in Figs. 4 and 10, various phosphate substrates should compete with one another for the active enzymatic site. Such mutually competitive patterns of inhibition among various combinations of phosphoryl substrates, in pairs and combinations of higher numbers, have been demonstrated experimentally (132, 145, 148, 197). And as pointed out in Section IV, C, 3, above, such competitions are observable even at higher pH values where phosphoanhydrides are poorly or totally nonfunctional as substrates. Such inhibitions with untreated microsomal suspensions (but not with nuclei) are highly detergent-sensitive (see Fig. 11). Stadtman (176a) previously has pointed out that the concerted action of hexokinase and glucokinase [Eq. (13)] plus glucose-6-P phosphohydrolase [Eq. (1)] constitutes a metabolically pointless, energetically wasteful drainage of potential chemical energy, the net result of which is the hydrolysis of ATP [Eq. (14)] (see Section IV, A, 2, b, above). It is now apparent that not only can ATP serve as initial phosphoryl donor in this multienzyme process, but also it can prevent the wasteful cycling by limiting the hydrolysis of glucose-6-P (142). Further, increased inhibition of glucose-6-P phosphohydrolase by a combination of ADP plus Pi, which would tend to accumulate at the expense of energy-rich ATP under conditions of accelerated gluconeogenesis, may serve to coordinate the rates of the energy-requiring gluconeo-

66

ROBERT C. NORDLIE UJ

o

80

·#--·- -·

70 60

ω

50

-

ι

40

-.£

^D—

30

X

P -er'

g

20

CO

10 1

1

1

1

I 0 0.02 0.040.06 0.08 0.10 I

Π0

I

I

I

L

1.0 2.0 3.0 4.0 DETERGENT CONC.

F I G . 11. Modifying effects of detergents on inhibition by A T P ( · , O , D ) or Pi ( ♦ ) of glucose-6-P phosphohydrolase at p H 7.5. Glucose-6-P concentration was 0.13 m l , that of A T P 6 m l , and Pi 10 m M . Palmityl coenzyme A ( □ ; axis of abscissas I I ) , lysolecithin (> ♦ ; a x i s II), or cetyltrimethylammonium bromide ( # ; axis I) were included in rat liver microsomal preparations at the indicated levels. Axis of abscissas I indicates %, w / v , of the detergent; I I indicates 105 X molar concentration of indicated detergent. From Nordlie et al. ill+2) (by permission of National Academy of Sciences). / TCA^ XYCLE/ T6ADP 6ADP '1+5 Pi ♦ 5Pj

Glucokinase,

ATP+ GLUCOSE

" — " · ^—> ADP

Λ....Ο bA,K

\"^^L

ESIs]I !LACTATE GLUCONEOGENESIS (GLC-6-p)

Kinase

GLUC0SE-6-P Glycolysis GLYCOGEN

F I G . 13. Proposed mechanism explaining the ability of bicarbonate to promote glycogen accumulation in liver slices. Inhibition by HCO^" of glucose-6-P phosphohydrolase and glucose-6-P dehydrogenase tends to divert glucose-6-P away from competing pathways and hence toward glycogen synthesis. Activation by HCO^" of glucose dehydrogenase activity of glucose-6-P dehydrogenase provides for continued generation of N A D P H under such conditions. Further details are given in the text.

rate of flow toward liver glycogen formation might then reasonably be expected in the presence of this anion. 7. ACTIVITY-DISCRIMINATING EFFECTS OF DIVALENT CATIONS

The general metabolic directive effects of various cations have received considerable recent attention. Various studies in our laboratory indicate interesting findings of considerable potential physiological significance relating to the effects of various divalent cations on hydrolytic and synthetic activities of glucose-6-phosphatase-phosphotransf erase. For example, various levels of Mg2+, as well as 6 vnM levels of Ba2+, Mn2+, Co2+, Ca2+, and Hg2+, have been found to inhibit CTP-glucose phosphotransferase activity extensively (133). Recent studies (92) indicate inhibition by Mg2+ of carbamyl-P:glucose phosphotransferase; glucose6-P phosphohydrolase,* however, was not affected by Mg2+. All these studies were carried out at constant ionic strength (μ = 0.1) by including relevant cation in assay mixtures along with substrate, buffer, supplemen* Various textbooks {117, 209) indicate that Mg 2+ is a required activator for 6-phosphatase. A thorough search of the literature by this author failed to any primary reference supporting this contention, however, and our own with a variety of enzymatic preparations indicate that added Mg 2+ is effect on such hydrolytic activity of either dialyzed or undialyzed preparations.

glucoseproduce studies without enzyme

MULTIFUNCTIONAL

GLUCOSE-6-PHOSPHATASE

69

tary NaCl, and enzyme. In other, complementary studies, preincubation of enzyme with Mg2+ prior to assay produced no detectable inhibition of phosphohydrolase activity, suggesting that this ion inhibits (when it does) by chelating CTP, carbamyl-P, PPi, or like substrate (but not glucose-6-P, for which it has a relatively lesser affinity), rather than by interacting with the enzyme per se. Further interesting studies by Mr. Thomas Johnson in our laboratory (92) indicate small but reproducible (25%) activation of phosphotransferase and inhibition (15%) of phosphohydrolase activities following preliminary incubation of the enzyme with relatively low (0.15 mM) levels of Cu2+. From these and other studies, it appears that certain divalent cations may exert a selective, activity-discriminating, directive role with hydrolase and transferase activities of this multifunctional enzyme. Such effects in turn may be related to (a) difference in affinity of various phosphate substrates for divalent cations, and (b) the initial presence of a divalent cation on the enzyme serving as a binding site for phosphate substrates (see Fig. 10). Further, metal ions appear to exert discriminating effects on various enzymes involved in glucose-6-P formation and utilization in gluconeogenic tissues. The two hitherto established hepatic glucose phosphorylating enzymes—glucokinase and hexokinase—both have an absolute requirement for added Mg2+, in contrast with the inhibitory effects of this cation on synthetic activity of glucose-6-phosphatase. Glucose-6-P dehydrogenase, another hepatic enzyme concerned with the metabolic dispensation of glucose-6-P, is activated by Mg2+, although it is not absolutely dependent upon addition of this cation, while both phosphoglucomutase and phosphohexose isomerase are dependent upon M2+ (see ref. 48). Generalizing, it appears that enzymes involved in glucose-6-P generation and utilization, exclusive of activities of glucose-6-phosphatase, are activated by divalent cations, while activities of the latter enzyme [which appears to contain tightly bound divalent cation in its active site (14$, 144)] are, except with Cu2+ (92), either unaffected (hydrolase) or inhibited (phosphotransferase) by such ions. The interesting activating effects of Cu2+ currently are being further investigated in our laboratory. Conceivably, they may be analogous in their potential for metabolic control to those of Fe2+ observed by Snoke, Johnston, and Lardy (171) with another key gluconeogenic enzyme—phosphoenolpyruvate carboxykinase. 8 . COMPARTMENTATION

AND M E T A B O L I C

CHANNELING

Glucose-6-P is a key metabolite in the liver (see Fig. 6). At least five possible mechanisms for its synthesis exist, specifically via glucose phos-

70

ROBERT C. NORDLIE

phorylation by glucokinase, hexokinase, and phosphotransferase activities of glucose-6-phosphatase; from noncarbohydrate precursors through the gluconeogenic pathway; and from glycogenolysis followed by phosphoglucomutase action. And at least five alternative hepatic metabolic fates are readily available for glucose-6-P in the liver—namely, hydrolysis through glucose-6-P phosphohydrolase action; glycolysis; incorporation into liver glycogen; oxidation via the hexose monophosphate shunt; and nucleotide-sugar formation preliminary to further, more exotic types of metabolism. Enzymes for these various processes are discretely located within (or strongly associated with) a variety of hepatic organelles and compartments, including cyto.sol, mitochondria, nuclear and endoplasmic reticulum membrane, and glycogen granules. The existence of morphologically separate, metabolically distinct pools of glucose-6-P, and of discriminant metabolic channeling between certain combinations of such reactions for giucose-6-P generation and for glucose-6-P utilization thus appears to be a distinct possibility of great relevance with respect to control of metabolism. And indeed, evidence both direct and more circumstantial for the existence within the liver cell of two (or more) distinct, noninterchangeable pools of glucose-6-P is present in the literature. For example, Cahill et al. (32) have demonstrated the quite different degrees of saturability with respect to glucose of systems for glycolysis and glycogenesis in liver slices. Figueroa and Pfeiffer (63), Smith et al. (170), Threlfall (192), Threlfall and Heath (193), and more recently Das et al. (44), have presented data, based on studies with isotopically labeled glucose, indicating that such labeled glucose does not pass through a single, homogeneous pool of glucose-6-P on its way to incorporation into glycogen in the liver or muscle. Heath (81, 82) has presented mathematical calculations supporting the presence in liver of at least two distinct, functionally separated glucose6-P pools. As indicated in Section IV, A, above, it is essential that control by one mechanism or another exist such that "nonsense cycling" involving concerted action of hepatic hexokinase or glucokinase plus glucose-6-P phosphohydrolase be precluded. One possibility for such control involves physical separation of glucose-6-P resulting directly from kinase action from glucose-6-P phosphohydrolase. Glucose-6-phosphatase is morphologically unique among enzymes of carbohydrate metabolism, being found exclusively either as a part of or extremely tightly bound to membranes, including those of endoplasmic reticulum (55, 132, 190), nuclei (79, 95, 96), and probably plasma membrane (52, 53) (see Section II, B, 1, a, above). In contrast, glucokinase

MULTIFUNCTIONAL

GLUCOSE-6-PHOSPHATASE

71

is cytosolic (200) and hepatic hexokinase is present both in cytosol and associated with mitochondria (see refs. 73, 159). Thus a physical separation of glucose-6-P involved with kinase as compared with phosphatase would seem quite possible. We have also suggested that glucose-6-P generated via synthetic activities of glucose-6-phosphatase under certain circumstances may be further directed in a highly discriminant metabolic fashion (127). The possibility that hydrolytic and synthetic activities of multifunctional glucose-6-phosphatase of smooth and rough endoplasmic reticulum may serve as a vectorially oriented permease, capable of channeling blood glucose into the hepatic cell under hyperglycemic conditions, and releasing free glucose to the blood stream when concentrations there are insufficient, is considered in detail in Section V, B, below. Similarly, such permease-type action of the enzyme of outer nuclear membrane has likewise been postulated. Such functions for this multifunctional catalyst would constitute metabolic channeling in its most sophisticated form, and would be highly consistent with the role of this enzyme in glucose homeostasis of the body (see Sections V, A and B, below, for an extensive discussions of the latter subject). 9. CATALYTIC ALTERATIONS EFFECTED THROUGH MEMBRANE MODIFICATIONS

Glucose-6-phosphatase is embedded in phospholipid-rich membranes of the smooth and rough endoplasmic reticulum and nucleus, and probably the plasma membrane (see Sections II, B, 1, a and IV, C, 8, above). It is clear at the present time that intimate interrelationships exist between the physical state of such lipid-containing membranes and catalytic behavior of this unique, complex, multifunctional enzyme, since a variety of conditions and manipulations which affect the morphology of these membranes also significantly modify the catalytic characteristics of the biological catalyst. Precisely what these interrelationships are, and mechanisms through which catalytic function is modified, are much less clear, however. A variety of descriptive, phenomenological data have accumulated relating to the effects on behavior of the enzyme of such membrane-modifying treatments as detergent supplementation (11, 17, 134, 1?®), exposure to high pH (180, 181, 184) or A1203 (35), the action of various phospholipases (7, 34, 50, 62, 173, 213), removal of phospholipids by gel filtration (69), supplementation with various phospholipids and phospholipid preparations (17, 34, 50, 62, 69, 173, 213), mechanical shearing under high pressure (138), glucocorticoid administration in vivo (7, 135, 138, 147, 157), deoxycholate acting in concert with phlorizin (114, 174), deter-

72

ROBERT C. NORDLIE

gents combined with various competitive inhibitors {142, 197), and the like. Much of the information thus gained has been confusing, and often conflicting. For example, detergents have been found to both activate and inhibit activities of the enzyme (11, 17, 134, 172). Phospholipids have been reported as essential for activity (50, 69), as necessary (34) or unnecessary (156, 213) for the purpose of stabilization, and as constituting a mechanism for the normal constraint of activities of the enzyme (127, 156, 173, 213). Phosphotransferase activities of the enzyme have been reported as normally latent (9, 127, 133, 173), or as essentially fully active (74, 139, 140), in untreated, isolated biological membranes. Obviously, the relationships involved are complex and probably multiple in nature. A primary source of confusion, this author believes, resides in the fact that the great majority of studies in this area have involved the use of liver microsomal preparations as the source of enzyme. And inherent here is the necessity to disrupt and alter drastically the naturally occurring membranes of the endoplasmic reticulum, and to manufacture and subsequently isolate a totally artificial particle, the "microsome," before manipulatory and analytical studies of any sort may be instigated. Thus a major artifact (74) is systematically introduced before studies on membrane modifications and catalytic behavior are even begun. As an approach to this forbidding problem, we (74, 139, 140) recently have undertaken systematic studies of relationships between membrane morphology and catalytic behavior of glucose-6-phosphatase-phosphotransferase of the hepatic nucleus. The multifunctional enzyme resides in the outer membrane of this organelle (19, 74, 95, 96) ; unlike endoplasmic reticulum, which must be fragmented before isolation is possible, this outer membrane may be isolated by differential centrifugation technique with membranes intact. With such preparations from avian liver, all known activities, including glucose-6-P phosphohydrolase, carbamyl-P: glucose phosphotransferase, mannose-6-P phosphohydrolase, and mannose-6-P: glucose phosphotransferase, were completely, or nearly completely ( > 9 0 % ) , manifest without the need for preliminary activation by detergents, high-pH-treatment, or other treatment (74). In such naturally occurring, intact membranes, all activities of the enzyme are thus functionally active rather than extensively latent. Further, disintegration of intact nuclear membranes, and isolation of the resulting micelles, was accompanied by the development of a significant degree of latency (i.e., detergent sensitivity) of all the mentioned activities. Some of these effects are illustrated in Fig. 14.

MULTIFUNCTIONAL

GLUCOSE-6-PHOSPHATASE

0

73

0.05 0.10 0.15 0.20 0.25 0.30 DEOXYCHOLATE CONC. (%.w/v)

FIG. 14. Differential latency of activities of intact nuclei and disrupted, isolated nuclear membrane preparations. Circles and triangles depict, respectively, glucose-6-P phosphohydrolase and carbamyl-P: glucose phosphotransf erase activities. From Gunderson and Nordlie (74) (by permission of Academic Press, Inc.).

In current studies with similar intact hepatic nuclear preparations from rabbit, rat, guinea pig, and pigeon, activities have been found somewhat more detergent-sensitive than those of nuclei from chicken. Even in these cases, however, all activities were more than 50% manifest in the absence of activating agents, and all (including glucose-6-P phosphohydrolase) were of nearly identical latency within a given species (74, 75) (see Table VI). All these observations are in marked contrast to those with hepatic microsomal preparations, where a differential, activity-discriminant latency (glucose-6-P phosphohydrolase is considerably less latent than other activities) is noted (9,127,133,172). Differences in the relative latency of the various activities also have been observed with microsomes originating from the smooth endoplasmic reticulum compared with rough endoplasmic reticulum, in studies by Stetten and Ghosh (182). Latency of PPii glucose phosphotransf erase activity of the former was considerably greater than with the latter preparations from the same liver. While interpretations at this stage are somewhat speculative, we do believe that certain tentative conclusions are justified from data currently at hand and should be of considerable value as a guide for further studies in this important area of metabolic control at the membrane level. (1) Intimate interrelationships definitely do exist between morphology of lipid-rich membranes and catalytic behavior of multifunctional glucose-6-phosphatase contained therein. (2) A quite specific requirement for a particular phospholipid—monounsaturated phosphatidylcholine—appears established from the work of Garland and Cori (69). (3) While requiring specifically the phospholipid indicated above, the

74

ROBERT C. NORDLIE

T A B L E VI COMPARATIVE LATENCY OF G L U C O S E - 6 - P PHOSPHOHYDROLASE AND CARBAMYL-P: GLUCOSE PHOSPHOTRANSFERASE ACTIVITIES OF INTACT N U C L E I , ISOLATED N U C L E A R M E M B R A N E , AND ISOLATED MICROSOMAL PREPARATIONS FROM L I V E R S OF VARIOUS SPECIES' 1

Relative manifest activity 6

Species Chicken Pigeon Rat Rabbit Guinea pig

Intact nuclei

Isolated nuclear membrane

Isolated microsomal preparation

Activity

(%)

(%)

(%)

Hydrolase 0 Transferase** Hydrolase Transf erase Hydrolase Transferase Hydrolase Transferase Hydrolase Transferase

93 101 69 64 60 82 61 56 81 70

61 54 37 38 48 28 26 42 42 38

42 17 72 13 38 13 50 17 47 8

a Activities were assayed as described by Gunderson and Nordlie (74). Activities were measured in the absence of detergent, and with preparations supplemented with deoxycholate, to the following concentrations: nuclei, 0.05%, w / v ; nuclear membranes, 0.05%, w / v ; and microsomal preparations 0.2%, w / v , which have been found maximally activating with each preparation (74, 75). b Relative manifest activity = 100 X activity observed in the absence of deoxycholate/activity observed in the presence of deoxycholate. This value represents the percentage of total activity which is manifest, under our in vitro assay conditions, without the need for prior dispersion of the enzyme preparation. c Hydrolase = glucose-6-P phosphohydrolase. d Transferase = carbarnyl-P: glucose phosphotransferase.

enzyme is quite susceptible to constraint of activities by phospholipids in general, as indicated by the work of Pollak and co-workers (156). Indeed, it appears from their studies that the enzyme normally may be rather extensively constrained (156). (4) Phospholipids may stabilize the enzyme under experimental conditions in vitro, if care is not taken otherwise to ensure stability of enzyme preparations as they are being manipulated in the laboratory (39, 69). (5) As normally present in intact membranes of certain cellular organelles—chicken hepatic nuclear membrane, for example—both synthetic and hydrolytic activities of the enzyme are nearly totally functional rather than latent (74, 139, 140).

75

MULTIFUNCTIONAL GLUCOSE-6-PHOSPHATASE

(6) A considerable degree of latency may be artificially conferred upon activities of the enzyme through destruction of the natural integrity of such membranes, as illustrated in the isolation of nuclear membrane preparations (74,139,140) (see Fig. 14). (7) A more extensive disruption and dispersion of such membranes— via detergent supplementation (11, 17, 134, 172) or exposure to high pH (180, 181, 184)—completely unmasks any residual latency, whether originally extant in membranous preparations or artifactually acquired as with isolated nuclear membrane micelles, and permits manifestation ef total intrinsic catalytic capacity of all activities of the enzyme. (8) Catalytic properties (apparent Km values, activity-pH profiles, Ki values for various inhibitors, activity ratios, and the like) observed with such maximally activated enzyme preparations from a variety of sources are identical with those observed with unsupplemented intact chicken hepatic nuclei, and undoubtedly reflect the intrinsic catalytic behavior of the enzyme when completely unconstrained (74, 139, 140) (see Tables VI and VII). (9) In vivo treatments and conditions (7, 135, 136, 138, 145, 182), as well as manipulations of various sorts in vitro (detergent supplementaTABLE VII A

COMPAKISON OF SOME K L N E T I C P A R A M E T E R S EVALUATED "INTACT

WITH

M I C R O S O M E S , " D E T E R G E N T - A C T I V A T E D MICROSOMAL

PREPARATIONS,

AND ISOLATED,

INTACT

NUCLEI«

Preparation studied

Parameter measured

Unsupplemented microsomes

Deoxycholatesupplemented microsomes

Untreated nuclei

^ C P , m i n i m u m ( m^ O

6.4 38 47 243 ND 1.9

1.5. 2.6 23 42 10 3.4

1.0 1.5 22 40 11 2.1

^ C P , m a x i m u m ( m^ O ■^•glc,minimum\JÜ-M ) ■**-glc,maximum vTO-lkZ )

KitATP(mM) V ,& r

max.trf

a Both nuclei (74, 75) and microsomes (93) were isolated from avian liver homogenates. Where indicated, microsomes were further supplemented with deoxycholate, to 0.2%, w / v , prior to assay. All assays were carried out at p H 7.0 and μ = 0.1 by methods described in Gunderson and Nordlie (74). ^ Ϊ , Α Τ Ρ values were determined with the carbamyl-P: glucose phosphotransferase system. Abbreviations used are: CP, carbamyl phosphate; glc, D-glucose; and trf, carbamyl-P: glucose phosphotransf erase; N D , not determined. 6 Kmax is expressed as micromoles per 10 minutes per milligram of microsomal or nuclear protein.

76

ROBERT C. NORDLIE

tion, high-pH treatment, etc.), also may tend to modify the relative latency of activities as measured with isolated microsomes. Classic examples {7, 135, 136, 138, 145) are glucocorticoid therapy, which appears to produce an activating, detergentlike effect in vivo, and experimental diabetes or fasting which induce a synthesis of new enzyme which appears (with hepatic microsomal preparations) to be more constrained than is constitutive enzyme originally present before induction (see Section IV, C, 1, above, and Fig. 7). (10) Again based on studies with microsomal preparations, phosphotransferase and glucose-6-P phosphohydrolase activities of the enzyme appear to be amenable to activity-discriminating modifications by the action of membrane-modifying natural or synthetic detergents. For example (see Fig. 15), palmityl-CoA concentrations between 10 μΜ and 30 μΜ markedly activate PPii glucose phosphotransferase while extensively (as much as 50%) inhibiting glucose-6-P phosphohydrolase activity {126, 1^2). And inhibition by phlorizin of glucose-6-P phosphohydrolase activity of such microsomal preparations is ameliorated by supplemental deoxycholate, while identical concentrations of this detergent markedly potentiate inhibition by this aglycone of phosphotransferase activity (114) (Fig. 16). The basic generalization here (that activity-discriminating responses may be elicited) is, we believe, more important than the specific nature of the compounds involved. (11) With microsomal preparations {142), but not with avian nuclear preparations {74, 139, 140), inhibition of activities of the enzyme by ATP 340

? >> 1< f-

LÜ

300 260 220 180

r

f^

/ " /'

-I

-4

100

en

60 20

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1

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140

> & -I

\

\

^ > ^ 1 1 1 1 1 1 1 1 1 1 1

0 20 4 0 6 0 80 100 120 PALMITYL CoA C0NC. (^Μ)

FIG. 15. Illustration of differential, activity-discriminating effects of palmityl coenzyme A on glucose-6-P phosphohydrolase ( O ) and PPt : glucose phosphotransferase ( Δ ) activities of rat liver microsomal preparations. Palmityl coenzyme A, at the indicated concentrations, was included in assay mixtures along with substrates and buffer. From Nordlie et al. (141) (by permission of American Society of Biological Chemists, Inc.).

MULTIFUNCTIONAL GLUCOSE-6-PHOSPHATASE PHOSPHOHYDROLASE

1PHOSPHOTRANSFERASE

I[_

]

\eo ]40 :

i :

20

o

^'^

77

^*

-^*

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0

l

0.4 0.8 1.2 PHL0RIZIN CONC. (m/tf)

0

0.4 0.8 1.2 PHLORIZIN C0NC.(m/tf)

FIG. 16. Contrasting modifications by supplemental cetyltrimethylammonium bromide of extent of inhibition by phlorizin of P P i ".glucose phosphotransferase and glucose-6-P phosphohydrolase activities of rat liver microsomal preparations. The detergent, in the indicated concentrations (%, w / v : O , none; □ , 0.05; Δ , 0.10; # , 0.20; ■ , 0.30), was added to microsomal preparations before assays were carried out. From Lygre and Nordlie {114) (by permission of Elsevier Scientific Publishing Co.).

and other nucleotides, and by Pi, is potentiated by detergent supplementation (see Fig. 11, for example). Mechanistic Interpretations. Two quite reasonable mechanistic interpretations appear to us to emerge from a consideration of available information. i. A "membrane sidedness" theory. It is proposed that a "sidedness" exists with respect to the placement of glucose-6-phosphatase-phosphotransferase within the membrane of the intact cellular organelle (see Fig. 17). The enzyme of membrane of intact nuclei of avian hepatic origin thus may be situated so as to be fully accessible to extranuclear (i.e., cytosolic) glucose-6-P, glucose, carbamyl-P, mannose-6-P, ATP, or other relevant substrate; hence little or no latency is seen with such preparations. When nuclear membranes are disrupted and fragments isolated, however, the possibility exists that membranes of such resulting vesicles are either "right-side-out" or "inside-out." Apparent latency which develops with the formation of such nuclear membrane vesicles is thus explained simply by this randomness of orientation of the enzyme concomitant with destruction of the originally present integrity of the nuclear membrane. Particularly relevant here, we believe, is the fact that latency of enzymatic activity which is artificially developed is identical with respect to all enzymatic activities studied, rather than discriminant with respect to glucose-6-P phosphohydrolase, as is the case with isolated microsomal preparations. ii. A theory involving membrane-regulated inter conversions of constrained and unconstrained conformers of the enzyme. Data obtained

78

ROBERT C. NORDLIE "RIGHT-SIDE OUT' ,0UTER NUCLEAR MEMBRANE

Glucose-6Phosphatase

Glucose-6Phosphatase

INTACT NUCLEUS

"INSIDE-OUT"

ISOLATED NUCLEAR MEMBRANE FRAGMENT MICELLES

FIG. 17. Theory of "membrane sidedness" to explain the development of latency of phosphohydrolase and phosphotransferase activities concomitant with the disruption of nuclei and isolation of nuclear membrane fragments. Details are given in the text.

with nuclei and nuclear membrane preparations, as described above, alternatively may be rationalized on the basis of the existence of both fully active (unconstrained) and relatively less active (constrained) conformational variants of the enzyme, which are reversibly interconvertible, as indicated schematically in Fig. 18. The former conformer is postulated to be fully operative without the need for any type of preliminary activation, to manifest ultimate intrinsic catalytic capacity and other characteristics, and may be typified by the enzyme of the membrane of the avian liver nucleus. In contrast, the constrained conformer is postulated to be relatively latent with respect to activity, and to exhibit relatively high Km values for phosphate substrates. Disruption of membrane integrity, as in the preparation and isolation of nuclear membrane (or in the manufacture of "microsomes" from cellular endoplasmic reticulum) is postulated to effect a relative increase in the ratio of constrained to unconstrained conformers. Addition of detergent to such preparations, exposure to high pH, phospholipase treatments, mechanical shearing, etc., would totally disperse such membranes or membranous fragments and thus convert such preparations totally to the unconstrained form. The reversible interconversion of constrained and unconstrained conformers is attributed to subtle conformational changes in the enzyme protein effected through morphological alterations in the phospholipid-rich membranes, and may be induced either in vivo (Table VI) or in vitro (Fig. 14).

79

MULTIFUNCTIONAL GLUCOSE-6-PHOSPHATASE

> All I

NUCLEAR

MEMBRANE

FRAGMENTS

F I G . 18. Schematic representation of some concepts involved in the theoretical rationalization of differential latency of various activities of different preparations of the enzyme on the basis of reversible, modulated interconversion of constrained ( T ) and unconstrained (■) forms of glucose-6-phosphatase-phosphotransferase. Details are given in the text.

According to this theory, and by analogy with the differential patterns of results obtained with intact nuclear membranes compared with isolated nuclear membrane preparations (see Fig. 14), it may be inferred that the enzyme may be predominantly in the unconstrained form in the membrane of the endoplasmic reticulum in situ, and that latency develops to a great extent as the equilibrium between unconstrained and constrained conformers is shifted in favor of the latter, concomitant with morphological changes accompanying fragmentation and isolation of such fragments (microsomes) of endoplasmic reticulum. Direct proof for this suggestion is needed, however. Latency observed experimentally with liver and kidney microsomal preparations differs significantly in one respect from that noted either with disrupted, isolated nuclear membranes or with membranes of intact nuclei from certain species (see Table VI and Fig. 14). While no discrimination with respect to the various activities of any of the nuclear, or nuclear membrane, preparations is observed, very distinct phosphate-substrate-discriminant specificity is apparent in isolated microsomal prepa-

80

ROBERT C. NORDLIE

£ 700 £ 600 p 500
80 GLUCOSE CONC. (mg/100 ml)

I

0

I

I

GLUCOSE CONC.imA/) I I I I 1 I I I 1—I

180 360 540 720 900 1080 GLUCOSE CONC (mg/100 ml)

F I G . 20. Various enzymatic activity values calculated for assumed physiological levels of various substrates (see text) and indicated, varying glucose levels. Activities are abbreviated as follows: G-6-P HY., glucose-6-P phosphohydrolase; C P G T , carbarn yl-P: glucose phosphotransf erase; GK, glucokinase; HK, hexokinase. Glucose6-P phosphohydrolase activity is presented both unadjusted (unadj.) and adjusted for inhibition by glucose (adj.). C P G T (adj.) indicates carbamyl-P:glucose phosphotransferase values calculated for physiological substrate concentrations and further adjusted for inhibitions by presumed physiological levels of A T P , Pi, HCOf, and glucose-6-P. Additional details are given in the text and in the Appendix.

86

ROBERT C. NORDLIE

but with varying glucose concentrations. Estimated levels of phosphoryl donors were as follows: 5 mM ATP for glucokinase and hexokinase; 0.2 m l carbamyl-P for carbamyl-P: glucose phosphotransferase. Levels of glucose-6-P phosphohydrolase determined under these same conditions, based on assumed physiological concentration of 0.13 m l (see Appendix) for this compound, also are included in Fig. 20, A-D for later reference. All activity data are adjusted to 37°. Further details of calculations employed here and below are presented in the Appendix. Normal rat was chosen for illustrative purpose as a representative species having a relatively high level of hepatic glucokinase (see Table I I I ) . Domestic chicken was similarly chosen as typical of species having relatively low normal levels of hepatic glucokinase and comparatively high normal blood glucose levels (see refs. 88, 188). Diabetic rat represents an artificially established situation characterized by a marked diminution in glucokinase activity, a significant increase in glucose-6-phosphatase, and concomitant extensive elevation in blood glucose level. Finally, ox was included as characteristic of yet another group of animals, the ruminants, in which relatively low normal levels of blood glucose (approximately 40 mg/100 ml) persist (20, 152). As can be seen from information in Fig. 20, A-D, hexokinase (Π) *s relatively low and is unaffected by variations in glucose concentrations [Km, glc ΕΞ 10-2 mM (73, 97) ] in the range considered. Nor do levels of hexokinase, a constitutive enzyme (47, 162, 166, 198), change in diabetes. On the basis of activity values calculated for presumed physiological levels of substrates, but otherwise unadjusted for inhibition by cellular metabolites, carbamyl-P:glucose phosphotransferase levels approximate those for glucokinase in normal rat liver (Fig. 20A). In the other three situations—diabetic rat (Fig. 20B), normal chicken (Fig. 20C), and ox (Fig. 20D)—such carbamyl-P:glucose phosphotransferase levels markedly exceed those of glucokinase and hexokinase. d. Relative Activity Levels Calculated for "Physiological" Concentrations of Substrates and Adjusted for Metabolite Inhibition. As indicated in Sections II, B, 2, C and IV, C, 4-6, activities of glucose-6-phosphatasephosphotransferase are subject to inhibition by a variety of metabolites. Since such inhibition is of considerable potential physiological importance, carbamyl-P: glucose phosphotransferase levels, which previously were adjusted on the basis of presumed physiological substrate concentrations and pH (see Section V, A, 1, c, immediately above), were further adjusted through calculations (see Appendix for complete details) taking into account inhibitions by ATP, Pi, HCO^, and glucose-6-P (estimated physiological levels are listed in the Appendix), all effective competitive inhibitors of phosphotransferase activity (with respect to carbamyl-P) at pH 7 (145).

87

MULTIFUNCTIONAL GLUCOSE-6-PHOSPHATASE TABLE IX I N H I B I T I O N S BY VARIOUS M E T A B O L I T E S UNDER " P H Y S I O L O G I C A L "

CONDITIONS' 1

Inhibition

Metabolite

Glucose-6-P phosphohydrolase (%)

Carbamyl-P: glucose phosphotransferase (%)

Glucose-6-P Carbamyl-P ATP Pi HCO; All relevant inhibitors combined 6

— 9 28 47 48 70

7 — 28 48 49 70

a

Inhibition by the various metabolites, if present alone, and finally, in total combination, is indicated. "Physiological" levels of the various inhibitors and substrates are presumed to be as indicated in Table X I I I . Further details of calculation are given in the Appendix. 6 Glucose-6-P + A T P + Pi + HCOj· for phosphotransferase; carbamyl-P + A T P + Pi + HCO-f for phosphohydrolase.

An indication of inhibitory potential of each metabolite, added alone in the absence of other inhibitors, and also present in total combination, is given in Table IX. Activity values thus obtained are plotted in Fig. 20, A-D. It is apparent that, even on such a restricted basis, carbamyl-P:glucose phosphotransferase activity levels exceed significantly those of glucokinase and hexokinase in the diabetic rat, or in bird or ox liver. In the case of the normal rat, in contrast, glucokinase exceeds that of carbamyl-P: glucose phosphotransferase by a factor of approximately 5 to 6 (depending, to a degree, on the concentration of glucose at which the comparison is made). It is thus apparent that, on this basis, the ratio, carbamyl-P'.glucose phosphotransferase/glucokinase, is considerably less than 1 in normal rat, but increases to values significantly exceeding unity as diabetes develops and glucokinase levels concomitantly drop as glucose-6-phosphatasephosphotransferase increases. Similarly, such activity values in excess of one appear to exist normally in bird and ox, based on presently available information. The significance of these observations will be elaborated upon below. 2. LIVER ENZYMES AND THE REGULATION OF BLOOD SUGAR LEVELS

Cahill et al. (31) in 1959 considered at length the role of the liver in maintenance of body carbohydrate homeostasis. They pointed out that

88

ROBERT C. NORDLIE

this organ has the capacity both to take up glucose from the blood and store it as glycogen when glucose is abundant (as after a meal) and to release such stored glucose to meet the body's energy requirements in times of need (as in fasting). The hepatic cell appears to be freely permeable to glucose (30), but is impermeable to the charged phosphate ester of this hexose—gluco.se6-P. Thus upon phosphorylation within the hepatic cell, glucose (as its phosphate ester) is effectively "captured." Release of free glucose from the hepatocyte requires the hydrolysis of glucose-6-P, the common product of glucose phosphorylation, gluconeogenesis, and glycogenolysis. It thus appears that a finely regulated balance between the rates of hepatic glucose phosphorylation and glucose-6-P hydrolysis is all-important in determining, under any particular set of ambient conditions, the direction and net rate of flux of glucose between the liver parenchymal cell and the blood. Concentrations of glucose in the blood, in turn, can affect both processes. Glucose is an effective inhibitor of glucose-6-P hydrolysis (see Section IV, C, 4, a, above) and, as substrate, directly determines the rate of glucose-6-P generation via the action of hepatic kinase. That concentration of glucose at which the rates of glucose-6-P hydrolysis and total glucose phosphorylation are equal will, according to this theory, approximate physiological blood glucose levels under any particular set of conditions (31). These concepts, as originally postulated by Cahill et al. (31), and modified by the present author for purposes of the present discussion, are presented schematically in Fig. 21. Glucose-6-phosphatase has, since the early work of the Cori's (40-43) and others (26, 4&&, 46, 58, 151, 189, 190), been established as the key enzyme for hepatic glucose-6-P hydrolysis (Reaction A in Fig. 21). Levels of this inducible (12, 31, 126) enzyme rise significantly under conditions dictating accelerated hepatic glucose production (fasting or untreated diabetes, for example), and fall concomitant with a diminution in the requirement for glucose (see Table V and Section IV, C, 1, above), consistent with such a key metabolic role for the hydrolase. The identity of liver enzymes responsible for glucose phosphorylation in such a controlled process (Reaction B, Fig. 21) is, however, more complex. The presence of nonspecific hexokinase in liver has long been established. However, this enzymatic activity of the hepatic cell is relatively low, and is constitutive rather than inducible (47, 162, 166, 198). Because of its relatively low levels of hepatic activity, its unresponsiveness to physiological stimuli, and its very low Km for glucose (approximately

MULTIFUNCTIONAL

GLUCOSE-6-PHOSPHATASE

ATP (CarbamylPPi.

etc.)

LIVER

89

Hexokinase ADP Glucokinase (NH. C0 o 3 (Phosphotransferase activities of Pi Glucose -6 - phosphatase) etc.) CELL

FIG. 21. Schematic depiction of interrelationships among liver, blood, and peripheral tissues with respect to glucose flow and utilization. Net rate and direction of flux of glucose between liver and blood is determined by the relative rates of glucose phosphorylation (B) and glucose-6-P hydrolysis (A), as generally postulated by Cahill et al. (81). The latter of these two processes (A) is catalyzed by hepatic glucose-6-P phosphohydrolase, while the former (B) involves hexokinase, glucokinase, and, we propose, transferase activities of glucose-6-phosphatase-phosphotransferase as well.

10"2 mM, refs. 73 and 97), hexokinase does not appear to be suited to play an important regulatory role. In contrast, the more recently discovered hepatic glucokinase possesses a complement of characteristics well suited for a prominent phosphorylative role in controlled hepatic regulation of body carbohydrate metabolism (47, 162, 166, 198, 199). This enzyme, most extensively studied in rat liver, is inducible (requires insulin), is present normally in quantities considerably higher than those of hexokinase (in certain species; see ref. 83 and Table I I I ) , and, because of its relatively high Km for glucose—10-20 mM (47, 199, 200)—is highly responsive to variations in glucose concentration in the physiological range. Some Quantitative Considerations. A quantitative evaluation of this general, theoretical role for key liver enzymes in the control of hepatic glucose uptake and release, under the modulating influence of varied glucose concentrations, may be made from the data of Fig. 20, A-D, where both rates of glucose-6-P hydrolysis and of glucose phosphorylative via glucokinase and hexokinase at "physiological" substrate concentrations are presented. Rates of glucose-6-P hydrolysis under such circumstances significantly exceed rates of glucose phosphorylation by glucokinase or hexokinase (or both). This is true whether or not the glucose-6-P phosphohydrolase

90

ROBERT C. NORDLIE

rates are adjusted for inhibition by increasing levels of glucose (see lines depicted by ■ — ■ and in Fig. 20). It is thus apparent on a quantitative basis that, if control of blood sugar concentrations at the hepatic level is to be manifest as generally outlined by Cahill et al. (31) and as described above, then either (a) additional mechanisms for phosphorylation of glucose in the liver must be postulated, and/or (b) further constraints must be imposed upon glucose-6-P phosphohydrolase. 3. INCORPORATION OF THE NEWLY DEFINED FEATURES OF GLLTCOSE-6-PHOSPHATASE-PHOSPHOTRANSFERASE INTO THE MECHANISM FOR HEPATIC CONTROL OF BLOOD GLUCOSE LEVELS

We believe that newly discovered features of multifunctional glucose-6pho sphat as e-phosphotr ans f erase, as described in some detail in Sections II-IV above, may operate in vivo, both in providing for the needed additional constraint of glucose-6-P phosphohydrolase, and to supplement the phosphorylation of glucose by hepatic glucokinase plus hexokinase. A quantitative reassessment of the calculations, initially considered in Fig. 20, A-D, is provided in Fig. 22, A-D. Here, additional constraints are incorporated on glucose-6-P hydrolysis, through inhibition by "physiological" levels of the metabolites ATP, Pi, HCO^, and carbamyl-P (see Table X I I I in Appendix for estimated "physiological" concentration values), as well as inhibition by increasing levels of glucose (107). Further, carbamyl-P: glucose phosphotransf erase activity of glucose-6-phosphatase, also adjusted for inhibition by these same concentrations of metabolites [glucose-6-P in place of carbamyl-P, but otherwise including ATP, Pi, and HCO^~ as for glucose-6-P phosphohydrolase activity (see Appendix, Table XIII)], is included along with calculated glucokinase and hexokinase levels. The rates of glucose phosphorylation, at any indicated level of glucose, by (a) glucokinase, (b) glucokinase plus hexokinase, and (c) glucokinase plus hexokinase plus carbamyl-P: glucose phosphotransf erase are directly indicated in Fig. 22, A-D. Under these conditions, which we believe on the basis of currently available information to most closely approximate the "physiological/' predicted intersections of experimental lines depicting the rates of glucose-6-P hydrolysis and of glucose phosphorylation by the combined kinases plus phosphotransferase are always observed. And these correlate very well with experimentally observed levels of blood glucose in the first three situations considered (Fig. 22, A-C). In the case of the normal rat (Fig. 22A), intersection of plots (see vertical arrows) is noted in the vicinity of 80-90 mg of glucose per 100

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F I G . 22. Activities of various hepatic enzymes, calculated as a function of varied glucose concentrations on the basis of presumed physiological levels of other substrates and further adjusted for inhibition by estimated physiological levels of various inhibitors. Such inhibitors include A T P , Pi, HCO^", and glucose-6-P for carbamylP : glucose phosphotransferase; and ATP, Pi, HCO^", and carbamyl-P for glucose-6-P phosphohydrolase activity. Hexokinase has been adjusted for inhibition by glucose and glucose-6-P. Details of calculation, and presumed levels of all relevant compounds are given in the Appendix and text. Abbreviations employed are as in Fig. 20. Vertical arrows designate crossover points, t h a t is, those concentrations of glucose at which rates of glucose-6-P hydrolysis and glucose phosphorylation (by hexokinase -fglucokinase + carbamyl-P:glucose phosphotransferase, or by a combination of the latter two, regulatory activities only) are equal.

ml; this equivalence glucose value is elevated to approximately 270 mg/100 ml (all 3 glucose phosphorylating systems considered) or 450 mg/100 ml (glucokinase plus carbamyl-P:glucose phosphotransferase, only, considered for glucose phosphorylation), in the diabetic rat (Fig. 22B). With the domestic fowl (Fig. 22C), which normally exhibits blood glucose levels in the vicinity of 250-300 mg/100 ml {187, 188), intersections are noted at 250 or 470 mg of glucose per 100 ml.

92

ROBERT C. NORDLIE

Considering the large number of experimental measurements, complex calculations, and assumptions involved in arriving at these proposed physiological activity values (see Appendix for precise details), we consider this correspondence of estimated crossover equivalence glucose values with the observed, blood glucose concentrations to be satisfactory. In the case of ox, which was included for purpose of contrast, a crossover of the relevant plots in the vicinity of 300 mg of glucose per 100 ml is apparent (see Fig. 22D). Normally ox, a ruminant, converts dietary carbohydrate to propionate and other volatile fatty acids before absorption takes place, and blood glucose levels are accordingly very low [in the vicinity of 40 mg/100 ml (20, 152)}. Data in Fig. 22D, however, suggest that even in this species, mechanisms at the hepatic enzymatic level do exist for placing an ultimate upper limit on blood glucose concentrations. Such mechanisms, although not normally operative, may be of importance in diabetes (119) in this species as well as in others, and may possibly also operate to control blood sugar levels during therapeutic glucose infusion (20) in the treatment of the acidotic, ketotic ruminant (see also Section V, B, 4, b).

4. A "TUNING-RETUNING HYPOTHESIS"

On the basis of information of the sort presented in Fig. 22, A-D, Tables I, III, V, and VI and at various other places throughout this chapter (references have been incorporated where appropriate), we hypothesize that synthetic (as well as more familiar hydrolytic) activities of glucose-6-phosphatase, along with hepatic glucokinase may indeed play key roles in determining ambient blood glucose levels. We further suggest that, because of the somewhat differing requirements for glucose in the two systems (see K'mglc values in Tables V, X, and XII), the ratio (carbamyl-P: glucose phosphotransferase activity)/ (glucokinase activity), is extremely important in establishing levels of blood glucose (often widely varying) among various species, in health and in disease. Marked differences in blood glucose levels normally present in various species (rats compared with birds, for example) may be rationalized on this basis. Because of the basic differences in inducibility of these two enzymes—glucokinase requires insulin (47, 162, 166, 198, 199) while phosphotransferase (and phosphohydrolase) activities of glucose-6-phosphatase increase significantly in insulin's absence (77, 83, 125, 126, 133, 146)—a "retuning" of blood sugar levels through hormonally nutritionally, or chemotherapeutically induced changes in such hepatic enzyme

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FIG. 23. Typical glucose tolerance curves for normal and diabetic individuals. Plots are composites based on the examination of a variety of such curves.

patterns is possible. This "retuning" may operate in vivo, under appropriate stress, to effect readjustments in blood glucose levels required for the continued existence of the organism under difficult (or at least radically altered) conditions. An example may be the development of untreated diabetes, as discussed below. a. Enzymatic Retuning of Blood Glucose Levels in Diabetes. "In all but the most severe states of insulin deficiency, a new equilibrium with essentially normal rates of peripheral assimilation—albeit obtained at much higher [than normal] blood-glucose concentrations—is reached'' (31). The differences in levels at which blood sugar levels equilibrate in the untreated diabetic compared with the "normal" individual are readily apparent from an examination of typical glucose tolerance curves (Fig. 23). The maintenance of elevated plateau levels of blood glucose appears essential in diabetes. Mediated transport of glucose, a prime source of metabolic energy, into peripheral tissue cells under normal conditions (i.e., blood glucose levels in the vicinity of 100 mg/100 ml) requires the presence of insulin. In diabetes, hyperglycemia promotes the continued passage, via a mass action effect on simple diffusion, of sufficient glucose to meet the needs of the cell in the absence of insulin. We believe that the required readjustment may be effected through a "retuning/' as described in general terms in Section V, A, 3, of the relevant hepatic enzymatic machinery as insulin production diminishes {83,125,126,133). In the normal rat, glucokinase may be the predominant activity for hepatic glucose phosphorylation, with carbamyl-P: glucose phosphotransferase (see Fig. 22A and Section V, A, 3) of lesser importance. Under these conditions, the kinetics of overall hepatic glucose phosphorylation reflect principally the characteristics of glucokinase, the rectangular hy-

94

ROBERT C. NORDLIE

perbola describing such activity considered as a function of varied glucose concentration (see Δ, Fig. 22A for example) rising fairly rapidly with increasing levels of the hexose substrate. Consequently such plots intersect with those depicting glucose-6-P hydrolysis at a point corresponding to relatively low levels of glucose (80-90 mg/100 ml in the example presented). In diabetes, the situation is quite different. Levels of the key gluconeogenic enzymes—phosphoenolpyruvate carboxykinase (149, 167) and fructose-diphosphatase (207), as well as glucose-6-phosphatase (12, 64, 77, 83, 133, 138, 147)—rise extensively, and glucokinase disappears (47, 162, 166, 198, 199). Increased concentrations of acetyl coenzyme A concomitantly active pyruvate carboxylase, another key gluconeogenic enzyme (196), while important glycolytic activities are diminished by inhibition exerted through accumulated fatty acds (206). Were these processes allowed to continue, glucose generation via such accelerated gluconeogenic processes would proceed unchecked in a continuously accelerating manner [note that the normally regulatory glucokinase is very low (Δ, Fig. 22B) ]. Loss of control of blood glucose concentrations at the hepatic level, which appears so elegantly operative under more normal conditions, seems to us improbable. We propose that under these circumstances phosphotransferase activities of glucose-6-phosphatase take over the physiological glucose phosphorylative role normally played in rat liver by glucokinase. This proposal derives support from the calculations in Fig. 22B, and from a considerable body of information currently in the literature and discussed more extensively in Section V, A, 5. In contrast with the situation in normal rats, as diabetes develops (see Fig. 22B) insulin-dependent glucokinase levels drop precipitously, and glucose-6-P phosphohydrolase and phosphotransferase levels rise considerably (see Tables IV and X). With the latter, the predominant mechanism of hepatic glucose phosphorylation, the rectangular hyperbola depicting total rate of hepatic glucose phosphorylation as a function of varied glucose levels (indicated by * or C in Fig. 22B) is flattened relative to that in the normal situation (compare Fig. 22A). And as a consequence of this flattening and the increase in glucose-6-P phosphohydrolase, the plots for rates of total glucose phosphorylation and glucose-6-P hydrolysis intersect (see vertical arrows) at a point corresponding to a glucose concentration that is considerably higher in the diabetic (Fig. 22B) as compared with the normal (Fig. 22A) animal. Thus, control of glucose release, at the hepatic level, is still maintained through the elegant mechanism initially generally outlined by Cahill et al. (31), but with the substitution of phosphotransferase activity of glu-

MULTIFUNCTIONAL GLUCOSE-6-PHOSPHATASE

95

cose-6-phosphatase* in place of insulin-dependent glucokinase as the major glucose phosphorylative activity. A subtle gradation of response to varying degrees of insulin insufficiency is also afforded by such a mechanism for "retuning" of blood sugar levels, since the ratio (phosphotransferase activity of glucose-6phosphatase)/(activity of glucokinase) is the key parameter here. Responses in levels of both activities, although in opposite directions, vary closely with the degree of diabetes encountered. b. Tuning and Retuning in Other Situations. Rather marked differences in blood sugar levels normally present in various species may, in part, be rationalized on such a basis as that described above. As already pointed out, the phosphotransferase/glucokinase ratio in bird liver is quite similar to that in the diabetic, rather than the normal, rat, correlative with the comparatively high levels of blood glucose normally encountered in birds capable of flight. We wish to reemphasize here that factors other than the hepatic system under discussion also, of course, contribute to the determination of glucose levels in the blood. What we are emphasizing with respect to the hepatic regulatory enzymatic system is its ability to define upper limits to such blood sugar levels, and to provide for the redefinition of such limits as circumstances dictate. The ox serves as an excellent example illustrative of this latter point. Here, it would appear that the regulatory mechanism as generally outlined is indeed present. But blood glucose levels are nowhere near the predicted maximum values (see Fig. 22D and accompanying text in Section V, A, 3, above). The point here is that blood sugar levels should plateau in the indicated range if the system is challenged by a sufficient glucose load.-f Normally it is not, for the reasons outlined in detail in

* Although it has been indicated (100) that separate enzymes may be needed for glucose phosphorylation and glucose-6-P dephosphorylation (81), we believe that multifunctional glucose-6-phosphatase-phosphotransferase may be suited to play either (or both) of such roles under appropriate conditions. This conclusion is based on the facts that (a) the enzyme appears to be under activity-discriminant control, and (b) reactions both in the direction of glucose-6-P synthesis and hydrolysis are highly exergonic. f The hepatic phosphorylation-dephosphorylation system, as we propose it, would function by analogy with a dam. The height of such a dam may determine the depth of the water behind it, provided that the inflow of water into such a lake or river behind this dam is sufficient to challenge it. If the latter condition is met, then the depth of the lake or river reflects the height of the d a m ; if inflow is insufficient, then the height of the dam is of no determining relevance with respect to the extant water depth.

96

ROBERT C. NORDLIE

TABLE X SUMMARY OF SOME H I G H L Y R E L E V A N T , CONTRASTING F E A T U R E S OF T H E T H R E E P R I N C I P A L ENZYMATIC ACTIVITIES FOR GLUCOSE PHOSPHORYLATION IN LIVER«

Enzymatic activity

Characteristic

CarbamylP : glucose phosphotransf erase

Glucokinase

Hexokinase

Constitutive or inducible? Response in diabetes

Inducible Increased 2-3-fold

Inducible Markedly decreased (>90%)

Constitutive Unchanged

Apparent Km or Km for glucose

25-40 m l (83)

10-20 m l (47, 199, 200)

10~2 m l (73, 97)

4 10 20 25

0.4 1 0.1 0.1

Observed levels in liver 6 Man c Rat, normal Rat, diabetic Chicken

0.4 0.2 0.2 0.4

α

Activity values, determined at physiological p H with constant levels of substrates, have been abstracted from Table I I I . R a t was chosen as representative of a species with a relatively very high level of hepatic glucokinase normally, chicken as a species with a relatively low level of glucokinase and a relatively high level of phosphotransferase activity of glucose-6-phosphatase normally, and m a n because of general, clinical interest. Note t h a t the patterns of enzymatic activities in the diabetic r a t resemble those of the chicken (see also Figs. 20 and 22). 6 Enzymatic activity is expressed as micromoles per minute per gram wet liver. c Autopsy specimens were employed for assay of human liver carbamyl-P: glucose phosphatase; activity presented thus m a y represent a minimum value.

Section V, A, 3, and relating to unique aspects of ruminant metabolism. We recently have suggested {128), on the basis of very preliminary studies (122), the possibility that a "retuning" of enzymes as outlined here may be involved in the tendency toward hyperglycemia associated with aging. Regulation of blood sugar levels in these and other situations through chemotherapeutically effected alterations in hepatic enzyme levels appears to be a distinct possibility, currently being explored experimentally. c. Summary of Factors Involved in the " Tuning-Retuning Hypothesis." A summary of some of the relevant characteristics of the various hepatic enzymes involved is presented in Table X.

MULTIFUNCTIONAL GLUCOSE-6-PHOSPHATASE

97

5. ADDITIONAL EXPERIMENTAL EVIDENCE SUPPORTING THE INVOLVEMENT OF PHOSPHOTRANSFERASE ACTIVITY OF GLUCOSE-6-PHOSPHATASE IN THE PHYSIOLOGICAL PHOSPHORYLATION OF GLUCOSE

In addition to (a) the demonstrated quantitative need for such additional hepatic glucose phosphorylative activities, and (b) the suitability of the carbamyl-P: glucose phosphotransferase activity of glucose-6-phosphatase for this function, a variety of additional information implicating phosphotransferase activities of this enzyme either directly or indirectly in physiological processes is accumulating in the literature. The results of some of these studies are reviewed briefly here. a. An extensive demonstration of the general occurrence of hepatic glucokinase in significant amounts has not been accomplished to date, either because of the pronounced instability of the enzyme (103) or because it is not generally present in relatively large amounts (see ref. 83). In contrast, phosphotransferase activities of glucose-6-phosphatase have been found in liver of all higher animals tested (83) (see Table I I I ) . b. Glucokinase does not appear in rat liver until more than 2 weeks after birth (199), raising the question of how blood sugar is regulated in the early stages of life. Glucose-6-phosphatase, in contrast, first appears at approximately day 19 of gestation in such animals (25, 44a), correlative with the marked increase in liver glycogen synthesis, which may be of significance in itself (see ref. 68) and is fully manifest with all its ramifications within a few hours (84) to 3 .days after birth (25). c. Friedmann et al. (68) have concluded that rates of synthesis of glycogen after oral administration of glucose to glucagon-treated fasted or diabetic rats was too great to be compatible with a rate-determining role for glucokinase. They have suggested the additional involvement of PPi-glucose phosphotransferase activity of hepatic glucose-6-phosphatase (see Fig. 24). Such a system would serve effectively to remove PPi generated at the UDPG synthetase step also involved in the overall glycogenic process and at the same time conserve a considerable portion of the intrinsic energy. d. Hornichter and Brown (89) have correlated elevated glucose tolerance curves with the virtual disappearance of hepatic glucokinase during starvation. Further, they have reversed both of these effects by refeeding glucose to such rats. However, fructose-Yefed rats still had very low levels of hepatic glucokinase activity, but did not show glucose intolerance. The authors pointed out that synthetic activities of glucose-6-phosphatase, as described by the present author and co-workers, may be involved as a compensatory mechanism for glucose phosphorylation under such con-

98

ROBERT C. NORDLIE Phosphotronsferase activity of Glucose-6phosphatase Λ A Ä GLUCOSE ^J" u » > GLUCOSE-6-P NI / ^ \ PhosphoglucoPj I \ \ mutase

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/

Glycogen n\£ Synthase^

' UTP / £·

UDPG Synthetase

(Glucosen.|) PRIMER GLYCOGEN FIG. 24. Cyclic utilization of P P i released at the U D P G synthetase step of glycogen synthesis for phosphorylation of glucose, the initial requisite step in glycogenesis. Modified from Friedmann et al. (68) (by permission of The Endocrine Society).

ditions. The hypothesis is strengthened, we believe, by the fact that levels of activities of glucose-6-phosphatase are increased in animals on a highfructose, as compared with a normal or high-glucose diet (25, 67). e. Studies in which glucose uptake and utilization in isolated perfused liver preparations (29, 70, 120, 121) or liver slices (14, 32) was examined as a function of variations in concentration of the hexose also support the participation of hepatic glucose phosphorylative mechanisms in addition to glucokinase and hexokinase. Indeed, some of these studies constitute the most convincing evidence yet available in support of such functions for transferase activities of glucose-6-phosphatase. They include the following: i. Some years ago, Cahill et al. (32) noted profound differences in the saturability by glucose of systems for glycogenesis in rat liver slices, in contrast with C0 2 production. The latter system was fully saturated with 15 mM glucose, while the rate of glucose incorporation into glycogen was directly dependent on the concentration of the hexose within the entire experimental range considered, 5 to 50 mM. Separate systems, with widely differing Km values for glucose phosphorylation, the compulsory first step in both glycolysis and glycogenesis, are thus suggested. ii. Although demonstrating the absence of glucokinase, Ballard and Oliver (14) have noted a direct dependence on glucose concentration, between 20 and 200 mM, in the rate of incorporation of labeled glucose into glycogen in sheep liver slices. in. Gordon (70) has observed a direct, linear relationship between perfusate glucose concentrations, between 100 and 500 mg/100 ml, and the rate of glucose uptake in isolated, perfused livers of rats which had been

MULTIFUNCTIONAL GLUCOSE-6-PHOSPHATASE

99

fasted for 18-20 hours and presumably were relatively low in glucokinase (see refs. 162, 198). Such studies have been extended in our laboratory, and the direct functional relationship (although nonlinear) was found apparently operative up to the maximum glucose level considered, 1200 mg/100ml (70 m l ) (15). iv. Very recently, Lowenstein and his group (29) have reported investigations in which the rate of fatty acid synthesis in isolated, perfused livers from both fed and fasted rats was studied as a function of glucose concentrations in the perfusate. They concluded that their observed correlation between rate and glucose concentration, over the entire studied range (4-25 m l ) , was inconsistent with known properties (Km, glc ^ 10 mM, for example) of hepatic glucokinase. v. Some of the concepts and factors governing hepatic glucose uptake or liberation have been subjected to more direct experimental analysis by McCraw, Peterson, and Ashmore (121) and McCraw (120), who utilized the isolated, perfused liver technique and studied the effects of varied perfusate glucose concentrations on both rates of glucose uptake and glucose production via glucongenesis from lactate. Such glucose levels were varied routinely between 50 and 600 or 1000 mg/100 ml in the various studies. To the surprise of these authors, glucose uptake remained a function of perfusate concentration, even at very high levels, with preparations from diabetic and fasted rats, where glucokinase is virtually absent. Further, McCraw et al. (121) actually assessed glucose "null-point" concentrations, that is, determined that concentration of glucose at which rates of glucose uptake and glucose production were identical, as predicted by the original theory of Cahill et al. (31). Such equivalence points were noted with an initial perfusate glucose concentration of about 200 mg/100 ml in the normal, fasted rat; 60 mg/100 ml with preparations from adrenalectomized rats; and 340 mg/100 ml with livers from alloxandiabetic rats. These authors suggest that their observations may indicate the involvement of PPi-glucose phosphotransferase activity of glucose-6-phosphatase in hepatic glucose phosphorylation (the carbamyl-P: glucose phosphotransferase activity had not yet been discovered at that time—1967). Correspondence of the null-point for the diabetic animals, as experimentally determined by these workers, with predicted values presented in the present chapter (see specifically Fig. 22B) would certainly suggest that phosphotransferase activities of the classical hepatic hydrolase may indeed be involved. Further, a replot (see Fig. 25) in double-reciprocal fashion (104) of data from Fig. 1 of McCraw's paper (120) is linear. Extrapolations of

100

ROBERT C. NORDLIE

0.04 0.03

FASTED

'v 0.02 /

/

0.01 - _ / S ^J9 1 iotin) = 10 - 1 5 M) with free d-biotin derivatives including the biotinyl prosthetic group of acetyl-CoA carboxylase (88, 187, 188). Although the mechanism by which cleavage of the y—P—0 bond of ATP is coupled to carboxybiotin formation has not been elucidated, it is evident that at some point during the reaction nucleophilic attack by the bicarbonate anion occurs at the γ-phosphoryl phosphorus atom of ATP. This is supported by the fact that one 18 0 atom from 18 0-labeled HCO^ is incorporated into orthophosphate for every two atoms of 18 0 incorporated into the carboxyl of the carboxylated biotin prosthetic group of propionyl-CoA carboxylase (132). This is consistent with other evidence (213) that bicarbonate, rather than C0 2 , is the active species in the acetyl-CoA carboxylation mechanism. On the basis of the isotopic exchange (ATP-ADP- 14 C and ATP- 32 Pi exchange reactions) experiments described above and because evidence to the contrary has been lacking, the ATP-dependent carboxylation of biotin by HCOj" is generally visualized (132) as a concerted (Fig. 5), rather than a step wise, process. In view of the fact that ATP-ADP- 1 4 C and ATP- 3 2 Pi exchanges, catalyzed by acetyl-CoA carboxylase, require all substrates, products, and cofactors (i.e., ATP, ADP, Pi, HCO^, and divalent cation) of the first half-reaction [Reaction (3) or (5)], it appears that ADP and Pi are released from the enzyme only after carboxybiotin is formed. However, a stepwise mechanism is not excluded by these findings, since sequential chemical steps could precede the release of both products (ADP and Pi). An alternative mechanism outlined below [Reactions (12)-(14)] was considered (210, 211, 213) which involves carbonic-phosphoric anhydride as a tightly bound enzyme intermediate.

159

ACETYL COENZYME A CARBOXYLASE

o Enz-biotin

+ HO-~-O-

+

o 11

.

II

P-OADP

_0 / ' " 0_ ATP

2

(HO-C-O-PO a-) Enz-biotin

II

o

o

Mg2+

~

Mg2+

~

2

(HO-C-O-PO a-) Enz-biotin

(12)

(-O-ADP)

0 11

Enz-biotin-C-O-

+ Ho-poi- + -OADP

(13)

(-O-ADP) Net:

HO-C02"

Mg2+

+ ATP + Enz-biotin ~Enz-biotin-C02

+ ADP + Pi

(14)

This mechanism accounts for the net incorporation of 180 from 180-labeled HCO into Pi as well as for the results of the isotopic exchange studies referred to above. While the expected instability (97) of carbonic-phosphoric anhydride [structure (II)] seemed to preclude its preparation and study in aqueous solution, the possibility that the closely related analog, carbamyl phosphate [structure (III)], might function in its place was investigated (210,211,213).

a

o II

HO-C-O-po:(II)

(III)

Polakis et ale (210, 211, 213) demonstrated that the biotin carboxylase component of E. coli acetyl-CoA carboxylase catalyzes a reaction [Reaction (15)] analogous to the first step [Reaction (12)] of the hypothetical biotin carboxylation sequence. More recently it was found that avian liver acetyl-CoA carboxylase (213) and kidney pyruvate

o 11

H 2 N-C-0-PO;-

0

Me2+

q-biotin

II

+ ADP;==::::=:: H N-C-0- + ATP

Carbamyl phosphate

Be

2

(15)

Carbamate

carboxylase (12) carry out the same reaction at a slower rate and in the absence of exogenous biotin. In this model reaction carbamyl phosphate serves as phosphoryl donor, and ADP as acceptor. Phosphoryl transfer, catalyzed by the E. coli biotin carboxylase is rapid, exhibiting a V max about one-half that for the carboxylation of free biotin. Of particular importance is the nearly absolute dependence of phosphoryl transfer on free d-biotin. This requirement for d-biotin is an interesting, if not unexpected, feature of the biotin carboxylase-catalyzed phosphotransferase

160

M . DANIEL LANE, J O E L MOSS, AND S. E F T H I M I O S

POLARIS

reaction, since biotin does not appear to participate chemically in the reaction per se. Chemical substitutions (CH3—-, CH 3 C(—0) and C H 3 0 — C ( = 0 ) — ) at the l'-N position of biotin, i.e., at the site of enzymatic carboxylation (see Section III, B), and other modifications of the imidazolidone and thiophane rings, reduce, but do not abolish the abilities of these derivatives to activate phosphoryl transfer. It would appear, therefore, that occupancy of the biotin-binding site of biotin carboxylase induces conformational change(s) at the adjacent binding sites for HCO^ and ATP, thereby activating phosphoryl transfer. Hence, premature splitting of ATP, i.e., ATPase activity, would be forestalled until the "CO2" acceptor—the biotin prosthetic group—is in the carboxylation site. 2. CARBOXYL TRANSFER FROM THE CARBOXYBIOTIN TO ACETYL-COA

The occurrence of the second half-reaction [Reaction (4) or (6) ] involving carboxyl transfer from carboxybiotin to acetyl-CoA is indicated by the demonstration (93, 116, 178, 180) that acetyl-CoA carboxylase catalyzes an avidin-sensitive exchange between malonyl-CoA and 14 C-labeled acetyl-CoA, which is independent of the components of the first partial reaction [Reaction (3) or (5)]. Resolution of the E. coli carboxylase into subunits made it possible to show that only the carboxyl transferase and biotin-containing carboxyl carrier protein components are required for this exchange (5, 100, 106, 213). In view of the fact that the biotin carboxylase and carboxyl carrier protein subunits are needed for the exchanges (see Section III, D, 1) characteristic of the first halfreaction [Reaction (3)], it is evident that biotin carboxylase and carboxyl transferase have distinct functions unique to the first and second half-reactions respectively, whereas carboxyl carrier protein serves a dual role participating in both processes. Additional evidence for the participation of the second half-reaction [Reaction (4) or (6)] in the overall carboxylation process was provided by the finding(s) that the labeled carboxyl group of isolated 14C-labeled A^-carboxybiotinyl enzyme can be stoichiometrically transferred to acetylCoA or acetylpantetheine (195, 204, 225, 240) or decarboxylated by addition of ADP, Pi, and Mg 2+ (239); CCP-biotin-14CO^" undergoes analogous reactions, but requires the presence of carboxyl transferase or biotin carboxylase, respectively (61, 213). Furthermore, transcarboxylation from malonyl-CoA to free d-biotin (or its derivatives) forming free d-biotinCO^, a model for the second half-reaction [Reaction (4) or (6)], is catalyzed by the carboxyl transferase subunit of the E. coli acetyl-CoA carboxylase (100, 103, 213).

161

ACETYL COENZYME A CARBOXYLASE

By virtue of the fact that iV-carboxybiotin derivatives are relatively unstable, particularly at acidic pH's {103, 187), all acetyl-CoA carboxylases act as sluggish malonyl-CoA decarboxylases {189, 225) via Reactions (16)-(18). E-biotin-CO^ (acetyl-CoA)

> C 0 2 + E-biotin + acetyl-CoA (18)

(17)|t E-biotin + malonyl-CoA v (16)

Enz-biotin (malonyl-CoA) (19)\ C 0 2 + acetyl-CoA + E-biotin

It is evident that the enzyme per se facilitates this process since the ti/2 for the decarboxylation of free carboxybiotin is considerably greater than that for enzyme-biotin-COj" {103, 187). In addition to the biotindependent decarboxylation of malonyl-CoA, avian liver acetyl-CoA carboxylase also catalyzes malonyl-CoA decarboxylation via a biotinindependent pathway [Reactions (16) and (19)] as evidenced by its insensitivity to avidin {189). This is compatible with the decarboxylation of malonyl-CoA catalyzed by the biotin-free carboxyl transferase subunit of the E. coli carboxylase {100, 102, 213). 3. INTERSUBUNIT TRANSLOCATION OF THE BIOTINYL PROSTHETIC GROUP

The two half-reactions [Reaction (3), carboxylation and Reaction (4), transcarboxylation] which comprise the carboxylation of acetyl-CoA share a common intermediate, carboxylated carboxyl carrier protein (CCP-biotin-CO^). Compelling evidence (see Sections III, D, 1 and 2) indicates that these half-reactions take place at separate catalytic sites and are functionally linked by the biotinyl prosthetic group, which acts as a mobile carboxyl carrier. Investigations in Vagelos' {5, 6, 61, 195) and Lane's {50, 100, 102, 103, 213) laboratories on the resolution of E. coli acetyl-CoA carboxylase (see Section II, B) into subunits and the assignment of function to each reveals that the two catalytic sites reside on different subunits. The acetyl-CoA carboxylase from E. coli is composed of three functional components: (a) carboxyl carrier protein (CCP) which contains the covalently bound biotin prosthetic group, (b) biotin carboxylase which catalyzes the ATP-dependent carboxylation of CCPbound [Reaction (4)] or free biotin, and (c) carboxyl transferase which catalyzes carboxyl transfer from the CCP-biotin-CO^ or free carboxybiotin derivatives to acetyl-CoA. Although neither catalytic component, i.e., biotin carboxylase {6, 49, 50) or carboxyl transferase {5, 100), contains covalently bound biotin, both catalyze model reactions with free

162

M . DANIEL LANE, J O E L MOSS, AND S. E F T H I M I O S

POLARIS

biotin, which account for their respective catalytic roles in the halfreactions [Reactions (3) and (4)]. Hence, both the biotin carboxylase and carboxyl transferase components must contain distinct binding sites for the bicyclic ring of the prosthetic group. Both sites exhibit a high degree of specificity for the biotinyl moiety, structural or stereochemical alterations to the naturally occurring bicyclic ring of d-biotin resulting in greatly reduced activity (50, 100, 103, 213). The fact that the catalytic sites reside on different subunits, distinct from the carboxyl carrier protein, indicates that either the prosthetic group must oscillate between sites or the subunits bearing these sites must move with respect to the prosthetic group. Since the functional bicyclic ring of the prosthetic group resides at the end of a flexible 14 A side chain which anchors it to the apo-CCP (240), the biotinyl group should be capable of oscillating between the remote carboxylation and transcarboxylation sites on biotin carboxylase and carboxyl transferase as visualized in Fig. 6 (94, 100).

FIG. 6. Model for intersubunit translocation of the carboxylated biotin prosthetic group of acetyl-CoA carboxylase. BC refers to biotin carboxylase, C C P to carboxyl carrier protein, and C T to carboxyl transferase. From Guchhait et al. (100).

163

ACETYL C 0 E N Z Y M E A CARBOXYLASE

The finding that the protomeric form of the avian liver carboxylase is composed of four nonidentical subunits, one of which contains a covalently bound biotin prosthetic group (see Section II, A, 2) (94, 95, 105, l^S), is compatible with the complex subunit pattern of the E. coli carboxylase system. However, in the case of the avian liver enzyme, the molecular weight of the carboxyl carrier protein is about 117,000 (94, 105) compared to 22,000 for the E. coli carboxylase (61, 63) (see Section II, A, 2 and II, B). Further investigations will be necessary before functions can be assigned to each of the subunits of avian liver acetylCoA carboxylase. The model proposed (Fig. 6), which corresponds to a classical twosite "Ping-Pong" mechanism (39-41), has been shown on first approximation to apply to the acetyl-CoA carboxylase-catalyzed reaction. Numa and co-workers (113-115, 203) have obtained kinetic evidence which indicates that the carboxylation of biotin proceeds by an ordered mechanism with ATP binding prior to HCO7 and following carboxybiotin formation, and with Pi release subsequent to ADP. The reaction is postulated to proceed via a "bi bi uni uni Ping-Pong'' mechanism as shown in Reaction (20). ATP Enz-biotin

HCO3-

ADP

Pi

AcetylCoA

MalonylCoA

Enz-biotin-C0 2

Enz-biotin

It should be pointed out that nonclassical "Ping-Pong'' kinetics have been observed for the reaction catalyzed by the related biotin-dependent enzyme, pyruvate carboxylase (15, 181). IV. Regulation of Catalytic Activity A. General Aspects The reaction catalyzed by acetyl-CoA carboxylase satisfies the accepted criteria for a regulatory step in a biosynthetic pathway (186). This reaction occurs early in the fatty acid synthetic sequence and is the first committed step, malonyl-CoA having no other apparent metabolic alternative in animal tissues and Escherichia coli (Fig. 7). Being the first step after the acetyl-CoA branch point, the carboxylase-catalyzed reaction affords the earliest unique point at which control of lipogenesis can be exerted. Moreover, it has been shown that the carboxylation of acetyl-CoA is effectively* the rate-determining reaction of fatty acid * Earlier reports that fatty acid synthetase activity greatly exceeds that of the fully activated carboxylase are in error because optimal carboxylase assay conditions at physiological p H had not yet been determined (87, 71, 175, 203).

164

M . DANIEL LANE, J O E L MOSS, AND S. E F T H I M I O S ANIMAL

TISSUES

POLAKIS

E. coli glucose pyruvote

acetyl CoA

\ vcar ° boxy läse malonyl CoA acety^S-ACP

J malonyl-S-ACP

\acetyl-CoA *(*]

Icarboxylase

/ m a l o n y l CoA

i ! \

\

fatty-acyl-S-ACP

fatty acid

N

\

fatty acyl CoA

triglyceride (storage or export)

phospholipids (membranes)

phospholipids (membranes) FIG. 7. Comparison of the pathways of complex lipid synthesis in animal tissues and Escherichia colt. OAA, oxaloacetate; acetyl-S-ACP and malonyl-S-ACP, acetyland malonyl-acyl carrier protein.

synthesis in animal tissues (37) and therefore has regulatory potential. The control of acetyl-CoA carboxylase activity by allosteric effectors reflects the physiological role of the products of the pathway, i.e., fatty acids, triglycerides, and phospholipids, in various organisms. An outline of the biosynthetic pathways operative in animal tissues and in E. coli is presented in Fig 7 (164). A few interesting differences should be pointed out. In animal tissues the direct precursor of cytoplasmic acetyl-CoA utilized in fatty acid synthesis is mitochondrial citrate (14@) > a n d in E. coli acetyl-CoA is derived directly from pyruvate. The major fraction of fatty acids synthesized by animal tissues, particularly liver and adipose tissue, is exported or deposited in the form of glycerides as a reserve source of energy. Only a minor part of the fatty acids synthesized are used as structural components of membranes in the form of phospholipids, glycerides, and cholesterol esters. Biosynthetic processes leading to the storage of energy are frequently regulated by feed-forward activation. The fatty acid biosynthetic pathway in animal tissues fulfills such a role, and existing evidence (H6) (see Section IV, B, 1) strongly suggests

ACETYL COENZYME A CARBOXYLASE

165

that this pathway is regulated by citrate activation at the step catalyzed by acetyl-CoA carboxylase. The rationale for feed-forward activation is that the citrate level in the cytoplasmic compartment serves to indicate that acetyl-CoA, reducing equivalents, and energy in the form of ATP are all available for the biosynthesis of fatty acids. In contrast to animal systems where fatty acids are used principally as a reserve source of energy, in E. coli fatty acids are primarily incorporated into phospholipids, which in turn are used as structural components of membranes (45). Hence, regulation of fatty acid synthesis in this organism would be expected to reflect the requirement for phospholipids in growth and cell proliferation. Consequently, a control mechanism that coordinates the rates of synthesis of the components of the cell membrane to the rate of growth would seem advantageous. As will be discussed, (p)ppGpp appears to be such a mediator, regulating fatty acid synthesis at the level of acetyl-CoA carboxylase. B. Animal Carboxylases 1. CITRATE ACTIVATION

In 1952, Brady and Gurin (23) observed that fatty acid synthesis from acetate in pigeon liver extracts was markedly activated by certain tricarboxylic acids. Nearly ten years later, investigators in several laboratories (1, 129, 173-175, 178, 179, 258, 259) independently demonstrated that the site of activation by citrate or isocitrate was at the level of acetylCoA carboxylase rather than fatty acid synthetase. Since that time, it has become apparent that virtually all acetyl-CoA carboxylases from animal tissues, including avian, rat, and human liver, bovine and rat adipose tissue, and rat and rabbit mammary gland {87, 92-95, 99, 134, 147, 178, 185, 190, 191, 201, 206, 225, 230, 231, 240, 255, 256) are activated by tricarboxylic acids. The animal carboxylases have the unusual ability to oscillate between catalytically active polymeric and catalytically inactive protomeric states. Thus, when allowed to depolymerize fully to the protomeric state in assay medium, prior to addition of citrate or isocitrate, these enzymes exhibit an absolute requirement for tricarboxylic acid activator {92, 94, 188, 191). Citrate and isocitrate are almost equally effective, having similar maximal velocities and activator constants; the KAS for citrate and DL-isocitrate are Z-A mill" for the avian liver enzymes (94) and 3-4 m l and 7-8 mM, respectively, for the bovine adipose tissue enzyme (191). On the other hand, certain di- and tricarboxylic acids, including malonate, methylmalonate, malate, and tricarballylate—a closely related analog of citrate and isocitrate—behave as "pseudo" activators under certain circumstances. These acids appear to

166

M. DANIEL LANE, JOEL MOSS, AND S. EFTHIMIOS POLARIS

activate when the assay is initiated with carboxylase in the active polymeric form, but in fact, merely retard the rate of transition of active polymer to inactive protomer and are unable to reverse this process (92-94, 146)· "True" activators, such as citrate and isocitrate, are able to shift the equilibrium toward the catalytically active state presumably by inducing a productive conformational change in the protomer which favors polymerization (92, 93, 146, 188, 191). r/ireo-D s -Isocitrate is as active as DL-isocitrate indicating that the Ds-isomer is the active form (93). Fluorocitrate (93) and (-)-hydroxycitrate (108), a potent inhibitor of the citrate cleavage enzyme (ATP:citrate lyase)(£70), also serve as activators. In addition to the "pseudo" activators mentioned above, orthophosphate and a large number of other mono- and dibasic acids are without activity (93, 94)· Thus, the structural requirements for activation are quite specific. a. Dependence of Catalytic Activity on Polymeric State. In 1963, Vagelos, Alberts, and Martin (256) reported that activation of a crude adipose tissue acetyl-CoA carboxylase preparation by citrate was accompanied by an increased sedimentation velocity in sucrose density gradients. This observation was subsequently confirmed by Numa et al. (200) with rat liver carboxylase and in Lane's laboratory (92, 95) with chicken liver carboxylase. Since the earlier investigations involved impure carboxylase preparations, it could not be concluded whether conformational changes per se, complex formation between carboxylase and another component in the preparation, or carboxylase aggregation per se was responsible for the sedimentation velocity effect. It was subsequently shown by Gregolin et al. (92, 95) that activation of homogeneous avian liver acetyl-CoA carboxylase by tricarboxylic acid activator (Fig. 8) was accompanied by an increased sedimentation velocity of carboxylase protein per se in sucrose density gradients containing the carboxylase assay reaction mixture components with or without activator; under conditions approximating those of the enzymatic assay, citrate or isocitrate induce a change in s20,2 from 13-15 S to 47-50 S (92, 94, 95, 134, W, 191) (Table I). Moreover, this change in sedimentation velocity corresponds to a transition from a protomeric to a polymeric filamentous form as judged by electron microscopy (92, 134, 191) (Fig. 9) and a large increase in intrinsic viscosity, i.e., from [η] of 11.3 for the protomer to 83 for the polymer (188). The slowly sedimenting (13-15 S) species of the avian liver carboxylase found in assay reaction mixture without activator corresponds to the protomeric form, s^.w = 13-1 S, obtained by dissociating the polymer in 0.5 M NaCl, pH 8.0 {95)) as determined by sedimentation equilibrium, this form has a molecular weight of 410,000, which is in good agreement

167

ACETYL C0ENZYME A CARBOXYLASE 30 S

0.6

(/>


X

I

ASSAY MIX + ISOCITR

o GO

12 S

I \

0.2 or < o

—ASSAY MIX (NO ISOCITR.)

UJ

o

or a.

-HV-Ä-

FRACTION NUMBER FIG. 8. The effect of tricarboxylic acid activator on the sedimentation velocity of avian liver acetyl-CoA carboxylase in assay reaction mixture. Sedimentation was carried out using 5 to 20% sucrose density gradients, total volume 4.5 ml. Sucrose density gradients contained the components of the carboxylase assay reaction mixture: 0.06 M Tris (Cl"), p H 7.5; 2 m l A T P ; 8 m M MgCl 2 ; 0.01 M K H C O . ; 0.2 m M acetyl-CoA; and 0.1 m M E D T A . Homogeneous acetyl-CoA carboxylase, 200 /Ag, in 0.2 ml of 50 m M potassium phosphate buffer, p H 7.0, 0.1 m M E T D A was applied to each gradient. Gradients were centrifuged at 39,000 rpm (SW 39 Beckman-Spinco rotor) for 60 minutes at 25°C. Where indicated, 20 m M DL-isocitrate was added. O — O refers to carboxylase activity, and bar graphs to carboxylase protein. From Gregolin et al. (92).

with the minimal molecular weight calculated from the biotin content of the enzyme and binding data (94, 95). Thus it appears that acetylCoA carboxylases from animal tissues exist in a catalytically inactive protomeric state and a catalytically active polymeric state—the position of the protomer-polymer equilibrium (Fig. 9) determining the level of catalytic activity. Tricarboxylic acid activators notably citrate and isocitrate, appear to induce a conformational change (see Section IV, B, 1, b and c) by binding to the protomer, causing a shift in the equilibrium toward the active polymeric form. The protomeric forms of acetyl-CoA carboxylases from liver (188) and adipose tissue (191) exhibit a marked propensity to aggregate into filaments, polymerization occurring in the presence of tricarboxylic acid activator either in dilute (1 μg/uύ) or concentrated (1 mg/ml) solutions of homogeneous enzyme. It is evident that the protomer-protomer interaction is specific and is not merely a property of the purified carboxylases, since self-assembly also occurs in the presence of a complex mixture of liver or adipose tissue proteins. Hence citrate-induced polymerization occurs readily in the post-mitochondrial supernatant fractions of 0.25 M

168

M . DANIEL LANE, J O E L MOSS, AND S. E F T H I M I O S

A

4*

*

POLARIS

::r> · ?\$

- ä. **'

; ':>?>*

r.^fv.'l

&.4Γ '.'···

v

/ν ί

:

' . * "\ ν·· ··.^^#"*'. i

* v

;

:^Ι^^^:^^^'''^:-·:

-^

FIG. 9. Protomer-polymer transitions of animal acetyl-CoA carboxylase. P R O T O M E R (inactive)

ζ^

P O L Y M E R (active)

Equilibrium toward pro tomer favored by:

Equilibrium toward polymer favored b y :

ATPMg2+ + HCOmalonyl-CoA fatty acyl-CoA alkaline p H NaCl ( > 0 . 2 M ) low enzyme concentration

citrate, isocitrate phosphate albumin p H 6.5-7.0 high enzyme concentration

sucrose homogenates of avian liver or bovine adipose tissue as judged by sucrose density gradient centrifugation and electron microscopy (188). It is significant that tricarballylate, Pi, and malonate, which are not activators, increase the sedimentation velocity of the enzyme in simple buffering media at neutral pH, whereas under carboxylase assay conditions only citrate and isocitrate are capable of maintaining the rapidly

ACETYL C0ENZYME A CARBOXYLASE

169

sedimenting polymeric form; tricarballylate, Pi, and malonate are not {94)- This is due to the fact that the protomer-polymer equilibrium is shifted toward the protomer by converting the enzyme to its carboxylated form, enzyme-biotin-CCKr (94, 188). Sucrose density gradient centrifugation conducted under assay conditions for the carboxylase-catalyzed forward reaction, ATP- 32 Pi and malonyl-CoA-acetyl-CoA- 14 C exchanges, all of which are activated by citrate or isocitrate, showed that there is a strict correlation between activation and the transition from protomeric to polymeric form (92} 94, 95, 188, 191). Interestingly, under these conditions none of the other factors, such as tricarballylate, Pi, or malonate (which are capable of maintaining the polymeric form in simple buffering media) could be substituted for citrate or isocitrate (94)· Moreover, none of these factors was capable of activating any of the above-mentioned reactions. It became apparent that some component(s) in the carboxylation, exchange, and decarboxylation reaction mixtures must promote disaggregation to the protomeric form. The causative agents were identified as those substrates and cofactors, or combination thereof, which were capable of carboxylating the enzyme to form "enzyme-biotinCO^" (94, 95, 188). Thus, introduction of a carboxyl function at the l'-iV-position of the biotinyl prosthetic group produces sufficient conformational strain such that only true activators—that is, citrate and isocitrate, but not tricarballylate, Pi, or malonate—are able to constrain the enzyme and prevent its depolymerization (94)- Malonyl-CoA which can carboxylate the enzyme is a competitive inhibitor of the overall reaction, Ki = ^ Ι Ο - 5 Μ, with respect to citrate (37, 95). This competitive kinetic relationship apparently results from the opposing effects of citrate and malonyl-CoA on the protomer-polymer equilibrium (Fig. 9). This effect has been verified by direct electron microscopic investigation as well. Avian liver acetyl-CoA carboxylase in 50 m l potassium phosphate buffer, pH 7.0, exists in the polymeric filamentous form; however, upon the addition of as little as 10 μΜ malonyl-CoA, which carboxylates the enzyme, there is an instantaneous (within seconds) depolymerization to yield protomeric forms having the typical protomer dimensions of 50-130 A (94). As pointed out (see Section III, B), conformational strain induced by carboxylation of the enzyme may be utilized for the electrophilic activation of the iV-carboxyl group of the biotinyl prosthetic group (a notoriously poor electrophile) by changing its bond angle or length as a result of strain or distortion induced by conformational changes at the active site. Thus, although carboxylation at the l'-N position of the biotinyl moiety induces sufficient conformational strain to cause depolymerization, it is evident that the binding of its allosteric activator, citrate, pre-

170

M. DANIEL LANE, JOEL MOSS, AND S. EFTHIMIOS POLAKIS

vents this dissociation presumably by constraining the enzyme in the active conformation compatible with the polymeric state. Under conditions where the enzyme is not carboxylated, the KD for citrate is 3 μΜ; however, under assay conditions, that is, where the enzyme is carboxylated, the activator constant for citrate, KA is 3 mM (94)- I t is suggested that the 1000-fold difference between KD and KA may reflect the binding energy needed for structural constraint to maintain the active conformation of enzyme-biotin-CO^. In this connection, Edwards and Lane (60) have found that avidin, which binds free- or enzymebound biotin with remarkable affinity (KD{Tee biotin = 10~15 M) (88), enhances the decarboxylation of enzyme-biotin-CO^; the ti/2 of enzymebiotin-CO^ at 2°, pH 7.5, of about 200 minutes is reduced to < 1 minute when treated with avidin. It is suggested that this effect may result from strain induced in the carboxyureido system owing to the extraordinarily tight binding of the carboxybiotinyl group by avidin. A number of other factors have been found to promote depolymerization, hence reversible inactivation of the enzyme. These include elevated salt concentration, e.g., 0.5 M sodium chloride, elevated pH, and a number of inhibitors of potential physiological importance, such as long-chain acyl-CoA derivatives {95, 200, 205) (see Section IV, B, 2). b. Kinetics of Protomer-Polymer Transitions and the Relationship to Catalytic Activity. Although the position of the protomer-polymer equilibrium appears to determine the level of acetyl-CoA carboxylase activity, it has been uncertain whether activation is a consequence of polymerization per se. Whereas citrate-induced polymerization and activation are complete within seconds, the methods that have been used to assess changes in polymeric state (sedimentation velocity, viscosity, and electron microscopy) are too slow to be of use in following the kinetics of polymerization. The depolymerization process, however, is several orders of magnitude slower and has been studied in relation to the loss of catalytic activity (188). By taking advantage of the slow rate of depolymerization of the filamentous form of the avian liver carboxylase in assay reaction mixture without activator (see Section IV, B, 1, a) at low temperature and high enzyme concentration, Moss and Lane (188) have obtained kinetic evidence for the coupling of the loss of activity to the polymer -» protomer transition. As illustrated in Fig. 10, the viscosity of the polymeric form of the enzyme added to assay reaction mix without activator undergoes first-order decay (t1/2 = 9.5 minutes) at a rate equal to that of activity decay (t1/2 = 10 minutes) indicating tight coupling of the polymer -> protomer transition with loss of catalytic activity. The initial value at t = 0 reflects only polymer, while the η&ν/0 obtained following extensive decay is due exclusively to protomer. The extrapolated value of 77Sp/c at t = 0 agrees well with the intrinsic viscosity ([η] = 83) of

171

ACETYL COENZYME A CARBOXYLASE

400 r h

\

200

14 0

>

Citrate Added

#

100 o 80

\

\

Γ '\ •V>

\

>

30 20

\Λ

8

#

\

\

viscosity

\

10

10

Q. C

1

20

u7

ωύ

0 o

6 ^ ^ £ x o

ΟΘ-

DD ° 4 a: * ^

\

< σ u .if

\

o v

\\_ \

o

1

30 MINUTES

3

I

^

V^ v o o

L_

Γ

· \

k —

6

fc

>I

activity

\Ä

\ · \ \\ \°

•"•H

20

> E 1-

^ 6 0 40 1

H

r-·-

Kp

1

E

\

2

21 ! \ L

1

40

*

Q. U

50

J

60

FIG. 10. Kinetic correlation of reversible viscosity and activity decay of polymeric acetyl-CoA carboxylase. The carboxylation reaction was initiated by the addition of 194 /-tg of homogeneous acetyl-CoA carboxylase in its polymeric state (in 50 m l potassium phosphate, p H 7.0) per milliliter of carboxylase assay reaction mixture (minus citrate) of the same composition as in Fig. 8. Viscosity measurements were made directly on the reaction mixture, and 0.1-ml aliquots were taken for the determination of 14 C-labeled bicarbonate fixation rates at the time indicated (times recorded for η^/c represent the midpoints of the determinations). The reaction mixture was held at 2°C during all operations. After 52 minutes potassium citrate (final concentration, 10 m l ) was added to the reaction mixture after which additional viscosity and carboxylase activity measurements were made. From Moss and Lane (188).

the polymeric form {188). Addition of citrate following inactivation (Fig. 10) causes an immediate rise in r/sp/c, as well as reactivation of the inactive enzyme; although some denaturation occurs, it is evident that the relative activity regain approximates that of relative ^ sp/c regain induced by citrate addition. Interestingly, the presence of avidin in the assay reaction mixture during decay does not alter the t1/2 for activity of viscosity decay. Avidin is known {188, 225) to instantaneously inactivate the protomeric form of the carboxylase by binding irreversibly to the biotinyl

172

M. DANIEL LANE, JOEL MOSS, AND S. EFTHIMIOS POLAKIS

prosthetic group: in contrast, the polymeric form in the presence of citrate, is completely resistant to avidin (188). Thus, during activity decay associated with depolymerization only that fraction of carboxylase activity remaining (i.e., that fraction still in the polymeric state) can be protected by citrate from further erosion of activity and inactivation by avidin (188). The fact that activity and viscosity decay, the kinetics of disappearance of the avidin-resistant polymeric form, and the rate of appearance of the avidin-sensitive protomeric species are closely correlated strongly indicates tight coupling between the loss of catalytic activity and the polymer —> protomer transition. Thus, a strong case is made for a causal relationship between the activation of animal acetylCoA carboxylases and a polymerization-associated conformational change (s) induced by citrate. As will be discussed later (see Section IV, B, 1, c), the inaccessibility of the biotin prosthetic group in the citrate-activated polymeric form of the enzyme indicates an activatorinduced conformational change in the vicinity of the active site (i.e., near the biotinyl prosthetic group). On the basis of the kinetics of viscosity and activity changes, Moss and Lane (188) suggest that the depolymerization mechanism involves a "one-shot" or concerted disruption of the filament structure with no, or only short-lived, intermediate oligomeric species. This model visualizes the occurrence of an initial break in the filament followed by a series of far more rapid cleavages which quickly lead to complete deploymerization. The absence of a rapid drop in viscosity prior to significant decay of activity, tends to negate a random cleavage mechanism or depolymerization in which smaller active oligomeric intermediates accumulate. c. Activation Mechanism. Citrate qualifies as allosteric activator (186) for animal acetyl-CoA carboxylases by virtue of the absolute dependence of the carboxylase-catalyzed reactions on tricarboxylic acid activator, its allo-, rather than iso-, steric structure compared to those of substrates for the reaction, and the striking conformational change (s) it elicits in the enzyme (see Section IV, B, 1, a and b). Moreover, there is a sound metabolic basis for regulation of the carboxylase by citrate. According to Monod, Wyman, and Changeux (186), two classes of allosteric effectors are likely to be encountered in enzyme systems, namely, those which promote F m a x or Km effects. In the case of the avian liver (93) and bovine adipose tissue (191) carboxylases, the principal kinetic effect of tricarboxylic acid activator appears to be on the F m a x for the reaction, the Km values for the substrates, A T P M g 2 + , HCO^~, and acetyl-CoA, not being materially affected by citrate or isocitrate. Further, the results of direct binding experiments show that the affinity of the avian liver enzyme for acetyl-CoA is not affected by citrate (94).

ACETYL C0ENZYME A CARBOXYLASE

173

In contrast, recent reports from Numa's laboratory (113, 115, 203) with rat liver enzyme suggest that citrate reduces the apparent Km value for acetyl-CoA. However, in these experiments assays were initiated with enzyme that had undergone preliminary activation with high levels of citrate (10 m l ) prior to starting the reaction. The fact that the polymerprotomer equilibrium is slowly attained when the reaction is initiated with polymer (see Section IV, B, 1, b and references 188 and 191) would be expected to lead to erroneous kinetic results. Failure to take this into consideration would result in hybrid reaction rates primarily characteristic of the polymer, even in the absence of citrate. In the experiments of Gregolin et at. (93) and Moss et al. (190, 191) in which Vm&yi, rather than Km effects were noted, care was taken to allow the carboxylase to equilibrate prior to initiating the reaction. It appears, therefore, that activation by citrate increases the reaction rate of bound substrate, rather than altering the affinity of the enzyme for substrates; hence citrate should be classified as a positive allosteric effector of the F m a x type (186). This is consistent with the findings that both partial reactions [Reactions (5) and (6)], which are involved in the overall carboxylation, are activated by citrate and isocitrate (93, 95, 96, 178). The fact that the exchanges that characterize the first half-reaction have no acetyl-CoA requirement (93, 96, 178) and those that characterize the second halfreaction have no ATP, ADP, Pi, Mg 2+ , or HCO^~ requirement, although both exhibit citrate activation, supports the view that a Fmax, rather than a Km, effect is of primary kinetic importance in the activation mechanism (93, 95, 178, 225). All the carboxylase-catalyzed reactions which involve participation of the biotinyl prosthetic group are activated by tricarboxylic acid activator. These include the overall forward and reverse reactions (92, 93), ATP- 3 2 Pi exchange (93, 178), ATP-ADP- 1 4 C exchange (96), malonyl-CoA-acetyl-CoA- 14 C exchange (93, 95, 178, 225), avidin-sensitive enzyme-biotin-CO^ decarboxylation (188, 225), and carboxyl transfer from enzyme-biotin-CCKT to acetyl pantetheine (240). In addition, the carboxylation of free d-biotin to form free l'-N-carboxyd-biotin, an enzymatic model for the first half-reaction [Reaction (5)] is markedly activated by citrate (240). These findings implicate a substituent at the active site of the enzyme, which is involved in the ratelimiting steps of both half-reactions, as the locus of the citrate-induced conformational change (14?)· The most obvious substituent common to both partial reactions is the biotinyl prosthetic group. Compatible with this view is the fact that evidence (147, 188, 225) has been obtained for a citrate-induced conformational change at the active site which is reflected in a change in the environment of the prosthetic group. The accessibility of the biotinyl group to avidin, the specific biotin-

174

M. DANIEL LANE, JOEL MOSS, AND S. EFTHIMIOS POLARIS

binding protein from egg white, has been investigated in Lane's laboratory (14?) 188, 225). Avidin inactivates biotin enzymes by binding almost irreversibly to the bicyclic ring of biotin, thereby blocking its functional ureido group (88, 187). It was found that the biotin prosthetic of the citrate-activated enzyme is completely inaccessible to avidin, whereas in the absence of the activator the biotinyl group is accessible to avidin (188). Moreover, acetyl-CoA can partially protect the carboxylase from avidin. In experiments in which the rates of inactivation were increased by raising the avidin to carboxylase ratio, the protection afforded by acetyl-CoA was synergistic with activator (147, 225). As will be discussed later, tricarboxylic acid and acetyl-CoA also act synergistically in the activation of enzyme-biotin-CO^ decarboxylation. The specificity pattern for the activation of the carboxylase and protection from inactivation by avidin are similar; citrate and isocitrate, which are activators, protect the enzyme from avidin, whereas tricarballylate neither activates nor protects (143, 225). Apparently, the biotin prosthetic group becomes shielded by neighboring groups as a result of activator-induced conformational changes at the active site. In view of the apparent changes in conformation near the biotin prosthetic group which accompany citrate activation, the possibility has been considered that the reactivity of the biotinyl- or A'-carboxybiotinyl group might also be altered. Studies on the effect of tricarboxylic acid activator on avidin-sensitive malonyl-CoA and enzyme-CO^ decarboxylation, both of which involve decarboxylation of the iV-carboxybiotinyl prosthetic group as the rate-limiting step, indicate that the activator greatly enhances the reactivity of this group (143, 147, 189, 225). In the course of investigations on the malonyl-CoA-acetyl-CoA- 14 C exchange, it was observed that acetyl-CoA carboxylase also catalyzes a citrate- and acetylCoA-dependent decarboxylation of malonyl-CoA (225); although this reaction is slow, it serves as an indicator of conformational changes at the active site of the enzyme. The carboxylase supports only a slow avidin-insensitive malonyl-CoA decarboxylation which is essentially unaffected by acetyl-CoA, but is increased somewhat by addition of isocitrate (189). Activation of the carboxylase-catalyzed decarboxylation occurred when both isocitrate (or citrate) and acetyl-CoA were present simultaneously. A synergism between citrate and acetyl-CoA was evident from the autocatalytic character of the kinetics of decarboxylation in the presence of citrate (225). That this is due to the generation of acetyl-CoA from the decarboxylation reaction per se was demonstrated by the reversion from autocatalytic kinetics to zero-order kinetics when an acetyl-CoA trapping system (arsenate and phosphotransacetylase or oxaloacetate and citrate synthase) was added. Like all the other acetylCoA carboxylase-catalyzed reactions, malonyl-CoA decarboxylation is

175

ACETYL COENZYME A CARBOXYLASE

not activated by tricarballylate (22). The citrate-activated and acetylGoA-activated decarboxylation of malonyl-GoA can be described by the following minimal reaction sequence: CO 2

biotin

I

E

+ M-CoA

biotin (1') ~

I

E(M-CoA)

1

4'

(2') ~

I biotin I

E(A-CoA)

1

5'

CO 2

I I

biotin (3') ~

E

+ A-CoA

16 '

CO 2

CO 2

CO 2

biotin

biotin

biotin

E(A-CoA)

E(A-CoA)

E

+

+

I

I

(21)

+ I

(E represents the enzyme; M-CoA, malonyl-CoA; and A-CoA, acetyl-CoA)

Malonyl-GoA-acetyl-CoA-14G exchange, which involves only the reversible Reactions (1'), (2'), and (3'), is considerably faster than the decarboxylation of malonyl-GoA under comparable conditions either in the presence or in the absence of citrate. Therefore, Reaction (4'), (5'), or (6'), each of which has been measured independently (143,147,189,225), must be rate-limiting for malonyl-GoA decarboxylation. Reaction (4') which is biotin independent (avidin-insensitive) (189) and Reaction (6') (143, 147, 225) are at least an order of magnitude slower than Reaction (5'). Reaction (4'Y, which as pointed out earlier (Section III, D) does not involve the biotin prosthetic group, is not activated by tricarboxylic acid activator. Since the rapid removal of acetyl-GoA by a trapping system drastically reduces the rate of malonyl-GoA decarboxylation [to a rate equal to that of Reactions (4') plus (5')], it is apparent that: (a)

CO 2

I

biotin

I

E(A-CoA)

undergoes decarboxylation [Reaction (5')] more rapidly than CO 2

I

biotin [Reaction (6')]

I

E

(b) Tricarboxylic acid and acetyl-GoA, the carboxyl acceptor substrate, activate the decarboxylation of the carboxybiotinyl prosthetic group synergistically.

176

M.

DANIEL L A N E , J O E L

MOSS, AND

S.

EFTHIMIOS

POLAKIS

TABLE III E F F E C T OF C I T R A T E AND A C E T Y L - C O A ON THE R A T E OF DECARBOXYLATION OF " Ε Ν Ζ Υ Μ Ε - Ο Ο Γ " "

Additions

Rate of enzyme-C0 2 decarboxylation 6 (min - 1 )

None + Citrate (10 m M ) + Acetyl-CoA (0.5 μΜ) + Citrate + acetyl-CoA (0.5 μΜ)

0.034 0.110(0.086) 0.063 (0.029) 0.635 (0.601)

a

From Lane et dl. {143). Determined at p H 7.5 and 25°C with the avian liver carboxylase. Values in parentheses are increments above control value. h

Investigations on the decarboxylation of malonyl-CoA indicated that tricarboxylic acid activator and acetyl-CoA activate the rate-limiting step, i.e., the decarboxylation of enzyme-biotin-CO^. These findings were corroborated in a more direct manner by following the rate of decarboxylation of enzyme-biotin-14CCKr per se in the presence and in the absence of citrate and/or acetyl-CoA (143, 147, 225). Citrate markedly activates the decarboxylation of enzyme-biotin-CO^ at pH 7.5 and 25° leading to a 4- to 5-fold increase in the first-order decarboxylation rate. The effect of citrate is greatly enhanced by the presence of less than saturating concentrations of acetyl-Co A, as shown in Table III. At an acetyl-CoA concentration of 0.5 μΜ, the addition of citrate activates enzyme-biotin-CO^ decarboxylation at least 10-fold. In these experiments, the equilibrium described by Reactions (Γ), (2')> and (3') (above) is rapidly established. Since the method used to follow the decay of 14COj" measures the sum of the rates of decay of enzyme-biotin- 14 CO^ and malonyl-CoA-3-14C, the effect of activator on the rate of decarboxylation of enzyme-biotin-14COj" is underestimated because the biotin-independent decarboxylation of malonyl-CoA is unaffected by activator (189). It is evident that the simultaneous presence of citrate and acetyl-CoA renders enzyme-biotin-CO^ more susceptible to decarboxylation, presumably because of conformational changes at the active site which enhance the reactivity of the carboxy group of carboxybiotinyl enzyme. One possible explanation for the citrate effect, illustrated in Fig. 11, is that the carboxybiotin prosthetic group may be brought into closer proximity to substrate binding sites by activator-induced conformational changes (147, 187). Insight into the effect of citrate on the biotin prosthetic group was gained in experiments in which "free" d-biotin served as a model for the covalently bound prosthetic group, i.e., in the ATP-

177

ACETYL COENZYME A CARBOXYLASE

♦ Citrate Enzyme

Enzyme

FIG. 11. Postulated scheme for the effect of tricarboxylic acid activator on the reorientation of the active site(s) of acetyl-CoA carboxylase. From Lane et al.

am. dependent carboxylation of ""free" biotin to yield carboxybiotin [Reaction (22)] and in carboxyl transfer from malonyl-CoA to "free" biotin to form carboxybiotin [Reaction (23)] which serve as model reactions for the halfreactions of acetyl-CoA carboxylation (see Section III, D). d-Biotin + HCO^ + ATP Malonyl-CoA + d-biotin

Mg2+

l'-iV-carboxy-d-biotin + ADP + Pi

(22)

± I'-iV-carboxy-d-biotin + Acetyl-CoA

(23)

Since secondary binding sites must be required to precisely orient the ureido ring system with respect to the substrates with which it must react, i.e., ATP-Mg 2 + and HCO^ at the biotin carboxylation site and acetyl-CoA at the carboxyl transfer site as visualized in Fig. 6 for the E. coli acetyl-CoA carboxylase, it would be expected that in the presence of citrate-free d-biotin and its closely related analogs would probably not compete favorably with the bicyclic ring of the prosthetic group at the carboxylation or carboxyl transfer site. Consistent with this prediction is the fact that the Km values for free d-biotin, d-homobiotin, and biocytin in the ATP-dependent carboxylation reaction at the biotin carboxylase site are markedly increased by citrate (see Table IV) {143, 240). A similar situation seems to prevail at the carboxyl transferase site. As shown in Table V, citrate nearly completely blocks carboxyl transfer from malonylCoA to "free" d-biotin and biocytin (189). On the basis of these findings, Lane and co-workers (143, 146, 147, 187, 189) have proposed that regulation of the animal acetyl-CoA carboxylases by citrate activation is mediated through a conformational change (s) which perfects the orientation of the biotin prosthetic group with respect to the biotin carboxylase and carboxyl transfer sites. d. Tissue Citrate Concentration. In order to attribute an effector role to citrate in the regulation of lipogenesis, the rate of fatty acid synthesis must reflect the citrate concentration at the specific site of synthesis. To date all investigations (14, 28, 52, 86, 89, 107, 123, 208, 234, ®37,

178

M . DANIEL

LANE,

JOEL

MOSS, AND S.

EFTHIMIOS

POLAKIS

T A B L E IV E F F E C T OF C I T R A T E ON THE Km

AND F m a x V A L U E S FOR THE

ATP-

D E P E N D E N T CARBOXYLATION OF F R E E B I O T I N D E R I V A T I V E S 0 · 6

Km ( m l )

F m a x (relative, %)

Derivative

Without citrate

+ Citrate

Without citrate

+ Citrate

d-Biotin d-Homobiotin Biocytin

10 19 12

45 200 50

11 10 17

100 100 151

a

From Lane et al. (147). F m a x is expressed as percent relative to t h a t for free d-biotin in the presence of citrate (5 nmoles per minute per milligram of avian liver carboxylase at 37°C). b

TABLE V E F F E C T OF C I T R A T E ON CARBOXYL T R A N S F E R FROM M A L O N Y L - C O A TO B I O T I N AND B I O C Y T I N 0 · 6

R a t e of acetyl-CoA formation (nmoles/min/mg enzyme) Additions

Without citrate

-f- Citrate

None d-Biotin Biocytin

22 71 86

19 18 30

° From Moss and Lane (189). Carboxyl transfer was determined using the avian liver carboxylase; citrate, d-biotin, and biocytin were added at a concentration of 9 m M . 6

252, 275-277) aimed at correlating acetyl-CoA carboxylase activity with hepatic citrate levels suffer the serious limitation that citrate concentrations have been determined for the whole tissue and not for the extramitochondrial compartment where fatty acid synthesis is localized. Cell fractionation procedures are presently too slow and citrate enzymatically too labile (234) to permit fractionation prior to citrate analysis. Since there is reason to believe (227) that the distribution of citrate markedly favors the mitochondrial compartment, correlations of the rate of fatty acid synthesis with total tissue concentrations may be grossly misleading. Moreover, conflicting results on the correlation of hepatic fatty acid synthetic rates to citrate concentration in different physiological states have been

ACETYL COENZYME A CARBOXYLASE

179

reported (14, 28, 52, 86, 89, 107, 123, 208, 234, 237, 238, 252, 275-277). For example, fat feeding (107) or the perfusion of liver with oleate (277) causes a 2- to 3-fold rise in hepatic citrate levels and a decreased rate of fatty acid synthesis. While differences in compartmentation may account for this apparent lack of correlation between citrate level and synthetic rate, it is conceivable that a dual regulatory mechanism is operative, i.e., an overriding of citrate activation through inhibition by fatty acids or their CoA derivatives whose concentrations rise under these circumstances (85, 86, 107). Several recent studies indicate that hepatic citrate concentration is markedly depressed by fasting (14, 28, 107, 237, 238) while certain other investigations show no effect. It has been suggested (238) that these discrepancies may in part result from differences between the reported length of fasting and the actual time at which feeding ceased. An oversight of this type would lead to an underestimate of hepatic citrate concentrations in fed controls. In experiments not subject to this limitation, the simultaneous administration of insulin and glucose to fasted rats which is known to promote lipogenesis leads to increased hepatic citrate levels (123). The citrate concentration of whole liver in the fed state is approximately 0.6-1.0 m l (107, 123, 234). This concentration is two orders of magnitude greater than the dissociation constant (94) found for citrate with the avian liver acetyl-CoA carboxylase (KD = 0.003 mM), but somewhat lower than the activator constant (93, 225) for citrate (KA = 2-3 mM). 2. INHIBITORS

Malonyl-CoA is a potent inhibitor of the avian liver acetyl-CoA carboxylase (37, 95), exhibiting a Ki of about 10-5 M. It has been established that inhibition by malonyl-CoA is competitive, both with respect to acetyl-CoA and tricarboxylic acid activator (95). While competitive inhibition by malonyl-CoA with respect to acetyl-CoA is of the "classical" type, the competitive relationship between malonyl-CoA and isocitrate is best interpreted in terms of the protomer ^± polymer equilibrium shown in Fig. 9. Citrate and isocitrate cause a shift in the equilibrium toward the catalytically active polymeric form, and malonyl-CoA is known to promote depolymerization (94, 95), thereby shifting the equilibrium toward the catalytically less-active protomer (see Sections IV, B, 1, a and b). The fact that the capacity of the carboxylase to generate malonyl-CoA approximately equals the capacity of the synthetase to incorporate malonyl-CoA into long-chain fatty acids suggests that circumstances arise in vivo, when the malonyl-CoA concentration would be sufficient to inhibit carboxylase activity (37). Inhibition of acetyl-CoA car-

180

M . DANIEL LANE, J O E L MOSS, AND S. E F T H I M I O S

POLAKIS

boxylase by malonyl-CoA may provide a safeguard against excessive acetyl-CoA utilization for malonyl-CoA production, thereby providing fine control over the committed step in the synthetic process. Long-chain fatty acyl-CoA derivatives, such as palmityl-CoA, stearylCoA, and oleyl-CoA, are inhibitors of hepatic acetyl-CoA carboxylase and are effective at extremely low concentrations, causing half-maximal inhibition at concentrations of 3· to 8 X 10~7 M (19, 200, 205). The corresponding free fatty acid salts are not inhibitory at similar concentrations. Since fatty acyl-CoA derivatives are end products of this biosynthetic process, a reasonable metabolic role can be visualized for these compounds as feedback inhibitors of an early step in the process. Such a role would be consistent with the observation that fasting (162, 176, 182, 203), fat feeding (124), and alloxan diabetes (21, 36, 38, 55, 272) which are known to depress hepatic fatty acid synthesis, lead to an increased long-chain fatty acyl-CoA concentration in liver (18, 20, 74, 75, 107, 251, 252). In the fasted and alloxan diabetic states fatty acids are mobilized from extrahepatic tissues and like fat feeding lead to an increased flux of fatty acids to the liver. Feeding carbohydrate to fasted animals restores fatty acyl-CoA levels to near normal values (107, 252). Like the inhibition of liver acetyl-CoA carboxylase by malonyl-CoA, inhibition by palmityl-CoA is competitive with respect to the activator, citrate, whereas, it is noncompetitive with respect to substrates (84, 156, 200, 205). The competitive kinetic relationship between palmityl-CoA and citrate and also the fact that palmityl-CoA prevents the citrate-induced increase in the s20w of the enzyme is most recently explained on the same basis as the inhibition by malonyl-CoA, i.e., through effects on the protomer-polymer equilibrium (Fig. 9) (see Sections IV, B, 1, a and b). In this case, however, palmityl CoA would be expected to promote depolymerization by binding to the catalytically less-active form of the carboxylase. It is known that long-chain acyl-CoA derivatives have strong detergent properties, e.g., palmityl-CoA has a low critical micelle concentration of 2 to 4 X 10~6 M (280). Moreover, palmityl-CoA inhibits a number of enzymes of wide diversity some of which are seemingly unrelated to this thioester in a regulatory sense (236, 243). For these reasons, as well as the fact that serum albumin or high protein concentration can prevent or reverse the inhibition (73, 84, 142, 157, 205, 273, 274), arguments have been raised against a role for fatty acyl-CoA derivatives as physiological inhibitors (54, 72, 142, 157, 236, 243, 274, 280). Serum albumin possesses hydrophobic sites capable of binding fatty acyl-CoA derivatives which would explain its protective effect (53, 67, 83, 109, 233). A physiological role for these inhibitors is not ruled out, however, on the basis of these arguments, rather the question must remain open until

ACETYL COENZYME A CARBOXYLASE

181

more definitive experiments are performed. In this connection Goodridge (84) recently has demonstrated that inhibition of the avian liver acetylCoA carboxylase by palmityl-CoA even in the presence of serum albumin can be reversed by citrate. Considered in light of the competitive character of inhibition by fatty acyl-CoA's with respect to citrate (84, 156, 200, 205), a rational argument can be made for dual control by these effectors based on their opposing effects on the protomer-polymer equilibrium (see Sections IV, B, 1, a and b; Fig. 9). There is abundant evidence indicating that a natural hydrophobic inhibitor (s) of acetyl-CoA carboxylase is present in crude enzyme extracts of liver and adipose tissue (69, 70, 90, 93, 172, 190, 191). The activating effect of (-(-)-palmityl carnitine on fatty acid synthesis in crude liver extracts and on impure acetyl-CoA carboxylase preparations has tentatively been ascribed to the displacement of hydrophobic inhibitors such as fatty acids or fatty acyl-CoA derivatives (69, 70, 90, 172, 190, 191). Inhibition of rat liver acetyl-CoA carboxylase by added palmityl-CoA can be reversed in part by (-f)-palmityl carnitine (70, 84). This activating effect is not specific with respect to (-f-)-palmityl carnitine in that cetyl trimethylammonium ion is also effective (90). Furthermore, impure preparations of acetyl-CoA carboxylase from adipose tissue or rat liver are markedly activated by serum albumin (79, 172, 191) or extensive dilution of the enzyme preparation prior to assay (190, 191). On the other hand, none of these agents [ (-(-)-palmrtyl carnitine, serum albumin or dilution], which activate the impure carboxylase, have an activating effect on the homogeneous acetyl-CoA carboxylases from adipose tissue or liver (190, 191, 226). It is evident that an inhibitory substance, apparently hydrophobic in nature, is removed either by purification of the enzyme or by the agents or treatments mentioned above. Several hypolipidemic drugs (2-methyl-2-phenoxypropionate derivatives) have been found to inhibit acetyl-CoA carboxylase (167-171). All appear to inhibit competitively with respect to acetyl-CoA and tricarboxylic acid activator and noncompetitively with respect to ATP and HCO~. These inhibitors, like malonyl-CoA and palmityl-CoA, are antagonistic to the activator in that they tend to reverse the activator-promoted aggregation of the carboxylase (see Fig. 9). Confirmation of the inhibitory effect of these drugs on lipogenesis at the carboxylase-catalyzed step in vivo would lend substantial support to the proposed regulatory role of the carboxylase. 3. COVALENT MODIFICATION

3',5'-Cyclic adenosine monophosphate (cAMP) or its iV 6 ,0 2/ -dibutyryl derivative inhibit the incorporation of acetate- 14 C or glucose-14C into

182

M . DANIEL LANE, J O E L MOSS, AND S. E F T H I M I O S

POLAKIS

hepatic fatty acids both in vivo (223) and with liver slices in vitro (4, 10, 11, 25, 26, 32, 33, 78, 153, 245). That this effect is due to a decreased rate of de novo fatty acid synthesis, rather than to a dilution of r e labeled intermediates of the pathway caused by cAMP-activated glycogenolysis, as suggested by Rous (223), is indicated by the finding that dibutyryl 3',5'-cAMP also blocks the incorporation of 3 H 2 0 into fatty acids (98, 245). Several recent preliminary reports (11, 32, 78, 153) propose that acetyl-CoA carboxylase may be regulated by a phosphorylation-dephosphorylation mechanism. Thus, Kim and Carlson (32) observed that carboxylase activity of crude rat liver extracts was activated by preliminary incubation wth Mg2+. Moreover, the carboxylase activities of rat liver (32) and rat adipose tissue (153) extracts were inactivated by incubation with ATP, these effects being independent of cAMP; although 32 P-labeling of protein by y-32P-labeled ATP occurred (32), definitive proof that the carboxylase per se was labeled is lacking. These results are not compatible with the finding of Alfred and Roehrig (11) that acetyl-CoA carboxylase activity is depressed in liver slices by cAMP. It should be noted that investigations by Guchhait and Lane (98) have been unable to confirm the above-mentioned effect of cAMP on acetyl-CoA carboxylase activity, nor has it been possible to demonstrate the phosphorylation in vitro of the carboxylase by y-32P-labeled ATP. It has been our experience, however, that, like crude carboxylase preparations, the homogeneous enzyme (which is free of protein kinase activity) undergoes a slow inactivation in the presence of ATP-Mg 2+ . However, this effect most likely results from the carboxylation of the enzyme yielding enzyme-biotin-CO~ which readily depolymerizes to form the relatively unstable inactive protomeric species (see Section IV, B, 1, a and b). Furthermore, Inoue and Lowenstein (126) have shown that although the homogeneous rat liver acetylCoA carboxylase contains one covalently bound phosphoryl group per enzyme subunit, the enzyme is fully active. It is evident that more definitive experiments will be required before a covalent modification mechanism can be implicated in the regulation of acetyl-CoA carboxylase. 4. POSSIBLE STRUCTURAL ROLE OF CARBOXYLASE FILAMENTS

Most intracellular enzymes are composed of subunits or protomers that interact to form oligomeric structures having "closed configurations" (135), thus a fixed number of subunits. This is in contrast to a unique class of filamentous enzymes, including animal acetyl-CoA carboxylases (see Section II, A) and kidney glutaminase (207), whose protomers or subunits aggregate to produce oligomers having "open configurations"

ACETYL COENZYME A CARBOXYLASE

183

and indeterminate length. In view of the fact that most proteins of this type (e.g., actin, myosin, collagen, certain viral capsular proteins, microfibril proteins of various types), appear to have structural functions, it is conceivable that the acetyl-CoA carboxylase filaments serve a structural purpose in addition to their known catalytic and regulatory roles. Although definitive proof is lacking, the authors' suggestion (146) that the carboxylase filaments might serve as an organizing matrix for a loose supramolecular complex of other enzymes of fatty acid synthesis and lipogenesis merits further study. Were this found to be the case, depolymerization of carboxylase filaments could lead to the disassembly of the fatty acid-synthesizing apparatus of the cell. In this regard, it is known (146) that the levels of the three key sequential enzymes of hepatic fatty acid synthesis, i.e., citrate cleavage enzyme, acetyl-CoA carboxylase, and fatty acid synthetase, fluctuate coordinately in response to alterations in physiological state which affect lipogenesis. Perhaps of significance in this connection is the finding in situ of cytoplasmic filaments (16, 228, 229) with dimensions similar to those of the citrate-activated bovine adipose tissue carboxylase. Sheldon and Ferguson (229) recently reported the occurrence of filamentous stuctures surrounding the fat droplets in thin sections of developing adipose tissue of yellow obese mice. This and the fact that purified acetyl-CoA carboxylases isolated from several sources [bovine adipose tissue (191), avian (92), and human (134) liver] are filamentous, suggests that these structures may have physiological relevance. C. Escherichia coli Carboxylase System In E. coli fatty acids are used almost exclusively for membrane synthesis (45) ; therefore, it might be expected that lipogenesis in this organism would be coupled to growth-related processes such as protein and RNA synthesis. The existence of such control mechanisms in E. coli was first suggested by the observation that amino acid starvation resulted in the cessation of RNA synthesis (56, 194, @®4) · This dependence of RNA synthesis upon protein synthesis, referred to as "stringency" and which is lost in "relaxed" (ret) mutants, is under genetic control of the rel gene (sometimes referred to the RC locus) in E. coli (56, 194, 224). Several reports have implicated the product of the rel gene in the control of lipid synthesis in E. coli (82, 232); however, these studies did not establish whether fatty acid synthesis per se is affected by amino acid deprivation. Recent investigations by Polakis et al. (212) showed definitively that stringent control is exerted on the rate of fatty acid synthesis. Rates of incorporation of glucose-U-14C, acetate-l- 14 C, and 3 H 2 0 into chloro-

184

M. DANIEL LANE, JOEL MOSS, AND S. EFTHIMIOS POLAKIS

20

40

60

Starvation time (min)

70

90

110

130

Time after 32 P addition (min)

1

2

3

[ppGppl ( m ^ )

FIG. 12. The effect of amino acid deprivation on (A) lipid synthesis (212) and (B) (p)ppGpp accumulation (35) in E. coli and (C) inhibition by ppGpp of E. coli carboxyl transferase-catalyzed malonyl-CoA-acetyl-CoA-[ 14 C] exchange and acetylCoA carboxylation (212). In (C) malonyl-CoA-acetyl-CoA-[ 14 C] exchange was determined with purified carboxyl transferase and carboxyl carrier protein plus Mn 2+ (O O ) °r with crude acetyl-CoA carboxylase plus ( # # ) and minus (Δ Δ ) Mn 2+ ; acetyl-CoA carboxylation, i.e., the overall reaction, was determined in the presence of Mn 2+ ( □ □ ) · Mn 2+ , when added, was equimolar with ppGpp except for measurement of acetyl-CoA carboxylation where additional Mn 2+ , equimolar with A T P was employed.

form-methanol soluble lipids ( > 9 5 % phospholipid) were instantly reduced 50-60% by leucine starvation of stringent (ret), but not of isogenic relaxed (ret) cells; the effect of amino acid starvation on the rate of incorporation of 3 H 2 0 into the lipid fraction of E. coli CP78 (rel+) is illustrated in Fig. 12A. That the depressed rate of lipid labeling was not due to leucine starvation-induced diversion of labeled fatty acyls into chloroform-methanol-insoluble form (e.g., lipopolysaccharide) is indicated by the fact that the rate of labeling from acetate-l- 14 C of the lipid extract of saponified cells was decreased to the same extent by leucine deprivation (212). With leucine-deprived ret cells the rate of acetatel- l f C incorporation into phosphatidylethanolamine and cardiolipin was more drastically curtailed than into phosphatidylglycerol, while the incorporation pattern in ret cells (±leucine) was similar to that of nonstarved ret cells. An elevated turnover rate of fatty acyl groups due to amino acid starvation cannot account for the decreased rate of lipid labeling by various precursors, since no detectable loss of 14C activity from fatty acyls labeled during growth or leucine deprivation occurs during subsequent growth or leucine starvation in the presence of unlabeled precursor (212). Only minor amounts of labeled lipid are secreted by stringent or relaxed cells grown in the presence of acetate-l- 14 C; leucine deficiency has no significant effect on the rate of labeling of these

185

ACETYL COENZYME A CARBOXYLASE guanme

guanme

'OH (3')

®®0>

®®OvJ

*0®®(3')

ppGpp

GDP

guanme

guanme

θ(Ρ)®(3')

*OH (3')

CPXPXP)0>sJ

®®®0

GTP

PPpGpp

FIG. 13. A. Mode of formation of (p)ppGpp (13, 111, 112β09, 222, B. Physiological transitions affecting (p)ppGpp concentration: Effect on [ppGpp] in cells Transition Amino acid starvation Amino acid resupplementation Diauxic lag Shift down Shift up Carbon deprivation Nitrogen deprivation Sulfur deprivation Uracil or guanine deprivation Phosphate deprivation Increase in growth rate Reduction in growth rate a 6

rel+

rel~

Reference

T 1

— —

T 1 T T T

τ·i

34, 35, 152 152 110, 152 110, 152 152 57,152 57,110 57 35,152 152 35 35

t

T T T T

—

—

I T

I T

b

6

Other than from amino acid-rich to amino acid-poor media. Sluggish and transient build-up of a relatively low (p)ppGpp level.

extracellular lipids. These results led to the conclusion that fatty acid synthesis per se is subject to stringent control being partially suppressed, i.e., 50-60%, by amino acid starvation in rel+ strains of E. coli. Cashel and Gallant (35) have demonstrated the appearance of two unusual nucleotides—ppGpp and pppGpp—in stringent, but not relaxed, strains of E. coli during amino acid starvation. The kinetics of formation of these nucleotides upon removal of a required amino acid (Fig. 12B) and of their disappearance upon resupplementation (34, 35, 152), are

186

M . DANIEL LANE, J O E L MOSS, AND S. E F T H I M I O S

POLARIS

compatible with their suggested role as mediators of the stringent response. Intracellular concentration of (p)ppGpp rises following amino acid starvation from 0.1-0.4 m l to 2-4 m l (125). In addition, the accumulation of these nucleotides during shift-down experiments or carbon source starvation (34, HO, 152), as well as the fact that (p)ppGpp concentration is inversely proportional to the RNA content of the cell (152) and growth rate (163), suggest that (p)ppGpp may be a growth modulator; the effects of these and other physiological transitions on (p)ppGpp levels are summarized in Fig. 13B. Finally, the recent interesting experiments of Haseltine et al. (112) and Haseltine and Block (111) have established that (p)ppGpp is formed on the ribosome by pyrophosphoryl transfer from ATP to GDP or GTP as an idling step in protein synthesis. The reaction (Fig. 13A) requires both the 30 S and 50 S ribosomal subunits, the stringent factor, mRNA, and an uncharged tRNA in the acceptor site of the ribosome. These observations also suggest that the mode of formation of (p)ppGpp is intimately connected to the cessation of protein synthesis and consequently to inhibition of growth, thus lending further support to the role of (p)ppGpp as growth modulator. Recent investigations in the authors' laboratory indicate that (p)ppGpp mediates stringent control of lipid synthesis in E. coli by acting as a negative effector at the committed step catalyzed by acetyl-CoA carboxylase (212) (Fig. 7). The maximal extent of inhibition of carboxylase activity, i.e., 50-60%, which is achieved at saturating concentrations of ppGpp or pppGpp (Fig. 12C), agrees well with the extent to which fatty acid synthesis is curtailed in vivo by amino acid deprivation (Fig. 12A). Most importantly, the maximal effect of ppGpp is manifested (212) at physiological concentrations of the nucleotide (less than 2 mM) (125). Of the two catalytic components of the acetyl-CoA carboxylase system, namely biotin carboxylase and carboxyl transferase (see Section II, B), only the latter component is inhibited by physiological concentrations (up to 4 mM) of ppGpp. Both partial reactions which characterize the carboxyl transferase-catalyzed step, namely the transcarboxylation from malonyl-CoA to biotin methyl ester [Reaction (24) ] and malonyl-CoA-acetyl-CoA- 14 C exchange [Reaction (4) ] are inhibited by ppGpp (Fig. 12C) (212). Malonyl-CoA + biotin methylester ^=± l'-iV-carboxy-d-biotin methyl ester + acetyl-CoA

(24)

The effect of (p)ppGpp is specific in that none of the other nucleotides tested were inhibitory at concentrations where the (p)ppGpp inhibition is maximal. Furthermore, it appears that the two inhibiting nucleotides, ppGpp and pppGpp, bind at a common site on the carboxyl transferase.

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These findings suggest that stringent control of fatty acid synthesis in E. coli is mediated through the inhibitory action of (p)ppGpp on the carboxyl tansferase component of the acetyl-CoA carboxylase system. V. Concluding Remarks It is evident that the mode of regulation of acetyl-CoA carboxylase reflects its physiological role in animal and bacterial cells. The carboxylase reaction, being the first committed step of the major energy storage pathway in animals, is regulated through feed-forward activation while the E. coli enzyme, being catalyst for the initial step of a biosynthetic pathway involved in membrane synthesis, is regulated by the inhibitory action of a growth modulator. The carboxylases from both animal and bacterial sources possess two catalytic sites: a biotin carboxylase site, at which the biotin prosthetic group is carboxylated, and a carboxyl transferase site at which the carboxyl group is transferred to acetyl-CoA to form malonyl-CoA. Regulation of the animal acetyl-CoA carboxylases by the feed-forward allosteric activator, citrate, appears to be mediated through a conformational change which perfects the orientation of the biotin prosthetic group with respect to both catalytic sites. The regulation of the bacterial enzyme is exerted through the inhibitory action of ppGpp, a growth modulator, on the carboxyl transferase component of the acetylCoA carboxylase system. ACKNOWLEDGMENTS The authors wish to thank Mrs. Norma Mitchell for her expert and tireless assistance in the preparation of this manuscript. REFERENCES 1. Abraham, S., Lorch, E., and Chaikoff, I. L., Biochem. Biophys. Res. Commun. 7, 190 (1960). 2. Abraham, S., Matthes, K. J., and Chaikoff, I. L., / . Biol. Chem. 235, 2551 (1960). 3. Abraham, S., Matthes, K. J., and Chaikoff, I. L., Biochim. Biophys. Acta 49,268 (1961). 4. Akhtar, J. B., and Bloxham, D. P., Biochem. J. 120, IIP (1970). 5. Alberts, A. W., Gordon, S. G., and Vagelos, P. R., Proc. Nat. Acad. Sei. U.S. 68, 1259 (1971). 6. Alberts, A. W., Nervi, A. M., and Vagelos, P . R., Proc. Nat. Acad. Sei. U.S. 63, 1319 (1969). 7. Alberts, A. W., and Vagelos, P. R., Proc. Nat. Acad. Sei. U.S. 59, 561 (1968). 8. Allen, S. H. G., Jacobson, B. E., and Stjernholm, R., Arch. Biochem. Biophys. 105, 494 (1964). 9. Allison, F. E., Hoover, S. R. ; and Burk, D., Science 78, 217 (1933).

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259. 260. 261. 262. 263. 264. 265. 266. 267.

Regulation of Lipogenesis in Animal Tissues I

SHOSAKU NUMA

I

SATOSHI YAMASHITA

I I I

Department of Medical Chemistry Kyoto University Faculty of Medicine Kyoto, Japan

I. Introduction I I . Regulation of the Enzymes Involved in F a t t y Acid Synthesis . A. Control of the Enzyme Quantity B. Control of the Catalytic Efficiency of the Enzymes . . I I I . Regulation of F a t t y Acid Esterification A. Control of F a t t y Acid Distribution in Glycerolipids . . B. Control of Glycerolipid Synthesis from F a t t y Acids . . References

197 200 200 211 220 221 234 239

I. Introduction Lipid is an essential component of all living cells, and a large group of lipids contain long-chain fatty acids in their molecules. Among fatty acid-containing lipids, glycerolipids are the most abundant; in them, fatty acids are esterified to glycerol. In triglycerides, the most common glycerolipids, each of the hydroxyl groups of glycerol is esterified to a fatty acid. Triglycerides are the most concentrated store of energy for the organism, yielding per gram over twice as many calories as does carbohydrate or protein. Phosphoglycerides also belong to glycerolipids. This class of lipids can be regarded as derivatives of phosphatidic acid, and it most frequently contains a nitrogenous base. It is well established that phosphoglycerides are essential components of nearly all biological membranes. The hydrocarbon chains of fatty acids contained in these compounds constitute the hydrophobic region of the molecule, while the polar headgroups of phospholipids form the hydrophilic part of the molecule. The presence of hydrophobic and hydrophilic portions within one molecule is apparently a prerequisite for integration of the membrane structure (see ref. 1). Figure 1 illustrates the pathway for lipogenesis from carbohydrates. Cytoplasmic acetyl-CoA, the precursor for fatty acid synthesis, is provided mainly from glucose via glycolysis in cytoplasm, followed by oxidative decarboxylation of pyruvate in mitochondria and transport of acetyl unit back into cytoplasm in the form of citrate (see ref. 2). The synthesis de novo of fatty acids from acetyl-CoA is catalyzed by two enzyme sys197

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YAMASHITA

1-Acylglycerophosphorylcholine (IX) ^ Phospnatidylcholine Phosphatidylethanolamine

Triglyceride > * Diglyceride

I (VII)

CDP-diglyceride

Phosphatidic acid

1-Acylglycerol-P (VI)\ -Glycerol-P

(VIII)

Acidic phospholipids

2-Acy4glycerol-P - Fatty acyl- Co A Fatty acids

Cytoplasm

Oxaloacetate Mitochondrion

FIG. 1. Pathway for glycerolipid synthesis from glucose. The enzymes discussed in this review are as follows: citrate cleavage enzyme ( I ) ; acetyl-CoA carboxylase ( I I ) ; fatty acid synthetase ( I I I ) ; hexose monophosphate shunt dehydrogenases (IV) ; malic enzyme (V) ; glycerophosphate acyltransferase (VI) ; 1-acylglycerophosphate acyltransferase ( V I I ) ; 2-acylglycerophosphate acyltransferase (VIII); 1-acylglycerophosphorylcholine acyltransferase ( I X ) .

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terns, acetyl-CoA carboxylase and fatty acid synthetase. These enzyme complexes are located in cytoplasm. The pathways that provide cytoplasmic NADPH, the reducing agent required for the fatty acid synthetase reaction, are also included in Fig. 1. The fatty acids formed are further incorporated into triglycerides and phosphoglycerides. In contrast to the enzymes involved in fatty acid synthesis, most of the enzymes responsible for glycerolipid synthesis from fatty acids are located in the membrane system, especially in microsomes. The major homeostatic function of lipogenesis is to store as triglycerides the chemical energy of foodstuffs ingested in excess of the immediate energy requirements of the organism. Therefore the lipogenic process must be precisely regulated in animals in response to their ever-changing energy needs and to the quantity as well as the quality of foodstuffs ingested. For instance, fatty acid synthesis is lowered in fasted or alloxan-diabetic animals and in animals fed a high-fat diet; in all these metabolic conditions, carbohydrate utilization is restricted. On the other hand, when fasted animals are refed a fat-free high-carbohydrate diet, more fatty acids are synthesized to replenish the triglyceride store, which has been depleted during starvation. In addition the importance of regulation of phosphoglyceride synthesis is evident, since this class of lipids represents major constituents of biological membranes, which play crucial roles in the living cells: they act as permeability barrier; they act as supports for catalytic functions; and they can be excitable. The acetyl-CoA carboxylase reaction is the first committed step leading specifically to fatty acid synthesis, since malonyl-CoA has no other apparent metabolic alternative. Hence it would be of teleonomic significance to regulate fatty acid synthesis at this carboxylase step. In fact, the tissue content of acetyl-CoA carboxylase changes with the rate of fatty acid synthesis in a variety of metabolic conditions (see Section II, A), and the catalytic activity of this enzyme is affected by a number of metabolites (see Section II, B). Although animal tissues contain a sufficient amount of acetyl-CoA carboxylase to match the catalytic capacities of citrate cleavage enzyme and fatty acid synthetase when the carboxylase is fully activated, it is suggested that in vivo the carboxylase does not exhibit its maximal catalytic efficiency, and that the carboxylase step is therefore the most likely site of regulation of fatty acid synthesis (see ref. 2). Although a number of intermediates of glycerolipid biosynthesis have been identified, and the pathway has been outlined, no enzyme involved in the process has been purified extensively and well characterized. Thus the knowledge concerning the control mechanism of glycerolipid synthesis is still scanty. The first part of the present review deals with the regulation of the

200

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YAMASHITA

quantity as well as the catalytic efficiency of the cytoplasmic lipogenic enzymes involved in fatty acid synthesis from acetyl-CoA. In the latter part of this review are discussed the enzymatic mechanism controlling the fatty acid distribution in glycerolipids and the regulation of glycerolipid synthesis from fatty acids. II. Regulation of the Enzymes Involved in Fatty Acid Synthesis A. Control of the Enzyme Quantity 1. COORDINATE RESPONSE OF THE ENYMES

The levels of acetyl-CoA carboxylase and fatty acid synthetase as well as those of citrate cleavage enzymes, hexose monophosphate shunt dehydrogenases, and malic enzyme, which are involved in the generation of the carbon precursor and the reducing agent required for fatty acid synthesis, undergo coordinate adaptive changes when the rate of fatty acid synthesis fluctuates under a variety of metabolic conditions. These conditions include dietary, hormonal, developmental, and genetic alteration, and generally the enzymes in the liver and adipose tissue are subject to these adaptive changes. Since details of these variations in the enzyme levels have recently been reviewed (see refs. 2-4), only those metabolic states will be referred to here which have been studied most extensively. The activity levels of the above-mentioned lipogenic enzymes in cytoplasm are lowered in starvation and restored upon refeeding fasted animals {5-28). The increase in the enzyme levels after realimentation is more marked on a fat-free diet than on a balanced diet. Fat-feeding results in a reduction of the enzyme levels {24, 29, 30). Diabetes mellitus is accompanied by a decrease in the enzyme levels (7, 8, 15, 20, 22, 31-36), whereas they are elevated in genetic obesity {12, 37-39). Weaning of rats (40-43) as well as hatching of chicks (44-4$) induces a marked rise in the enzyme levels. This rise is apparently associated with the animal's change from high-fat to relatively low-fat nutrition at the time of weaning or hatching. All these changes in the activity levels of the lipogenic enzymes are in accord with changes in the rate of fatty acid synthesis under the various metabolic conditions. 2. CONTENT, SYNTHESIS, AND DEGRADATION OF THE ENZYMES

Previous studies in this direction, however, were based exclusively on measurements of catalytic activity, which did not differentiate between changes in the number of enzyme molecules, i.e., enzyme quantity, and changes in catalytic efficiency per enzyme molecule. Recently an immuno-

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chemical approach has been undertaken by several groups to answer the question, whether the observed changes in the activity levels of the enzymes are actually determined by changing quantities of the enzyme proteins. Studies on acetyl-CoA carboxylase will be taken as an example and discussed below in some detail. It is of interest to compare two pathological conditions, diabetes mellitus and obesity, with respect to deranged lipogenesis. In fact, a number of studies on this subject have been carried out with the use of alloxandiabetic rats and genetically obese hyperglycemic mice (C57BL/6J-o6), which represent experimental models for human diseases. It is well known that fatty acid synthesis is depressed in diabetic animals {^9-52), whereas it is elevated in obese mice {5, 53). In accordance with the changes in fatty acid synthesis, the level of hepatic acetyl-CoA carboxylase activity is likewise decreased or increased in these animals {33, 38). Figure 2 shows the results of immunochemical titrations of liver extracts derived from normal and alloxan-diabetic rats (A) as well as from normal and obese mice (B). Increasing amounts of the liver extracts were added to a fixed amount of antiacetyl-CoA carboxylase γ-globulin,

=>

E

c o "o c w. Φ

o.

3 to

< 0

50

100

150

200

Activity added (mU) FIG. 2. Immunochemical titration of hepatic acetyl-CoA carboxylase derived from: (A) normal ( O O ) and alloxan-diabetic rats ( # · ) ; (B) normal ( O O) and obese mice ( # # ) . Increasing amounts of liver extracts containing the carboxylase activities indicated were added to a fixed amount of antiacetyl-CoA carboxylase. After completion of precipitation, the supernatant fluids were assayed for carboxylase activity. From Nakanishi and Numa {89, 5^).

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YAMASHITA

FIG. 3. Ouchterlony double diffusion analysis of hepatic acetyl-CoA carboxylase. (A) Derived from normal rats (N) and alloxan-diabetic rats (D). (B) Derived from normal mice ( M ) , obese mice ( 0 ) , and normal rats ( N ) . Ab denotes antibody against rat liver acetyl-CoA carboxylase. From Nakanishi and N u m a {39, 54).

and, after completion of precipitation, the enzyme activity remaining in the supernatant fluids was determined. In this particular experiment, the specific activity of hepatic acetyl-CoA carboxylase was decreased 2.8-fold in diabetic rats and increased 3.0-fold in obese mice as compared with normal values. Despite this wide fluctuation in the level of enzyme activity, the equivalence point, i.e., the point at which the enzyme activity first appeared in the supernatant, was identical for normal and pathological liver extracts when based on the amount of enzyme activity added. In other words, the changes in the level of enzyme activity were accompanied by proportionate changes in the quantity of immunochemically reactive protein. These results indicate that the catalytic efficiency per enzyme molecule is not changed either in diabetic or in obese animals, and therefore that the observed changes in the activity level can be ascribed to changes in the quantity of the enzyme protein, which is indistinguishable from that derived from normal animals. This conclusion is further supported by the results of Ouchterlony double-diffusion analysis and of kinetic and heat inactivation studies. Figure 3 shows Ouchterlony patterns. The completeness of connections of the precipitin bands indicate that enzyme molecules derived from livers of normal and diseased animals are immunologically indistinguishable. The data of Table I reveal that the enzyme preparations obtained from obese and normal mice exhibit no qualitative differences with respect to kinetic properties and heat stability. Thus neither the alloxan-diabetic condition nor the mutation causing obesity appears to affect the structure of hepatic acetyl-CoA carboxylase, but they alter the enzyme content of the liver.

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203

TISSUES

TABLE I P R O P E R T I E S OF H E P A T I C A C E T Y L - C O A CARBOXYLASE FROM O B E S E AND NORMAL M I C E 0

Property

Obese

Normal

Michaelis constant: A T P Acetyl-CoA Citrate Palmityl-CoA concentration required for 50 % inhibition ti/2 of heat inactivation at 45°C

12 μΜ 32 μΜ 4.3 m l

12 μΜ 19 μΜ 5.0mM

5μΜ 6 min

6μΜ 6 min

α

From Nakanishi and N u m a (39).

The quantity of an enzyme is affected by changes in the rates of its synthesis and/or degradation. Under steady-state conditions, the content of an enzyme is related to these rates as follows: E = k8/kd

(1)

where E is the content of enzyme per mass, ks is a zero-order rate constant of synthesis per mass, and kd is a first-order rate constant of degradation expressed as reciprocal of time (see ref. 55). In order to examine whether the variations in the quantity of hepatic acetyl-CoA carboxylase observed in alloxan-diabetic and obese animals are due to changes in the rate of enzyme synthesis or in that of enzyme degradation, combined immunochemical and isotopic studies were carried out. The rate of synthesis of hepatic acetyl-CoA carboxylase was measured by injecting animals with a dose of leucine-3H and shortly thereafter determining the extent of isotope incorporation into the protein that is precipitated by antibody specific to the enzyme. The results of such experiments are shown in Table II. Since the extent of labeling of total soluble liver protein is reflected in enzyme labeling, the ratio of the radioactivity incorporated into the enzyme to that incorporated into total soluble protein (a/b) was calculated as a measure of the rate of enzyme synthesis. It is evident from Table II that this relative rate of enzyme synthesis was decreased 1.7-fold in diabetes and increased 7.7-fold in obesity. The rate of enzyme degradation was measured by following the loss of isotope from the prelabeled enzyme. The data are shown in Fig. 4. The decay of radioactivity in the enzyme followed first-order kinetics. The rate of loss of isotope, expressed as half-life (t1/2), was 59 hours both in normal and in diabetic rats, and 67 hours and 115 hours in normal and in obese mice, respectively. In the experiments to determine the rate of enzyme degradation, reutilization of leucine-3H may lead to an overestimation of the half-life (56). However, this does not appear to be of

204

SHOSAKU N U M A AND SATOSHI

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TABLE II R E L A T I V E R A T E S OF H E P A T I C A C E T Y L - C O A CARBOXYLASE SYNTHESIS IN A L L O X A N - D I A B E T E S AND O B E S I T Y 0

Acetyl-CoA carboxylase in liver extract

Animal Normal rats Alloxan-diabetic rats Normal mice Obese mice 1

Leucine- 3 H incorporation

(&)

(a) Acetyl-CoA carboxylase per liver (cpm)

Total soluble protein (cpm/mg)

a/b

Mean weight (g)

Specific activity (mU/mg)

Total activity per liver (U)

180

8.58

4.18

19,000

9,250

2.05

250 24 40

3.79 4.7 16.7

2.20 0.36 3.66

5,850 1,830 15,800

4,860 15,100 16,900

1.20 0.12 0.93

From Nakanishi and N u m a (39, 54). 5.000 4.000 3,000

40

80

120

160

Time after leucine-8H injection (hr) FIG. 4. Kinetics of degradation of hepatic acetyl-CoA carboxylase in alloxandiabetes and obesity. (A) Normal ( O O ) and alloxan-diabetic rats ( · #). (B) Normal ( O O ) and obese mice ( # # ) . From Nakanishi and N u m a (39, 54).

major significance in the above experiments, since there was no essential difference in the half-life for degradation of total soluble liver protein, estimated simultaneously by means of this isotope, between normal and diseased animals.

REGULATION OF LIPOGENESIS I N ANIMAL

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205

It is concluded from the results mentioned above that the decrease in the enzyme content in alloxan-diabetic rats is attributed to retarded synthesis of the enzyme rather than to its accelerated degradation, and that the increase in the enzyme content in obese mice is due mainly to a rise in the rate of synthesis, and in a minor degree, to a decrease in the rate of degradation. Normal and obese animals as well as animals suffering from prolonged diabetes can be assumed to be in steady states. Thus the changes in the enzyme content in diseased animals, predicted from the ratio ks/kd according to Eq. (1), should be a 1.7-fold decrease in diabetic rats and a 13-fold increase in obese mice. The predicted values are in fairly good agreement with the 1.9-fold decrease and the 10-fold increase actually found. Analogous studies were carried out also with rats subjected to different dietary manipulations, such as fasting and refeeding with a fat-free diet (54, 57, 58). The variations in the level of acetyl-CoA carboxylase activity in liver extracts derived from these animals were likewise shown to be ascribed to changes in the quantity of the enzyme. Furthermore it was concluded that the increase in the enzyme content in fat-free refed rats is due to accelerated enzyme synthesis, while the decrease in the enzyme content in fasted animals is attributed both to retarded enzyme synthsis and to accelerated enzyme degradation. Recent studies have shown that the alimentary, hormonal, and developmental variations in the activity levels of the other lipogenic enzymes, including fatty acid synthetase, citrate cleavage enzyme and malic enzyme, are likewise accompanied by proportionate changes in content of the enzymes {60-6S). In order to delineate the mechanisms underlying the changes in enzyme content, the rates of synthesis and degradation of these enzymes were measured by isotopic amino acid incorporation studies combined with immunological precipitation or enzyme purification. Kinetic analysis of the time course of changes in the activity level (see ref. 55) was also employed to estimate the rates of synthesis and degradation of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results of these studies, together with the aforementioned data on acetyl-CoA carboxylase, are summarized in Table III. It is noteworthy that the contents of hepatic enzymes responsible for fatty acid synthesis respond strikingly to various dietary, hormonal, genetic, and developmental alterations, whereas the content of the brain enzyme does not change upon dietary manipulation but does change during development (see also ref. 65). Table III reveals also that the changes in enzyme content can always be accounted for at least in part by changes in the rate of enzyme synthesis. In most cases, the enzyme con-

Streptozotocindiabetic, fast Streptozotocindiabetic,

Fast-+ fat-free diet Suckling Weaning —> fat-free diet Adult

Fat-free diet Fast

Balanced diet

Balanced diet Fat-free diet Fast Fast-> fat-free diet Alloxan-diabetic Normal Genetically obese

Conditions

0.59 1 7.75 (2.69)

0.53 1 10.2 (3.54)

1 2.50

1

4.08

14.9 1

0.18 (0.55) 14.3 (5.84) 1 13.4

0.13 (0.23) 16.6 (4.61) 1 78.2

1

0.54 4.05

0.28 3.76

1

1

(*.)

Rate of synthesis 6

1

Enzyme content 6 (E)

55 67

0.82

3.72 1.00 1

18 69

1.03

59 67 115

69 or 71 or 67 65

1

1.00 1 0.58

59 or 50 48 31 or 18 55

tl/2

1 1.04 1.90 or 2.78 1.07

Half-life (hours)

CONDITIONS

Rate constant of degradation (kd)

UNDER V A R I O U S M E T A B O L I C

4.98

1

(0.15) 14.3

1

0.59 1 13.4 (4.64)

0.28 3.78

1

ka/kd

Immunoprecipitation and isotope incorporation (89). Values in parentheses are per unit weight of organ Enzyme purification, immunoprecipitation and isotope incorporation (59-61). Values in parentheses are per unit weight of organ (61) Immunoprecipitation and isotope incorporation (61). Values are per unit weight of organ Enzyme purification and isotope incorporation (62)

Immunoprecipitation and isotope incorporation (54-, 58)

Method of study, remarks, and references

SHOSAKU N U M A AND SATOSHI

R a t liver fatty acid synthetase

R a t liver fatty acid synthetase

R a t liver fatty acid synthetase

Mouse liver acetyl-CoA carboxylase

R a t liver acetyl-CoA carboxylase

Enzyme

TABLE III

C O N T E N T , SYNTHESIS, AND DEGRADATION OF THE ]ENZYMES INVOLVED IN FATTY ACID S Y N T H E S I S

206 YAMASHITA

Rat intestine glucose-6phosphate dehydrogenase Rat liver 6-phosphogluconate dehydrogenase

Rat liver glucose-6phosphate dehydrogenase

Balanced diet Fast

Rat liver citrate cleavage enzyme

Balanced diet Fat-free diet Fat-free diet + insulin

fat-free diet Fast

Fast~

fat-free diet Insulin Thyroxine Balanced diet Fat-free diet Fat-free diet + insulin

Fast~

fat-free diet Adult

Weaning~

fat-free diet Suckling

Fast~

fat-free diet Balanced diet Fast

Rat brain fatty acid synthetase

Rat brain fatty acid synthetase

fast~

fast ~ fat-free diet Streptozotocindiabetic + insulin,

1 4.60 4.60 4.60

1.54 2.36 1 38.2 103.0 73.6

1 8.21 22.1 15.8

1 1.56 2.71

1 5.93 5.93

3.24

14

1.93

1 9.27 15.9

1

27

1

Kinetic analysis (26). Values are per unit weight of protein 81 14 14

TISSUES

(continued)

Kinetic analysis (64)

Kinetic analysis (27). Values are per unit weight of protein

Immunoprecipitation and isotope incorporation (61). Values are per unit weight of organ Immunoprecipitation and isotope incorporation (63)

Immunoprecipitation and isotope incorporation (61)

6

15

69 15 15

2.03

1 0.35 6.26

1 0.11 2.33

1.54

0.30

1

0.61

46

1

0.46

154 149 151

1 1.03 1.02

1 0.82

N o change

23.6

1 0.72

N o change

I I

19.8

REGULATION OF LIPOGENESIS I N ANIMAL

207

fat-free diet Insulin Thyroxine Neonatal 3 Days of age 8 Days of age 11 Days of age Single glucose meal, neonatal Fast, 10 days of age

Fast~

Balanced diet Fast

high-fat diet Fast

Fast~

fat-free diet

Fast~

Conditions 1.5.9 5.73

2.60 0.96

1 0.43 5.23 1.31 3.18 1 30.0 42.4 53.6 2.60 17.6

1 0.48 1.40

1 23.3 58.3 62.5 2.00 34.2

(E)

28

55 55

6.36 6.36 12.5

350

b

1.41

6.67 8.42

1

2.83

34

1.85

1

1

ks/k d

63

8

14

14

Half-life t1l2 (hours)

1

5.93

5.93

Rate constant of degradation (k d )

Immunoprecipitation and isotope incorporation (28). Values are per unit weight of protein

Immunoprecipitation and isotope incorporation (63)

Kinetic analysis (64)

Method of study, remarks, and references

SHOSAKU N U M A AND SATOSHI

a E, k s , k d , and ks/k d are given as values relative to those under certain standard conditions. Per organ unless otherwise stated under "Remarks."

Chick liver malic enzyme

Rat intestine 6-phosphogluconate dehydrogenase Rat liver malic enzyme

Enzyme

Rate of synthesis b (k s)

Enzyme contentb

TABLE III (Continued)

208 YAMASHITA

REGULATION OF LIPOGENESIS I N ANIMAL

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209

tent is determined exclusively or principally by changes in the rate of synthesis. The only instance in which a change in the rate constant of degradation plays an important regulatory role, is the state of starvation. In fasted animals, hepatic acetyl-CoA carboxylase, fatty acid synthetase and malic enzyme are degraded 2- to 4-fold more rapidly than in fed animals, and the decrease in the content of these enzymes upon starvation is effected both by retarded synthesis and by accelerated degradation. Starved animals are not in a steady state, while the other animals in which the rate of degradation was measured, except growing rats and chicks, can be assumed to be in steady states. Thus it is suggested that the control of the rate of enzyme degradation makes a significant contribution to the regulation of enzyme content only when the animal deviates from a steady state for adjustment to a new environment. This concept is supported also by the following findings of Schimke {66). Rats maintained on diets containing 8, 30, and 70% casein show different steady state arginase contents but essentially the same rate constants of arginase degradation. Upon changing rats from a diet containing 70% protein to one containing 8% protein, the rate constant of arginase degradation increases during the first 3 days. However, it then gradually approaches the value seen in the steady state, as the enzyme content attains a new steady state level lower than the initial one. When rats are fasted, arginase degradation ceases, and the enzyme content rises concomitantly. The adaptive increase in the levels of the hepatic lipogenic enzymes upon fat-free refeeding {11, 21, 67-70) and upon weaning {71) is blocked by injecting animals with inhibitors of protein synthesis including actinomycin D, puromycin, and ethionine. Furthermore, actinomycin D prevents the recovery of fatty acid synthesis following treatment of alloxan-diabetic animals with insulin {72, 73). These findings support the aforementioned conclusion that the increase in the enzyme levels under these conditions is due principally to accelerated enzyme synthesis. It is of interest that most of the hepatic enzymes involved in fatty acid synthesis exhibit a half-life of 50-70 hours under steady state conditions when it is measured by decay of the radioactivity incorporated into the enzymes. This suggests a considerable degree of coordination in the control of degradation of these enzymes. Another peculiar feature is the unusually slow turnover rate (t1/2 = 350 hours) exhibited by hepatic malic enzyme of unfed neonatal chicks. The degradation of brain fatty acid synthetase is much faster in 4- to 6-day-old rats (t1/2 = 46 hours) than in the adult {t1/2 = 149-154 hours). The accelerated turnover of the synthetase in the young brain is accompanied by faster degradation of the soluble protein in general. The striking decrease in the rate of degradation of the brain enzyme during growth of the animal is distinctly

210

SHOSAKU N U M A AND SATOSHI

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different from the situation in the liver. Tweto et al. (74) showed that the subunits of the rat liver fatty acid synthetase multienzyme complex are degraded with half-lives which are related to their molecular weight in a manner similar to that found by Schimke and his associates (75, 76); subunits of greater molecular weights are degraded more rapidly. The rates of turnover of all the subunits appear to be slower than the rate of exchange of the prosthetic group, 4'-phosphopantetheine. The mechanism responsible for the independent control of synthesis and degradation of these lipogenic enzymes remains to be elucidated. Some findings of interest in this context are described below. Muto and Gibson (24) reported that oral administration of methyl esters of polyunsaturated fatty acids, such as linoleic, linolenic, and arachidonic acid, to rats maintained on a fat-free high-carbohydrate diet results in a progressive diminution of the levels of the hepatic lipogenic enzymes, acetylCoA carboxylase, fatty acid synthetase, citrate cleavage enzyme, malic enzyme, and glucose-6-phosphate dehydrogenase. Methyl esters of saturated and monounsaturated fatty acids exhibited little damping effect. The lipogenic enzymes behave as a constant proportionality set regardless of the kind of nutritional manipulation. The polyunsaturated fatty acid content of liver lipids and free fatty acids reflects the exogenous fatty acid inrjart. Thus there is a reciprocal relationship between the levels of the lipogenic enzymes and the polyunsaturated fatty acid content of the liver. Recent studies of Goodridge (77) showed that the increase in the levels of hepatic acetyl-CoA carboxylase, fatty acid synthetase, and malic enzyme which occurs in neonatal chicks given a normal mash diet or a single glucose injection is accompanied by a decrease in the concentration of free fatty acids and fatty acyl-CoA thioesters. Jacobs et al. (78) showed that human skin fibroblasts grown with fetal calf serum contain a smaller amount of acetyl-CoA carboxylase than cells grown with lipid-deficient serum. The effect of the lipid-deficient medium is reversed by the addition of normal serum or by the addition of any of the fatty acids tested including palmitic, stearic, oleic, linoleic, and arachidonic acid. Kitajima et al. (79) also found analogous effects of triglycerides and fatty acids using rat hepatocytes maintained in a lipidand protein-free synthetic medium. These in vivo and in vitro studies support the concept that fatty acids or a substance metabolically related to them might be a factor responsible for the control of synthesis of the lipogenic enzymes. As shown in Table III, Lakshmanan et al. (62) described that the increase in the level of hepatic fatty acid synthetase which normally occurs upon fat-free refeeding of fasted rats is abolished in streptozotocin-diabetic animals. Insulin specifically restores this increase by enhancement of the rate of enzyme synthesis. On the other

REGULATION OF LIPOGENESIS I N ANIMAL

TISSUES

211

hand, glucagon or cyclic AMP (cAMP) partially block the adaptive increase upon refeeding. Similarly, the increase in the level of hepatic glucose-6-phosphate dehydrogenase upon refeeding is prevented by glucagon or cAMP (79a). These results suggest that the synthesis of the lipogenic enzymes is under the control of the relative concentrations of insulin and glucagon through the action of cAMP. Evidence indicating the involvement of the cyclic nucleotide in the control of the catalytic efficiency of the lipogenic enzymes will be discussed in Section II, B, 4. 3. Loss

OF CONTROL IN HEPATOMAS

One of the striking features of minimum deviation hepatomas is their failure to regulate fatty acid synthesis upon dietary alteration (80, 81). Majerus et al. (82) demonstrated that the levels of acetyl-CoA carboxylase and fatty acid synthetase in hepatomas, in contrast to those in host livers, do not respond to fasting and fat-free refeeding of the tumor-bearing animal. The tumor carboxylase is essentially identical with the liver enzyme in terms of kinetic and other properties. Preliminary experiments indicate that the level of carboxylase activity in tumors parallels the content of immunochemically reactive protein, and that no change in the rate of enzyme synthesis occurs in tumors after fat-free refeeding. These results suggest that the defect of control of fatty acid synthesis is due to the failure of tumors to regulate the quantity of the carboxylase rather than to structural alteration of the enzyme. Using hepatic tissue transplanted to a subcutaneous environment and receiving no portal blood, Bartley and Abraham (83) showed that in the autografts the levels of lipogenic enzymes including acetyl-CoA carboxylase and fatty acid synthetase vary in response to fasting and refeeding in essentially the same manner as in normal liver. Since hepatic autotransplants with a blood supply comparable to that of hepatomas are able to respond to dietary manipulation, the site and blood supply of the tumor cannot explain the absence of dietary control in the neoplastic tissue. B. Control of the Catalytic Efficiency of the Enzymes 1. SHORT-TERM vs LONG-TERM CONTROL

As discussed above, the wide variations in the activity levels of the lipogenic enzymes in liver extracts observed under various metabolic conditions are due to changes in the quantity of these enzyme proteins. However, several lines of evidence indicate that the rate of fatty acid synthesis in vivo is controlled also by changes in the catalytic efficiency of these enzymes, especially of acetyl-CoA carboxylase. Korchak and Masoro

212

SHOSAKU N U M A AND SATOSHI

YAMASHITA

(84) pointed out that at an early stage of fasting the fatty acid-synthesizing capacity of liver slices is more depressed than can be accounted for by the level of acetyl-CoA carboxylase in liver extracts; after 24 hours of fasting, the enzyme level falls only by 50%, whereas a 99% depression in fatty acid synthesis in liver slices is observed. Analogous discrepancy was observed also with alloxan-diabetic rats (85) as well as with rats fed a single dose of fat (29) at an acute or early stage. In view of the half-life for degradation of hepatic acetyl-CoA carboxylase ranging from 1 to 5 days (see Table I I I ) , the content of this enzyme cannot change rapidly, since theoretically the time required for the content of an enzyme to change to one-half of the final change at a new steady state is equal to the half-life of the enzyme (see ref. 86). Even at later stages, the acetyl-CoA carboxylase level in liver extracts is generally less reduced than fatty acid synthesis in liver slices, although this discrepancy is not so marked as at early stages. These observations suggest that other factors, in addition to the enzyme content, are involved in the regulation of fatty acid synthesis in intact cells. In fact, the catalytic activity of acetyl-CoA carboxylase of animal origin is affected by various metabolites as positive and negative effectors. Tri- and dicarboxylic acids, most notably citrate and isocitrate, activate mammalian and avian acetyl-CoA carboxylase (87-98). In contrast, long-chain acyl-CoA thioesters (99-103), malonyl-CoA (89, 104-106), and some metabolites of tryptophan including kynurenate and xanthurenate (106) are inhibitors of the carboxylase from rat and chicken liver. In view of the fact that citrate is a precursor of acetyl-CoA, which is a substrate for this enzyme, a regulatory role of citrate as a positive feedforward activator is conceivable. On the other hand, the inhibition by long-chain acyl-CoA can be regarded as a negative feedback mechanism due to end-product inhibition. Changes in the tissue contents of citrate (88, 107-110) and long-chain acyl-CoA thioesters (111-115) in various metabolic states associated with elevated or lowered lipogenesis are generally consistent with the proposed regulatory roles of these allosteric effectors. In recent studies of Nishikori et al. (116), an attempt was made to evaluate the relative importance of the control of the acetyl-CoA carboxylase content and that of its catalytic efficiency for the increased fatty acid synthesis which occurs upon refeeding rats previously fasted with a fat-free diet. For this purpose, studies were made on the time course of changes in the levels of hepatic acetyl-CoA carboxylase, citrate and long-chain acyl-CoA thioesters as well as in the rate of fatty acid synthesis in liver slices. The results of these studies are illustrated in Fig. 5.

REGULATION OF LIPOGENESIS IN ANIMAL TISSUES

E

co-i

E

f

t

213 i400

37 °C

O

2

1

4°C

,

11

Jk-

1

|_

1

4

1

1 A^-f^^J, 10

1

12

PH

Fio. 11. Effect of p H and temperature on the first-order rate constant for the conversion of subfraction Cy I into Cy I I ( A and # ) and of Cy I I into Cy I I I ( O ) · Formate and borate buffers of ionic strength 0.1 were used. The figure is from Flatmark (59).

270

ARTHUR B. ROBINSON AND COLETTE J . 1

6

1i

1

—

*

RUDD

·

2 T o a> (days"1) FIG. 18. P/T vs 1000/P for GlyPheGlnGlyGly, GlylleGlnGlyGly, GlyTyrGlnLeuGly, and GlyThrGlnAlaGly. P is the deamidation half-time in days in p H 7.4, 37.0°C, ionic strength 0.2 phosphate buffer, and T is the deamidation half-time in days in 37.0°C Joklik modified minimum essential tissue culture medium. The straight line was fitted by the method of least squares to the experimental measurements ( O ) shown. The line has a correlation coefficient of 0.997 and an equation such that T = F/(9.08 - 3781/P). The figure is from Robinson and Scotchler (177).

V. Molecular Clocks A. Hypothesis Why are two of the commonly occurring residues in peptides and proteins inherently unstable in aqueous medium? This property of glutaminyl and asparaginyl residues is especially puzzling, because their static structural properties are not particularly unique and could be provided by stable amino acid residues. The answer may be that the instability of glutaminyl and asparaginyl residues is their primary biologically important property {170, 174) · The deamidations of glutaminyl and asparaginyl residues may serve as general molecular timers of biological processes. These molecular clocks may serve as timers of development, turnover, and aging in proteins, cells, and organisms. As mentioned in Section II, A, for single amino acid residues in model peptides under physiologically interesting solvent conditions, the distribution function of deamidation half-times has a width of at least 6 days to 9 years (see Table I ) . The in vivo behaviors of cytochrome c, aldolase, and lysozyme are consistent with the deamidation half-times of peptides with the same amide sequences as these proteins. Correlation between the in vivo lifetime of proteins and their total amide content has been found (see Fig. 10). Elec-

DEAMIDATION OF GLUTAMINYL AND ASPARAGINYL RESIDUES

285

trophoretic heterogeneity caused by deamidation has been found in a large number of protein preparations (see Table I I ) . B. Settings Available Each individual glutaminyl or asparaginyl residue has its own characteristic deamidation half-time. This deamidation half-time is determined by the primary sequence of residues adjacent to the amide residue, by the nature of residues that are held close to the amide residue through secondary and tertiary protein structures and intermolecular interactions, by the general properties of the solvent surrounding the amide residue, and by the specific nature of certain solutes that may be present in the surrounding solvent. It is known (see Sections II, III, and IV) that the available range of individual molecular timer settings extends from 6 days to 9 years, and it is to be expected that the range available in vivo is somewhat wider. C. Evolutionary Adjustment 1. LONG-TIMED INTERVALS

Let us assume that there is still a large degree of randomness in protein structures. Rates of mutation acceptance have been calculated for 17 types of proteins (32). These rates vary from 9.0 paulings to 0.006 pauling with a median of 1.0 pauling and are therefore consistent with our assumption. Figure 10 shows the in vivo lifetime versus percentage amide residues for all proteins for which both values were found during a review of the literature (122). Although some of these values are probably in error, none that were found have been excluded from Fig. 10. The general trend is probably correct. There are fewer amides in the proteins with in vivo lifetimes greater than 30 days than would be expected from random mutation. Figure 4 shows that only a few peptides may be expected to have deamidation half-times of less than 30 days, but that above the value of 30 days a large percentage of amide sequences are included in the distribution function. Amide sequences with deamidation half-times less than the optimum biological lifetime of a protein would be unacceptable for inclusion in that protein. Therefore, a part of the distribution shown in Fig. 4 is unavailable to a long-lived protein, and the percentage of amide residues is accordingly lower. It has been suggested (170) that the amide content of a long-lived protein molecule is determined by evolutionary rejection of amide sequences that have deamidation half-times shorter than the optimum biological lifetime for that protein.

286

ARTHUR B. ROBINSON AND COLETTE J .

RUDD

2. SHORT-TIMED INTERVALS

The turnover and other timed functions of a short-lived protein must occur within the lifetime of the protein. However, the distribution function shown in Fig. 4 does not include amides with deamidation half-times that are as short as the in vivo lifetime of many short-lived proteins. If extra amides were accumulated in these proteins, the summations of their respective deamidation half-times could determine short-timed intervals. Figure 10 shows that there has been a marked accumulation of amide residues in short-lived proteins. Most of this accumulation has been of asparaginyl residues which have generally shorter deamidation half-times than do glutaminyl residues (see Fig. 4). It has been suggested (170) that the amide content of short-lived protein molecules is determined by evolutionary accumulation of amide sequences that have moderately short deamidation half-times. These may function synergistically for the regulation of short-timed intervals. It was suggested (180) that the protein lysozyme has undergone this kind of amide accumulation. 3. SPECIAL TIMED INTERVALS

In addition to the setting of molecular timers by general rejection or accumulation of amide residue sequences, it may be expected (170) that many molecular timers are set by special intermolecular and intramolecular interactions. These interactions may markedly alter the sequence-determined deamidation half-times of one or more amide residues in a particular protein. As mentioned in Section III, B, this kind of amide has been shown to be responsible for the second in vitro deamidation of beef heart cytochrome c (see Section IV) and may be responsible for the setting of many other molecular timers. D. Implementation The timed change of uncharged glutaminyl and asparaginyl residues into negatively charged glutamyl and aspartyl residues would be expected to have a profound effect on the structure of peptides and proteins. The change in hemoglobin that occurs when a single, negatively charged residue is converted to a neutrally charged residue in the disease sickle cell anemia demonstrates the profound effect that this kind of alteration can have on the structure of a protein (154) · As the time settings on the molecular amide clocks run out, the proteins in which they are embedded may change in structure in such a way that they lose their biological activity or gain a new biological activity. They may become more or less susceptible to degradative processes within the living organism and more or less strongly associated with other macro-

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molecules and cellular organelles important to their biological function {170). Most biological processes are carried out by peptide and protein molecules whose structural information has been stored in the DNA of their respective organisms. The great diversity of protein functions that has been accomplished by variations in the combination of the 20 naturally occurring amino acids shows the versatility with which changes in these residues can be used. Changes from neutral hydrogen-bonding residues, like asparaginyl and glutaminyl residues, to negatively charged residues, like glutamyl and aspartyl residues, have serious consequences for protein structures. They are clearly capable of alterations that can have biological importance. E. Protein Turnover There are many biological processes that might be timed by the change of uncharged residues into acidic, negatively charged residues. One example is protein turnover. It has been suggested (170, 174) that the increased negative charge might increase the susceptibility of a protein molecule to proteolytic degradation by opening the protein structure or causing the protein to dissociate from another cellular component. This mechanism would allow for the planned obsolescence of individual protein molecules that is required for optimum cellular and organismic health. Human carbonic anhydrase C has been shown to be resistent to chymotryptic digestion of the sequence LeuLysAsnArg but to be easily digested after deamidation of this sequence to LeuLysAspArg (121). Changes in susceptibility to proteolytic digestion after deamidation have been reported in cytochrome c and probably occur in a variety of other proteins. The Asp-His bond in aldolase is resistant to carboxypeptidase A unlike the Asn-His bond, which is sensitive (109). F. Organismic Development During the development of an organism, large numbers of different chemical processes must be timed to occur in the right sequence and for the right duration. It may be (170, 174) that simply through variation of the sequences of residues around asparaginyl and glutaminyl residues, timed changes that would be useful during development can be programmed. Particularly important might be changes in chromosomal proteins (178) as mentioned in Section III, E. G. Cellular and Organismic Aging In Section V, E, the possibility that the planned obsolescence of individual protein molecules that is required for optimum cellular and organismic health may be timed by deamidation is reviewed. It may be

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argued (170, 174, 176) that a similar mechanism effects the planned obsolescence of cells for the good of an organism and the planned obsolescence of organisms for the good of a species, aging. Age-dependent changes in degree of deamidation of glutaminyl and asparaginyl residues have recently been observed in a large number of proteins (see Table I I ) . Deamidation may play a general role in the aging of living organisms (170,174,176). VI. Summary The occurrence of glutaminyl and asparaginyl residues in peptides and proteins is so widespread that these residues have become known as 2 of the 20 most commonly occurring amino acid residues. It has been hypothesized that these residues play static roles in protein structure and also play dynamic roles as molecular timers of biological events. Deamidation of glutaminyl and asparaginyl residues has been responsible for artifacts in peptide and protein chemistry. The rate of deamidation of these residues has been shown to be dependent upon primary sequence, secondary and tertiary structure, temperature, pH, ionic strength, and special intermolecular interactions. Deamidation has been found to occur in a wide variety of different proteins and may be expected to occur in most proteins under physiological conditions. Many of the essential processes in living things may be timed by deamidation of glutaminyl and asparaginyl residues. These molecular timers are under simple genetic control, are adjustable to many different timed intervals, and are easily implementable by means of the important changes that they can effect in the proteins in which they are included. Furthermore these amides are certainly causing timed changes in the structures of most protein molecules in vivo. If these timed changes are not being put to biological use, then they are surely damaging to the order of biological systems and should have been evolutionarily eliminated long ago (170). ACKNOWLEDGMENTS We thank Mrs. Karen Irving, Mr. Mark Legaz, Dr. Jim McKerrow, and Dr. Jim Scotchler for communication of their results before publication and Professor Torgeir Flatmark for permission to reproduce his data in Figs. 11 and 12. We thank Mrs. Laurelee R. Robinson for careful reading of the manuscript and for carrying out the estimated distribution function calculations shown in Fig. 1. We also thank Mr. S. L. Richheimer. The reference to his work was added in proof. REFERENCES

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216. Titani, K , Ishikura, H., and Minakami, S., J. Biochem. (Tokyo) 46, 151 (1958). 217. Trippi, M. V. S., Bmlfert, J., and Plantefol, M. L., C. R. Acad. Sei. 272, 1093 (1971). 218. Van Kamp, G. J., Schats, L. H. M., and Hoenders, H. J., Biochim. Biophys. Ada 295, 166 (1973). 219. Vauquelin, L. N., and Robiquet, P. J., Ann. Chim. (Paris) [1] 57, 88 (1806). 220. Velick, S. F., Biochim. Biophys. Ada 20, 228 (1956). 221. Vickery, H. B., Pucher, G. W., Clark, H. E., Chibnall, A. C , and Westall, R. G., Biochem. J. 29, 2710 (1935). 222. Wagner, V. F., Hofmann, K. D., Preibosh, W., and Koob, G., Zentralbl. Gynaekol. 92, 1060 (1970). 223. Wellner, D., and Hayes, M. B., Ann. N.Y. Acad. Sei. 209, 34 (1973). 224. Westall, F. C., Ph.D. Thesis, University of California, San Diego (1971). 225. Westall, F. C , personal communications (1971, 1973). 226. Westall, F. C , J. Theor. Biol. 38, 139 (1973). 227. Westall, F. C , Robinson, A. B., Caccam, J., Jackson, J., and Eylar, E. H., Nature (London) 229, 22 (1971). 228. Wetter, L. R., and Deutsch, H. F., J. Biol. Chem. 192, 237 (1951). 229. Wherrett, J. R., and Tower, D. B., J. Neurochem. 18, 1027 (1971). 230. Williamson, A. R., Eur. J. Immunol. 1, 390 (1971). 231. Williamson, A. R., Salaman, M. R., and Kreth, H. W., Ann. N.Y. Acad. Sei. 209, 210 (1973). 232. Wright, H. T., Ph.D. Thesis, University of California, San Diego (1968). 233. Zeelon, P., Gershon, H., and Gershon, D., Biochemistry 12, 1743 (1973). 234. Zloch, Z., and Ginter, E., Z. Klin. Chem. Klin. Biochem. 8, 302 (1970).

Pasteur Effect and Phosphofructokinase

I. II. III. IV.

V.

VI.

VII.

VIII.

I

ABBURI RAMAIAH

I I I I

Biochemistry Department All-India Institute of Medical Sciences New Delhi, India

Introduction Definition and Physiological Importance Methods for Demonstrating the Pasteur Effect . . . . Inhibitors of the Pasteur Effect A. Inhibition by Cellular Damage B. Inhibition by Potassium and Ammonium . . . . C. Inhibition b y Miscellaneous Agents Mechanism of the Pasteur Effect A. Reversible Oxidative Inhibition Theory . . . . B. Resynthesis Theory of Meyerhof C. Pasteur Enzyme Theory of Warburg D . Phosphate Competition Theory of Lynen and Johnson . Phosphofructokinase as the Site of the Pasteur Effect . . A. Early Observations on Phosphofructokinase as the Possible Site B. Phosphofructokinase as the Site of Pasteur Effect Based on Analysis of Intermediary Metabolites of Glycolysis after a Change in Glycolytic Flux Properties of Phosphofructokinase and Their Relation to the Pasteur Effect in the Cell A. Control of Phosphofructokinase by Adenylate Energy Charge B. Activators, Inhibitors, and Deinhibitors of Phosphofructokinase from Various Sources C. Role of Various Effectors of Phosphofructokinase in Regulating Its Activity and Glycolysis D . Activation of Phosphofructokinase Is Coupled to the Activation of Hexokinase E. Activation of Phosphofructokinase Is Coupled to the Activation of P y r u v a t e Kinase F . Inhibitors of the Pasteur Effect Lead to Activation of Phosphofructokinase Analysis of the Multiplicity of Effectors of Phosphofructokinase A. A T P , A D P , 5'-AMP, and Pi B. Citrate C. 3',5'-Cyclic A M P D . Fructose 1,6-Diphosphate E. Ammonium 297

298 299 299 302 302 303 303 303 303 304 305 305 306 306

307 308 308 310 311 311 313 314 315 315 315 316 316 317

298

ABBURI RAMAIAH

I X . Systems in Which the Pasteur Effect Is Absent . . . . A. Retina B. Intestinal Mucosa C. Striated Muscle D. Tumor Cells E. Embryonic Tissue F. Leukocytes G. Red Blood Cells H. Renal Medulla X. Molecular Properties of Phosphofructokinase and the Mechanism of Action of Effectors A. Effectors Mainly Change (F6P) 0 . 6 Value . . . . B. Mechanisms by Which Effectors Change (F6P) 0 .5 Values . C. Subunit Structure of Phosphofructokinase . . . . X I . Specificity of Effectors and Substrates of Phosphofructokinase A. Specificity of Effectors B. Substrate Specificity of Phosphofructonkinase X I I . Sedoheptulose 7-Phosphate Kinase Activity of Phosphofructokinase X I I I . Conclusions References

317 318 319 322 325 326 327 328 330 331 331 332 333 333 333 334 335 336 337

Scientific conclusions may be but ever shifting hypotheses. Abburi Ramaiah

I. Introduction Louis Pasteur, during his classical studies on yeast fermentation in 1861, observed that under anaerobic conditions much more sugar was taken up and fermented per quantity of yeast present than was consumed in presence of air (204) · This original observation of Pasteur on yeast cells was shown in the last 112 years to be true in a wide variety of tissue preparations and organisms that have both respiratory and fermentative systems. This is perhaps one of the earliest known examples of regulation of energy metabolism by which the fermentative capacity of a cell is blocked in the presence of sufficient oxygen and the energy is supplied almost exclusively by a far more efficient respiratory apparatus. The mechanism (s) by which presence of oxygen inhibits fermentation or glycolysis* is not understood completely. This review includes a brief history of this phenomenon and discusses various theories that were put forward over the years and their inadequacies in explaining the Pasteur effect. The literature is reviewed to support the hypothesis that phosphofructokinase plays a central role in the regulation of glycolysis and that its properties can account to a major extent for the phenomenon of the Pasteur effect. * The terms "fermentation" and "glycolysis" are used synonymously in this article.

PASTEUR EFFECT AND

PHOSPHOFRUCTOKINASE

299

Alcoholic fermentation by yeast was the first example of anaerobic fission of a carbohydrate to be discovered (204, 205), and Pasteur was the first to realize that the presence of oxygen reduces the destruction of sugar by living organisms. He showed that the ratio of sugar destroyed to yeast substance formed could be varied from 126:1 in complete absence of air to 4:1 under marked aeration. Under conditions of high aeration, the power of fermentation is thus most inhibited. But it was Meyerhof (178) who proved conclusively in the case of yeast that the rate of sugar destruction in a given time is less in the presence of air than in its absence (205). This phenomenon was called "Pasteur effect" by Warburg (282) in honor of the first investigator of anaerobic metabolism. II. Definition and Physiological Importance The Pasteur effect can be defined in two parts: (1) as the action of oxygen in diminishing the carbohydrate uptake and its catabolism; (2) as the action of oxygen in suppressing or decreasing the accumulation of the products of anaerobic metabolism. Glycolysis, or variations thereof, is the primitive mode by which organisms synthesize ATP and is almost universally present in all living systems, whereas the respiratory mechanisms evolved after the introduction of oxygen into the earth's atmosphere. Of these two mechanisms, respiration is a more efficient process; thus the oxidation of 1 mole of glucose to C0 2 and H 2 0 leads to the synthesis of 36 moles of ATP, as compared to 2 moles of ATP by fermentation. In addition to this lack of efficiency, the end products of fermentation—for example lactic acid or alcohol— may be harmful. Lactic acid formation can decrease the cellular pH to an inhibitory range. Ethanol at high concentrations · has narcotic effects. The existence of a Pasteur effect in most of the systems therefore means that, whenever 0 2 is available, respiration plays a major role as a more economical and potentially less harmful mechanism for the synthesis of ATP. III. Methods for Demonstrating the Pasteur Effect For demonstrating the presence of the Pasteur effect in any system, one can make use of either part (1) or part (2) of the definition of the Pasteur effect. The direct method would be to show that the rate of glucose flux in any tissue or organism in presence of oxygen is less than in its absence. This direct method was adopted by few workers (70, 186, 246). The other method most generally used in demonstrating the Pasteur effect is the measurement of the Meyerhof quotient, defined by Warburg etal (286) as: Anaerobic fermentation — aerobic fermentation oxygen uptake

Kidney (rat) Thyroid gland (rat) Liver (rat) Brain cortex (rat) Lactic acid bacteria, Bacterium cereale Bacterium delbruekii Lactobacillus casei Propionic acid bacteria Propioni bacterium pentosaceum Sperm 6

Tissue 1

TABLE I

15 1

4 6.5

79 287

0.0 0.0 0 . 6 (0) 2.5 49

21 13 12 11 189 109 0

^co2 3

Respiration Qo 2 2

Aerobic glycolysis

20 8.0

188 316

3 2 3 19 305

4

QS5,

Anaerobic glycolysis

1.06 1.5

—

1.0

0.143 0.154 0.2 1.5 1.35

J

87

80 18.8

9

58

84

J

1|

\

80 (100) f

\

1

100

100 1UU

100

A

Pasteur effect (%)

Qco2 ~~ Qcc>2 Qc6 2 - Qcp2 0N2 Qo 2 ^co2 6 5

Meyerhof quotient

E X T E N T OF P A S T E U R E F F E C T IN V A R I O U S T I S S U E S AND ORGANISMS 0

87 161

54, 55

36, 282

References 7

300 ABBURI RAMAIAH

93 65. 100 100 100 100 33 20 33

1.34 2.06 0.286 0.57 0.32 0.21 1.2 0.86 1.9

260 274 1 0.8 1.6 2.7 36 31 34

18 95 0 0 0 0 24 25 17

180 87 3.5 1.4 5 12.7 10 7 9

36, 282

90

178

° Usual conditions: 37°C, p H 7.7, 0.2% glucose, 25 m l N a H C 0 3 Ringer's solution. Qo 2 = microliters of O2 respired per milligram of tissue or organism dry weight per hour. Qgo2 = microliters of lactic acid or fermentation carbon dioxide produced aerobically per milligram of tissue or organism dry weight per hour. Qc 0 2 = microliters of lactic acid or fermentation carbon dioxide produced anaerobically per milligram of tissue organism dry weight per hour. (One mole of lactic acid or substance referred to is considered as gas occupying 22.4 liters at normal temperature a n d pressure. Thus Qco 2 = 1 means 1/22.4 μΐηοΐβ of lactic acid; i.e., 0.004 mg of lactic acid was produced.) 6 Qo2, and Q°o 2 a n d Qco 2 were expressed per 10 8 cells.

Wild yeast Baker's yeast Plants Lathyrus plant sprouts Leaf Algae, Chlorella pyrenoidea Coelastrum proboscideum, malignant tissues Bladder carcinoma (man) Flexner Jobling sarcoma (rat) Jensen sarcoma (rat)

PASTEUR EFFECT AND PHOSPHOFRUCTOKINASE

301

302

ABBURI RAMAIAH

where the rate of fermentation is expressed in the same units as the oxygen consumption. If oxygen has no effect on glucose flux, the formation of lactic acid (or other fermentation product) in the presence of oxygen would be less than the formation in its absence merely by an amount of equivalent lactic acid or carbohydrate oxidized. Since 1 mole of lactic acid requires 3 moles of oxygen for its complete oxidation, the decrease in lactic acid production in the presence of oxygen would be one-third of the respiration, or oxygen consumed. Therefore the Meyerhof quotient would be 0.33. It is assumed that only carbohydrate is oxidized and the respiratory quotient (RQ) is 1.0 under these conditions. It is thus clear that if the Meyerhof quotient is 0.33, there is no Pasteur effect. If the Meyerhof quotient is greater than 0.33, the Pasteur effect is present (72). Accordingly, the higher the Meyerhof quotient, the higher is the extent of Pasteur effect in any system. The work of Warburg and his colleagues has demonstrated by this method the presence of a Pasteur effect in numerous animal tissues {282). Another way of expressing the presence and extent of Pasteur effect, perhaps the most direct one, is to express the percentage inhibition by oxygen of the accumulation of the fermentation proouct(s) or of glucose catabolic products. Table I lists some of the systems where the Meyerhof quotient and percentage inhibition by oxygen in the accumulation of fermentation product(s) are determined. From Table I, it is clear that the Pasteur effect is exhibited by a variety of systems including bacteria, plant, and animal systems. The extent of Pasteur effect as judged from the percentage inhibition of the Pasteur effect (column 6 of Table I; Meyerhofs quotient is not a proper measure of Pasteur effect, as seen in column 5), bears a relationship to the magnitude of respiration. The systems described in Table I are intact cell systems or tissue slices. It was once thought that the Pasteur effect could not be demonstrated in broken-cell preparations or in cells subjected to other types of cellular injury (282). IV. Inhibitors of the Pasteur Effect A. Inhibition by Cellular Damage Negelein (187) found that when embryonic tissue is allowed to remain for some time in Ringer's solution aerobic glycolysis develops without any fall in respiration. This does not occur when the tissue is kept in serum. Nakashima (184) showed that the Meyerhof quotient of fish retina is reduced by high temperature even when there is actual rise in respiration. Similar effects were described by Dixon (71) for brain cortex. Baker's yeast when ground was found to have aerobic alcoholic fermentation al-

PASTEUR EFFECT AND PHOSPHOFRUCTOKINASE

303

though the respiration was increased (247). The Pasteur effect is also decreased in cells subjected to osmotic or other types of cellular injury (282). However, later experiments indicate that the Pasteur effect is demonstrable even in homogenates. For instance, Terner (261) demonstrated Pasteur effect in cell-free preparations of yeast and mammary gland. B. Inhibition by Potassium and Ammonium Ashford and Dixon (9) found that the addition of 0.1 M KC1 to the Ringer bicarbonate buffer in which mammalian brain cortex slices were suspended causes an increase in aerobic glycolysis and respiration. Rubidium and cesium bring about similar effects (62, 73). That the Pasteur effect is really inhibited by the addition of potassium ions was shown by Dixon (70), who demonstrated that the disappearance of carbohydrate in the presence of excess potassium is raised to the normal anaerobic level. The potassium effects was originally shown where glucose was the substrate supplied to the cells and was not seen in absence of the substrate (69). Similar effects of potassium are also seen in muscle (103). In addition to potassium, low concentrations of ammonium also inhibit the Pasteur effect in certain tissues (291). C. Inhibition by Miscellaneous Agents Cyanide, azide, carbon monoxide, and other compounds that interfere with respiration; nitrophenols and phenosafranine which uncouple phosphorylation to oxidation (152) ; and other compounds that inhibit the Pasteur effect are listed in Table II. V. Mechanism of the Pasteur Effect Ever since the original observation of Pasteur that oxygen inhibits fermentation, many theories were put forward to explain the mechanism of Pasteur effect (for reviews, see 36, 60, 72, 131, 149). Only brief descriptions of a few of the many theories and the reasons for their failure to explain the phenomenon will be given. A. Reversible Oxidative Inhibition Theory Lipmann (148) and Hahn et al. (99) suggested that the simple presence of oxygen inactivates glycolysis by reversible oxidation of SH groups of enzymes in the glycolytic pathway. Lipmann listed a number of partial enzymes of glycolysis that were thus susceptible to inactivation (149). This suggestion could not be accepted since glycolysis in cells and tissues is actually increased in the presence of oxygen and dinitrophenol (74) or cyanide (177) and abolish the Pasteur effect.

304

ABBURI RAMAIAH

TABLE II I N H I B I T O R S OF THE P A S T E U R E F F E C T "

N a m e of the compound or agent Phenosafranine derivatives of quinoline acridine, and phenazine-containing base constituents Heat Carbon monoxide

Low Po 2 Dinitrocresol 2,4-Dinitrophenol Ethylcarbylamine Cyanide

Sodium azide, phenylurea, sodium arsenate, propionitrile, acetonitrile, p-nitrophenol Guanidine compounds Reduced glutathione Reduced glutathione and cysteine

System where demonstrated

References

Brain, tumor

57, 58

Tumor Yeast Retina, chorion, liver, embryonic a n d tumor tissues Embryo Retina, chorion, liver Tumors and kidney Muscle Various tissues, tumors Chopped muscle Tumors, embryo Retina Sweet pea seedlings Yeast

61 281 140

R a t brain cortex, yeast, tumor cells R a t sarcoma Baker's yeast

59

286 HO 74 77 280 174, 177 280, 286 139 90a 246

34 207

a

These are compounds t h a t decrease the percentage inhibition of anaerobic glycolysis by the presence of oxygen.

B. Resynthesis Theory of Meyerhof Meyerhof {175, 176) put forward a theory that the gross rate of carbohydrate catabolism is same in the presence as in the absence of oxygen, but that respiration causes resynthesis of part of the anaerobic products of carbohydrate catabolism to glucose and glycogen. This results in an apparent decrease in the net rate of carbohydrate catabolism in the presence of oxygen and is known as the Meyerhof cycle, which may be diagrammed as follows: Anaerobic phase Lactate

Carbohydrate

i Oxidation

Aerobic phase

C0 2 + H 2 0

PASTEUR EFFECT AND PHOSPHOFRUCTOKINASE

305

Thus it was established that the nonappearance of the products of fermentation in the presence of oxygen could not be caused by their complete oxidation as suggested by Fletcher and Hopkins (83, 84). On the basis of this theory, the rate of disappearance of lactic acid in the presence of oxygen should be the same as the difference in the rate of lactic acid formation in the absence and in the presence of oxygen. In fact, the rate of disappearance of lactic acid in the presence of oxygen in brain cortex and chick embryo is about one-ninth of the difference in the rate of production of lactate in the absence and in the presence of oxygen (69). Thus this theory is inconsistent with the available facts. C. Pasteur Enzyme Theory of Warburg Warburg (280) produced the first clear proof of the separation of the processes of glycolysis and respiration in tissue slices. He found that ethyl isocyanide at a concentration of 10~3 M increased aerobic glycolysis to the level of anaerobic rate. Warburg visualized that this compound inhibits a particular reaction catalyzed by the "Pasteur enzyme." Selective inactivation of the Pasteur enzyme by ethyl isocyanide was attributed to its chelation with heavy metal presumed to be present in the Pasteur enzyme. Stern and Melnick (245) indicated that the Pasteur effect is controlled by a specific iron-containing catalyst closely resembling the cytochrome c oxidase in structure. But no such enzyme was ever characterized. Phosphofructokinase may be visualized as the Pasteur enzyme, but for reasons not related to chelation by ethyl isocyanide, which are discussed in Sections VI and VII. D. Phosphate Competition Theory of Lynen and Johnson Lynen (157) and Johnson (115) independently suggested that inorganic phosphate may be the key substance in controlling the rate of glycolysis since glycolytic and oxidative phosphorylation systems compete for inorganic phosphate under aerobic conditions. If oxidative phosphorylation can proceed at lower concentrations of phosphate than the glycolytic process, oxidative phosphorylation would compete successfully for Pi; therefore, in the presence of oxygen, the glycolytic rate will be reduced. However, inorganic phosphate concentrations are high in many tissues. Its concentration, expressed as micromoles per gram wet weight, is 3.5 for rat liver (95), 4.7 for mouse brain (156), 0.24 for cerebrum (251), 10.4 for skeletal muscle of rat (264), 4.1 for frog sartorius muscle (105), and 9.5 for intestinal mucosa (258). Thus the concentration of Pi is high compared to the concentration required for maximum glycolytic rate in tissue extracts (146). Moreover, Ochoa and Stern (192) pointed out that the difference in the level of Pi in the steady state of

306

ABBURI RAMAIAH

respiration and fermentation in the experiments of Lynen and Koenigsberger (160) is small and cannot possibly be the sole cause for different rates of glycolysis in the presence and in the absence of oxygen. In addition, the demonstration of a Pasteur effect in cell-free preparations of yeast and mammary gland at high initial phosphate levels and short incubation periods that avoid undue depletion of P i ? argues against Pi as a controlling factor in the Pasteur effect (261, 262). Perhaps the most important weakness of this theory is that it cannot account for the inhibition of glucose phosphorylation in the Pasteur effect, because that reaction requires neither Pi nor ADP. VI. Phosphofructokinase as the Site of the Pasteur Effect A. Early Observations on Phosphofructokinase as the Possible Site Engelhardt and Sakov were the first to suggest that the Pasteur effect must operate at the phosphofructokinase step (78), based on the fact that fructose-1,6-diphosphate is readily fermented in the presence of agents, including oxygen, that inhibit glucose fermentation. They suggested that inhibition of fermentation by oxygen could be due to oxidative inactivation of phosphofructokinase although at that time little was known of the varied mechanisms that exist for the regulation of enzyme activity. Other experiments suggesting that phosphofructokinase may be the rate-limiting step of glycolysis in muscle and other mammalian tissues were carried out by Cori and Cori (47). They observed that in intact muscle tissue fructose 6-phosphate does not react rapidly with ATP, as shown by the fact that considerable increase in fructose 6-phosphate concentration under certain experimental conditions does not lead to increase in the formation of fructose 1,6-diphosphate or lactic acid. Aisenberg and Potter (5, 6) studied the Pasteur effect by adding varying amounts of mitochondrial preparation from various tissues to a glycolytic system of rat brain or tumor tissue in the presence or in the absence of hexose diphosphate and inhibitors of respiration. They came to the conclusion that oxygen, acting through the cytochrome chain and cytochrome c reductase of mitochondria, brings about the formation of some high-energy intermediate of oxidative phosphorylation and that this compound in some manner is able to inhibit the phosphofructokinase reaction and perhaps hexokinase, thereby inhibiting the utilization of glucose. However, the mechanism by which the phosphofructokinase reaction is inhibited was entirely obscure at that time. About a year before this observation, Lardy and Parks had noted that ATP itself is a strong inhibitor of partially purified phosphofructokinase from skeletal muscle of

PASTEUR E F F E C T AND

PHOSPHOFRUCTOKINASE

307

rabbit (138). Therefore, the hypothetical high-energy intermediate proposed by Aisenberg, Reinafarje, and Potter, which may inhibit phosphofructokinase in their system, could have been ATP itself. In this context it is of great interest to refer to the studies of Ostern and Mann (194), who found that the addition of adenosine triphosphate to mashed muscle depressed aerobic glycolysis and raised the Meyerhof quotient from 2.2 to 4.0, indicating a more pronounced Pasteur effect. Later, Lennerstrand (145) discussed the possibility that with aerobic overphosphorylation of adenylic acid, the ratio of ATP to adenylic acid may become unfavorable for efficient fermentation. B. Phosphofructokinase as the Site of Pasteur Effect Based on Analysis of Intermediary Metabolites of Glycolysis after a Change in Glycolytic Flux The measurement of concentration of intermediates of a metabolic pathway can be used to identify control sites of that pathway, using the crossover theorem (40-42) and the principle suggested by Krebs (ISO). Phosphofructokinase is a nonequilibrium reaction under in vivo conditions (106, 108, 218, 299), and a decrease in fructose 6-phosphate concentration in a tissue with increased glycolytic flux indicates that phosphofructokinase is a regulatory enzyme of glycolysis. Lowry et al. (156) had shown that in the mouse brain ischemia induced by decapitation results in a 4- to 7-fold increase in glycolytic flux. This Pasteur effect is associated with a decrease in the intracellular concentration of glucose, glucose 6-phosphate, and fructose 6-phosphate. Similar studies were done in other systems. Investigations on the isolated perfused rat heart in the presence of insulin demonstrated that anoxia caused an activation of phosphofructokinase (190, 191, 217, 299, 300). Extensive studies on the kinetics of changes in glycolytic intermediate concentrations following an increased flux induced by the aerobic-anaerobic transition, i.e., during the release of the Pasteur effect in yeast (93, 158, 159), ascites tumor cells (162), potatoes, apples, and peas (20), perfused rat hearts (197, 217, 299), rat diaphragm and isolated perfused rat heart (190), slices of kidney cortex, Novikoff hepatoma and adenocarcinoma (303), and locust flight muscle (85), indicated that phosphofructokinase and hexokinase are activated during anoxia. Determination of glycolytic intermediates following increased glycolytic flux induced by introduction of glucose to Ehrlich's ascites tumor cells (107, 151) and increase in glycolytic flux induced by contraction of muscle (104, 224) and in a host of other systems (see for reviews, 10, 230, 242) also indicated that phosphofructokinase and hexokinase are activated during increased glucose flux. Phosphofructokinase is central to the phenomenon of the Pasteur

308

ABBURI RAMAIAH

effect although hexokinase activity is also altered under these conditions. One may argue that if the Pasteur effect were due to inhibition of phosphofructokinase in the presence of oxygen and its activation in the absence of oxygen, as shown to be the case in a number of systems enumerated above, one would still expect the consumption of glucose because phosphofructokinase catalyzes the conversion of fructose 6-phosphate to fructose 1,6-diphosphate. Thus inhibition of phosphofructokinase would not explain the decreased rate of glucose utilization in the presence of oxygen that characterizes the Pasteur effect. Inhibition of hexokinase by its product, glucose 6-phosphate, provides the explanation. Inhibition of hexokinase activity by glucose 6-phosphate was first shown for brain hexokinase (48, 292) and schistosoma (32) and later for hexokinases from many sources (277). In the case of yeast, where hexokinase is not inhibited by glucose 6-phosphate, glucose 6-phosphate inhibits the uptake of glucose (238). Under aerobic conditions, when phosphofructokinase is inhibited the substrate of this enzyme, fructose 6-phosphate, accumulates. Owing to the high activity of hexosephosphate isomerase, the accumulation of fructose 6-phosphate results also in the accumulation of glucose 6-phosphate. Thus the inhibition of phosphofructokinase automatically generates the inhibitor of hexokinase; this decreases the phosphorylation of glucose, and hence its utilization, or in the case of yeast its uptake from the medium (238). It could thus be argued that phosphofructokinase is the central site for the operation of Pasteur effect (210). VII. Properties of Phosphofructokinase and Their Relation to the Pasteur Effect in the Cell The determination of in vivo levels of glycolytic intermediates in a variety of systems after an increased glycolytic flux, described in the preceding section, indicates that phosphofructokinase plays a central role in the regulation of glycolysis and in the operation of the Pasteur effect. The mechanisms of its activation under anaerobic conditions are described in this section. Previous theories on the Pasteur effect phenomenon failed for various reasons described in Section V. Any new theory must be able to explain the direct relationship between the extent of the Pasteur effect and respiration as presented in Table I. It should also explain the inhibition of the Pasteur effect in the presence of uncouplers of oxidative phosphorylation, inhibitors of respiration, and other agents listed in Table II. A. Control of Phosphofructokinase by Adenylate Energy Charge In view of the fact that one of the physiological functions of both respiration and glycolysis is to produce ATP—the energy currency of the cell

PASTEUR EFFECT AND PHOSPHOFRUCTOKINASE

309

and the universal stoichiometric coupling agent (13)—and because the Pasteur effect is in essence a phenomenon for the regulation of ATP synthesis, it would be logical to expect ATP to act as the regulatory metabolite of glycolysis and the citric acid cycle. It is an end product of these metabolic pathways. Therefore, like other metabolic pathways controlled by feedback inhibition of the first enzyme of the pathway by the end product (269, 305), accumulation of ATP might be expected to inhibit the first committed enzyme of the glycolytic sequence, phosphofructokinase, and the first enzyme of the citric acid cycle, citrate synthetase. This was indeed found to be the case (102,114,138). Since adenine nucleotides are universal stoichiometric coupling agents between anabolic and catabolic segments of metabolism, and since many enzymes are regulated by these nucleotides, Atkinson (10) advanced the adenylate control hypothesis. This states that the concentrations of adenine nucleotides in a cell are important parameters in the regulation of sequences that lead to the regeneration of ATP or to the production of storage compounds. Atkinson introduced the term "adenylate energy charge," defined as half the number of anhydride-bound phosphate groups per adenine moiety (17). In terms of concentration of individual components, the adenylate energy charge can be expressed as: (ATP + 3^ADP)/(ATP + ADP + AMP) A generalized response to the energy charge by enzymes involved in regulation of ATP regenerating (R) and ATP utilizing (U) sequences or metabolic pathways are shown in Fig. 1 (11). Since phosphofructokinase is a rate-regulating enzyme of glycolysis, an ATP-regenerating metabolic sequence, it responds to changes in energy charge as indicated by curve R in Fig. 1 (234). Thus low concentrations of ATP relative to ADP and AMP expected under anaerobic conditions will tend to activate the enzyme, thus explaining the activation of phosphofructokinase and increase in glycolytic flux during anaerobic conditions, i.e., the Pasteur effect. The inhibition of phosphofructokinase from skeletal muscle by high concentrations of ATP was first shown by Lardy and Parks (138), and the reversal (deinhibition) of ATP inhibition by ADP and AMP was shown by Passonneau and Lowry (201). Passonneau and Lowry (201) were the first to suggest that these properties of phosphofructokinase may provide the explanation of the phenomenon of the Pasteur effect. The work of Passonneau and Lowry (203) added fructose 1,6-diphosphate, and ammonium and potassium ions as activators of the enzyme, and the studies of Mansour added 3',5' cyclic AMP to the list (163).

310

ABBURI RAMAIAH

Energy charge

FIG. 1. Generalized response to the adenylate energy charge expected for enzymes involved in regulation of ATP-regenerating (R) and ATP-utilizing (U) sequences. From Atkinson (11); reprinted with permission of the American Chemical Society.

B. Activators, Inhibitors, and Deinhibitors of Phosphofructokinase from Various Sources The effectors of phosphofructokinase were classified as inhibitors, deinhibitors, and activators by Passonneau and Lowry (203) and are listed in Table III. Further studies on phosphofructokinases of yeast {210, 274), TABLE I I I E F F E C T O R S OF PHOSPHOFRUCTOKINASE"

Activators 6

Inhibitors ATP Citrate Mg2+ Ca 2 + P-creatine 3-P-glycerate P-enolpyruvate 2-P-glycerate 2,3-di-P-gly cerate

NH+ K+ Pi

5'-AMP 3',5'-cyclic A M P ADP Fructose 1,6-di-P

Deinhibitors of A T P , citrate, or Mg 2 + C Fructose 1,6-di-P 3',5'-cyclic A M P 5'-AMP ADP Fructose 6-P Pi

Glucose 1,6-di-P

a Taken from Tejwani (258); adopted from Passonneau and Lowry (203). b Activators increase the velocity of the phosphofructokinase reaction at noninhibitory concentrations of A T P . c Deinhibitors increase the velocity of phosphofructokinase reaction at inhibitory concentrations of A T P .

PASTEUR EFFECT AND PHOSPHOFRUCTOKINASE

311

E. coli (16), the liver fluke Fasciola hepatica (167), heart (168), intestinal mucosa (110, 259), brain (155), liver (121), and liver, brain, heart, and skeletal muscle of rabbit (211), as well as phosphofructokinases from microorganisms, higher plants and many other sources (153) indicate that the phosphofructokinases exhibit many similar, though not identical, regulatory properties. The activators, inhibitors, and deinhibitors of phosphofructokinases from various sources are listed in Table IV. C. Role of Various Effectors of Phosphofructokinase in Regulating Its Activity and Glycolysis From Table IV certain generalizations can be made. Citrate and ATP at high concentrations are inhibitors of phosphofructokinase from a majority of the sources so far studied. P E P is an inhibitor in certain cases, and 2,3-diphosphoglycerate inhibits the phosphofructokinase from red blood cells and skeletal muscle. AMP, P b and NH 4 are usually either deinhibitors or activators, while ADP is a deinhibitor of phosphofructokinase in about half of the cases studied and an inhibitor or without effect on phosphofructokinases from the other sources listed in Table IV. Fructose 6-phosphate, the substrate of the reaction, is an activator in every case with only one exception. By inspection of Table IV, it may be generally stated that activators of phosphofructokinase, such as AMP, ADP, Pi, fructose 1,6-diphosphate are those that tend to accumulate during anaerobic conditions whereas the inhibitors of the enzyme, such as ATP and citrate, are those that tend to increase on transition from anaerobic to aerobic conditions. The consequences of such alteration in the concentration of effectors of phosphofructokinase are such as to activate the enzyme under anaerobic conditions and inhibit it under aerobic conditions. The Pasteur effect phenomenon can thus be visualized as due to the activation of phosphofructokinase under anaerobic conditions and inhibition of its activity under aerobic conditions. There are certain organisms (Table IV) where phosphofructokinase activity is not affected by adenine nucleotides and Pi, thus its activity will not alter on transition from aerobic to anaerobic conditions. But these exceptions do not invalidate the above generalization, since in these organisms either the Pasteur effect is not observed or glucose is not the major source of energy, hence regulation of energy metabolism must be located elsewhere. D. Activation of Phosphofructokinase Is Coupled to the Activation of Hexokinase Activation of phosphofructokinase under anaerobic conditions is also coupled to activation of hexokinase because a decrease in the concentra-

I I I I NE NE NE NE NE

I I I I I I I I I I A**

ATP

NE NE NE I I NE

NE

NE

I

I

I

NE NE

I

NE NE I

I

PEP

I I NE NE I I

Citrate

NE I I,A

A A A A NE

I I I I NE I I

A A

ADP

NE A A A NE A NE I I

A A A A I I I NE A I NE

AMP

NE I NE

NE

NE NE A

NE

NE I NE NE

A A NE A

A NE NE

I I A NE

NE

A A NE A A A NE A I

Effectors b cAMP Pi

A A A A

A A

A

A A A A A A A A A A NE

F6P

I

NE

I

NE

I I

A

FDP

NE A* A A

-

A*

A

A A NE NE

NHt

306,307

16, 27, 153 153 153 226 81 272 75 75

267 23

18,210,274-

153 153

120

153,203

31, 167 278 278 56

References

b

a

Taken from Tejwani (258). Abbreviations: cAMP, cyclic adenosine monophosphate; I, inhibitor; A, activator (increases the velocity of enzyme at inhibitory or noninhibitory concentrations of ATP); NE, no effect; FDP, fructose diphosphate; -, not determined; PEP, phosphoenolpyruvate; F6P, fructose 6-phosphate; A *, absolutely necessary for the enzyme activity; A**, ATP does not inhibit, but overcomes the inhibition caused by ADP, fructose 1,6-di-P, and pyrophosphate.

Mammalian Schistosoma Desert locust (fat body) Desert locust (flight muscle) Brussels sprouts Pea seeds Avocado Parsley Yeast Neurospora crassa Slime mold (Dictyostelium discoideum) Escherichia coli Clostridium perfringens Staphylococcus aureus Aerobacter aerogenes A rthrobacter crystallopoietes Clostridium pasteurianum Lactobacillus casci Lactobacillus plantarum Flavobacterium thermophilium

Source

TABLE IV THE ACTION OF VARIOUS EFFECTORS ON THE ACTIVITIES OF PHOSPHOFRUCTOKINASES FROM VARIOUS SOURCES a

312 ABBURI RAMAIAH

PASTEUR EFFECT AND PHOSPHOFRUCTOKINASE

313

tion of fructose 6-phosphate resulting from the activation of phosphofructokinase leads to a decrease in the concentration of glucose 6-phosphate, because of the high activity of glucose-6-phosphate isomerase. Glucose 6-phosphate is an inhibitor of hexokinase from various sources {277), and its removal results in the activation of hexokinase. In addition, Pi {221, 273) and K+ {183) increase the activity of hexokinase as well as that of phosphofructokinase. ATP 4 - at high concentration is an inhibitor of hexokinase {96, 97), similar to its effect on phosphofructokinase {154). Thus both phosphofructokinase and hexokinase are activated or inhibited under similar conditions leading to increased or decreased glucose utilization. E. Activation of Phosphofructokinase Is Coupled to the Activation of Pyruvate Kinase The other enzyme to play an important regulatory role in glycolysis, based on its displacement from equilibrium under in vivo conditions {108, 156, 203, 219, 220, 300) and on thermodynamic considerations {132), is pyruvate kinase. The activity of this enzyme in various cell types appears to be proportional to the glycolytic capacity {236). The activation of phosphofructokinase is also coupled to the activation of pyruvate kinase, for the following reasons. Pyruvate kinase is inhibited by concentrations of alanine and ATP that occur in the cell (see review 232a). The activation of this enzyme under anaerobic or other conditions that activate phosphofructokinase is due to an increase in the steady state level of fructose 1,6-diphosphate, which is an activator of pyruvate kinase from yeast {89, 109), liver {256), and other sources {232a). The importance of fructose 1,6-diphosphate in the activation of pyruvate kinase under in vivo conditions is illustrated in the case of yeast (Table V). The enzyme is activated during glycolysis following addition of glucose to nongrowing cells of Saccharomyces cerevisiae, as judged by the decreased level of phosphoenolpyruvate under these conditions. The level of fructose 1,6-diphosphate under these conditions is about 100-fold higher than the level under nonglycolytic conditions. Pyruvate kinase, in addition, is activated by potassium and ammonium, which are also activators of phosphofructokinase (see 232a). Thus the effectors of phosphofructokinase also modulate the activity of hexokinase and pyruvate kinase, the other two regulatory enzymes of glycolysis, in the same direction as their action on phosphofructokinase. Numerous experiments have been recorded which show that the postulated changes in the concentrations of the effectors of phosphofructokinase, hexokinase, and pyruvate kinase are correlated with the rate of glycolysis {156, 189, 214, 300, 301).

314

ABBURI RAMAIAH

TABLE V M E T A B O L I T E L E V E L S DURING E N D O G E N O U S M E T A B O L I S M , GLYCONEOGENESIS, AND GLYCOLYSIS"- 6

Glyconeogenesis, minutes after addition of ethanol Metabolite 0 G6P F6P F6P/G6P FDP PEP

Endogenous

>o >0

—

0.01 1.8

Glycolysis, minutes after glucose addition

10

11

12

10

11

12

0.6 0.18 0.3 0.03 0.21

0.59 0.18 0.31 0.02 0.23

0.54 0.16 0.30 0.02 0.23

1.84 0.46 0.25 2.45 0.12

1.87 0.44 0.24 2.2 0.13

1.86 0.45 0.24 2.4 0.13

° From Barwell and Hess {22). Reproduced with permission from the copyright owner. 6 Values for metabolite levels are expressed as micromoles per gram wet weight of nongrowing cells of Saccharomyces cerevisiae. c G6P, glucose 6-phosphate; F 6 P , fructose 6-phosphate; F D P , fructose diphosphate; P E P , phosphoenolpyruvate.

F. Inhibitors of the Pasteur Effect Lead to Activation of Phosphofructokinase The mechanism by which the inhibitors of respiration or oxidative phosphorylation listed in Table II decrease the Pasteur effect can now be understood in terms of the properties of phosphofructokinase. Since the rate of ATP synthesis and the level of citrate decrease when the respiration rate is low, the activity of phosphofructokinase and the rate of glycolysis will be inversely related to the rate of respiration; this explains the decrease in the extent of Pasteur effect with decrease in respiratory quotient (Table I) and or by inhibitors of respiration and uncouplers of oxidative phosphorylation (Table II). Other factors which are neither uncouplers of oxidative phosphorylation nor inhibitors of respiration and which inhibit the Pasteur effect are N H | and K+ and reduced glutathione. NHj" and K + have been shown to be activators of phosphofructokinase from various sources (see Table IV). In some cases NH|" acts as a deinhibitor of ATP inhibition (121). In addition, its synergistic action with other positive effectors of phosphofructokinase in increasing phosphofructokinase activity (discussed in Section VIII, E) may account for the increase in aerobic glycolysis and the decrease in the Pasteur effect. However,, no simple explanation is available for the inhibition of the Pasteur effect by reduced glutathione, although phosphofructokinase activity is stabilized by SH compounds in vitro.

PASTEUR E F F E C T AND

PHOSPHOFRUCTOKINASE

315

In systems where glycolysis and gluconeogenesis both occur, as in liver, their simultaneous operation would lead to futile cycles (230). Such futile cycles are prevented by the inhibition of the key glycolytic enzymes, phosphofructokinase, hexokinase and pyruvate kinase, by gluconeogenic signals (290). VIII. Analysis of the Multiplicity of Effectors of Phosphofructokinase A. ATP, ADP, 5'-AMP, and P, Phosphofructokinase is an ATP-utilizing enzyme, but in contrast to other kinases its activity responds to changes in the adenylate energy charge as illustrated in curve R of Fig. 1 rather than in the direction of curve U. This is consistent with its role in the glycolytic sequence and the synthesis of ATP, and the regulation of this process by the adenylate energy charge. Thus, the increases in ADP, AMP, and Pi and the decreases in ATP under anaerobic conditions are ideal signals for activating phosphofructokinase, the rate-limiting step in the ATP regenerating glycolytic sequence, so as to counteract the decrease in the energy charge. The concentrations of ADP, AMP, and Pi change in parallel, and small changes in these concentrations can be more effective if they all serve as effectors, than if only one were to act as the signal affecting phosphofructokinase activity. B. Citrate In a cell metabolizing glucose, the function of glycolysis is not only to regenerate ATP but also to provide biosynthetic intermediates, such as pyruvate, oxaloacetate, acetyl-COA; the glycolytic pathway is thus an amphibolic sequence in the terminology introduced by Davis (53). Consequently regulation of the glycolytic sequence through the modulation of phosphofructokinase activity by energy charge alone could severely depress the levels of the biosynthetic intermediates when the energy charge is high. A plentiful supply of energy might thus depress rather than enhance biosynthetic activity. This undesirable result is prevented in the case of glycolysis by modulation of phosphofructokinase activity by citrate, which may be considered to monitor the level of biosynthetic intermediates in a cell. The effect of citrate on the response of phosphofructokinase to the energy charge is shown in Fig. 2 (23·4). Citrate as an inhibitor of phosphofructokinase may thus have an important physiological function.

316

ABBURI RAM AI AH r

i

1 '

I

:==z

!

I

I

]

^::x^

\10 I

l r -i 0.6

»i

i 0.8

Energy

1

charge

i1 1.0

FIG. 2. Interaction between energy charge and citrate concentration in the control of phosphofructokinase activity. Rate of the reaction catalyzed by rabbit muscle phosphofructokinase as a function of energy charge; effect of citrate. Mixtures of A T P and A M P of the desired energy charge and at a combined concentration of 6 m l were added to the components of the ADP assay for phosphofructokinase (coupled to lactate dehydrogenase). The concentration of fructose 6-phosphate was 0.5 m l , and the Mg 2+ concentration was 6 m M . Citrate was added as indicated by dotted line. From Shen et al. (234)', reprinted with permission of the American Chemical Society.

C. 3',5'-Cyclic AMP 3',5'-Cyclic AMP (cAMP), the second messenger in the action of many hormones (253), may be viewed as a component of an override mechanism by which local control, e.g., control from the energy charge, is partially superseded by a signal reflecting the wide needs of the organism (284). D. Fructose 1,6-Diphosphate The effect of fructose 1,6-diphosphate in rendering phosphofructokinase less sensitive to inhibition by ATP provides a positive feedback mechanism that may have particular significance in the activation of glycolysis in cardiac and skeletal muscles (1). Fructose 1,6-diphosphate is cleaved by aldolase and hydrolyzed by fructose-1,6-diphosphatase. An increase in 5'-AMP during muscular contraction inhibits fructose-1,6-diphosphatase (253a) thus enhances the increase in phosphofructokinase activity. In resting muscle, a decrease in 5'-AMP should allow fructose-l,6-diphosphatase to decrease the steady state level of fructose 1,6-diphosphate and thus ensure almost complete stoppage of glycolysis at the fructose 6phosphate level. This interpretation has been suggested (188a) to account for the existence of fructose-1,6-diphosphatase in muscle, where gluconeogenesis does not occur to a significant extent. However, recent studies of Clark et al. (43a) indicate that the existence of fructose-1,6-diphosphatase in muscle helps in the production of heat by cycling of fructose 6-phosphate through phosphofructokinase and fructose-1,6-diphosphatase (futile cycle in other systems).

PASTEUR EFFECT AND PHOSPHOFRUCTOKINASE

317

E. Ammonium Addition of NH+ ions to yeast cells oxidizing glucose increases aerobic glycolysis. About 60-90% of NHJ disappearing in 30-45 seconds after addition can be accounted for by reductive amination of a-ketoglutarate, the concentration of which decreases correspondingly in the cells. This may be an important mechanism by which the energy supply rate is enhanced at the time when building units for protein synthesis are synthesized {111). The activation of phosphofructokinase of yeast by N H | {171, 183, 209) explains its activation of glycolysis. In mammals, three systems known to function in the fixation of NH 3 are glutamate dehydrogenase, glutamine synthetase, and carbamyl phosphate synthetase, substrates of which are α-ketoglutarafe, NADH, ATP, and C0 2 . These are derived either from the glycolytic pathway or from the citric acid cycle. Thus it seems reasonable to assume that the activation of phosphofructokinase by N H | enhances the utilization of carbohydrate to produce the compounds essential for its own fixation and the maintenance of its tissue concentration at a nontoxic level {225). Contraction of muscle is also associated with an increased formation of ammonia {199). The activation of phosphofructokinase, both by reversing the inhibition of the enzyme by excess ATP {211) and by increasing its maximal velocity, would result in the activation of glycolysis in muscular contraction. The importance of ammonium in activating phosphofructokinase of muscle and intestinal mucosa is discussed in Sections IX, B and C. Thus the modulation of phosphofructokinase activity by a multiplicity of effectors can serve effectively to regulate the supply of ATP and biosynthetic intermediates under a variety of physiological conditions. IX. Systems in Which the Pasteur Effect Is Absent Although the Pasteur effect was observed in numerous animal tissues, plants, and bacteria {282) (see Table I ) , there are exceptions in which the Pasteur effect is absent or in which more glycolysis occurs in the presence of oxygen than in its absence. Some examples are listed in Table VI. The absence of the Pasteur effect in retinas and intestines of various species is striking, and in some tissues, such as the mouse jejunum, a considerable predominance of aerobic glycolysis over anaerobic glycolysis has been reported (35, 68,150, 291). The observations of Burk {35) and Weil-Malherbe (291) that the Pasteur effect was absent in intestinal mucosa has been amply confirmed (65, 150, 244, 302) over the years. However, in a recent report Lamers and Hulsmann (135) suggested that an insufficient supply of oxygen to

318

ABBURI

RAMAIAH

T A B L E VI SYSTEMS LACKING T H E P A S T E U R E F F E C T "

Tissue or organism Retina (rabbit) Retina (chick) Retina (pigeon) Bottom yeast 16-Day-old r a t jejunum Intestinal mucosa Mouse jejunum

-Qo2 27 Very small 8 10 15.2 10 18.3

Pasteur effect (% inhibition of glycolysis b y oxygen

References

+Q&,

+ Qco2

33 109

37 106

10.8

183 150 13.1

187 170 13.9

— —

129 178 160

13 23.1

14 16.2

— —

85 150

H

129

12

° Respiration and glycolytic values of various tissues are taken from literature. The units of Q are defined as in Table I.

jejunum incubated under aerobic conditions in vitro might explain this high rate of aerobic glycolysis. They observed a Pasteur effect in the perfusates of rat small intestine when they added fluorocarbon to the perfusion medium. However, it is difficult to accept this explanation for the lack of Pasteur effect observed for intestinal mucosa by previous workers, since under similar conditions of incubation a Pasteur effect could be observed in parts of intestine other than the jejunum of rats. Lohmann et al. (150) employing similar conditions of incubation of jejunum observed a Pasteur effect in the small intestine of the mouse, rat, golden hamster, chicken, and sparrow, while high aerobic glycolysis was found in jejunum of the rat and mouse. Absence of Pasteur effect in the retinal and intestinal mucosa and high aerobic glycolysis are not perhaps artifacts as was suggested by Warburg (284), since determination of lactate in portal blood and vitreous body indicate much higher levels than in blood (124,131). A. Retina The high aerobic glycolysis in retina that serves as a major source of energy is related to a reduced number of mitochondria per cell, which in turn may be correlated with the absence of blood vessels (131). If respiration were to be the major energy source in retina as in other tissues, light absorption by the red blood cells and decrease in transparency

319

PASTEUR EFFECT AND PHOSPHOFRUCTOKINASE

of the retina by light scattering caused by mitochondria would interfere with visual perception. Thus high aerobic glycolysis in retina has evolved perhaps because it confers a decisive biological advantage. B. Intestinal Mucosa Similarly, high aerobic glycolysis in the intestinal mucosa may have an important biological function since considerable amounts of glucose and as much as 50% of fructose absorbed from the lumen appear as lactate in the portal blood (124). However, the mechanism by which high aerobic glycolysis is obtained in the intestinal mucosa was not clear. As in many tissues and organisms the phosphofructokinase of jejunal mucosa was shown to be one of the rate controlling enzymes of glycolysis in this tissue (240). Therefore in view of the association of properties of phosphofructokinase with the Pasteur effect (Sections VI and VII), it is possible that the properties of the enzyme from this tissue may be significantly different from those of the enzyme of other tissues. However, studies of the properties of phosphofructokinase from this tissue (110, 259) indicated that they are essentially the same as those of other mammalian phosphofructokinases except (259) that ammonium ion is a very potent activator and acts synergistically with other positive effectors in deinhibiting the enzyme activity by excess ATP. The remarkable syner-

T A B L E VII E F F E C T OF A M P ,

ADP,

Pi,

AND NH+

SEPARATELY AND IN

COMBINATION ON T H E ACTIVITY OF PHOSPHOFRUCTOKINASE FROM T H E M U C O S A OF R A T

JEJUNUM"

Percentage of maximum activity 6

AMP (ml)

ADP (ml)

Pi (ml)

NH+ (ml)

With 0.195 m M ATP

0 0.20 0 0 0 0.20 0.20

0 0 0.76 0 0 0 0.76

0 0 0 1.0 0 1.0 1.0

0 0 0 0 2.0 2.0 2.0

0.0 4.3 3.5 8.7 3.5 53.5 55.2

With 1.56 m.i ATP 0.0 0.0 1.7 1.7 0.0 40.9 51.3

" F r o m Tejwani and Ramaiah {259). Reproduced with permission from the copyright owner. b The concentration of fructose 6-phosphate was 0.2 m M .

320

ABBURI RAMAIAH

gism of these effectors in activating and deinhibiting the enzyme activity is shown in Table VII. It was shown that these effects are related to their synergistic effect in decreasing the apparent Km value (F6P)0.5 for fructose 6-phosphate by about 20-fold (259). A survey of literature indicates that the ammonium ion level in this tissue may indeed be high in view of the presence of the enzyme urease in these cells (123, 252) and also because of high content of adenosine deaminase activity in the intestinal mucosa (227). The lack of Pasteur effect may thus be related to the favorable ratio of activators to inhibitors of this enzyme under aerobic conditions. The concentrations of ATP, ADP, AMP, Pi, N H | , and fructose 6-phosphate in the jejunum of rat were therefore determined, and the values are shown in Table VIII (258). The concentrations of adenine nucleotides observed in the jejunum of rat are in agreement with those obtained by Parsons (200) but differ from the reported values for mucosa (113), which are indeed low, with an energy charge of 0.46 that may not be compatible with maintenance of life (14)A recent analysis of adenine nucleotides in rat jejunum (136) reported much lower values for all the nucleotides, but the energy charge was about 0.9. This value is in the range observed for many systems (14)The N H | concentration in jejunum (1.65 μΐηοΐββ per gram wet weight of jejunum) is considerably higher than in brain (0.41 μηιοΐββ/^ιη wet weight) (29), resting muscle of rat (0.33 μιηο1β8 per gram wet weight of tissue) (91) or rat liver (0.36 μΐηοΐββ/^ηι wet weight) (95). Also the concentration of Pi in jejunum (9.5 μΐηοΐββ^ηι wet weight) is much higher than in rat liver (3.5 μΐηοΐββ^ηι wet weight) (95), mouse brain (4.3 μΐηοΐββ/^ηι wet weight) (156), or frog sartorius muscle at rest (4.1 μΐηοΐββ per milliliter of intracellular water) (104) (1-3 μΐηοΐβθ per gram wet weight of frog muscle) (119, 232). The activity of the partially purified phosphofructokinase of rat jejunal mucosa was estimated at the concentration of effectors as given in Table VIII at various concentrations of ATP. These results are shown in Fig. 3 (259a). The enzyme under these conditions is uninhibited by 1.69 m l ATP, the level reported for intact jejunum (258). Addition of citrate at a concentration of 0.25 mM observed in intact jejunum (243) also had no effect on the enzyme activity under conditions described in Fig. 3. Increasing the concentration of ATP to 3.0 mM decreases the enzyme activity by only 25%. Thus the lack of a Pasteur effect and high aerobic glycolysis in this tissue may be due to a favorable ratio of activators to inhibitors of phosphofructokinase. The glycolytic rate in this tissue is simply controlled by the concentration of fructose 6-phosphate. This simple control may be well suited to the function of glycolysis since one transport mechanism for glucose and fructose from the intestine into the blood stream has been reported to be via lactic

PASTEUR E F F E C T AND

321

PHOSPHOFRUCTOKINASE

TABLE VIII CONCENTRATIONS OF VARIOUS M E T A B O L I T E S IN THE INTACT J E J U N U M OF R A T 0 - 6

Metabolite ATP ADP AMP Glucose 6-P NH+ Pi

Energy charge 0 Fru 6-P d

Expt. 1

Expt. 2

1.18 0.63 0.18 0.039 1.58 10.35 0.75

1.32 0.66 0.20 0.044 1.73 8.65 0.75

—

—

Average of Expt. 1 and 2 1.25 0.645 0.19 0.0415 1.65 9.50 0.75 0.014

a

From Tejwani {258). All values are expressed as micromoles per gram wet weight. In each experiment jejuna from five animals were pooled. The values given are the average values of two or more analyses done for each metabolite, in each experiment. The duplicates varied by less than 5 %. c Energy charge = (ATP) + Y2 ( A D P ) / ( A T P ) + (ADP) + (AMP). d F r u 6-P was calculated from Glu 6-P on the assumption t h a t Glu 6 - P : F r u 6-P is 3 : 1 . b

acid {124)- This conclusion is further supported by the fact that glycolytic enzymes in this tissue are inducible. The activities of hexokinase, glucokinase, fructokinase, phosphofructokinase, and pyruvate kinase decrease on fasting and increase on feeding glucose or fructose (240, 248, 249). It may seem unreasonable that so many activators and inhibitors have evolved to regulate phosphofructokinase of this tissue only to be canceled out under in vivo conditions. In most of the tissues the function of glycolysis is to provide biosynthetic intermediates and ATP, which is then stringently controlled to suit the immediate demands of the cell, while in the case of mucosal cells its additional functions in transport require control by the substrate levels in the diet. The favorable ratio of activators to inhibitors of phosphofructokinase in the jejunum of rat may be correlated with the decreased oxygen tension in the intestinal fluid surrounding the mucosa, similar to the correlation of the high aerobic glycolysis in the medulla of kidney and cartilage to the relatively poor blood supply to these tissues (63, 64)-

322

ABBURI RAMAIAH 0.1

0.08 c

'ε ^0.06 c O