Supramolecular organization of glycolytic enzymes

On the basis of the analysis of the data on adsorption of glycolytic enzymes to structural proteins of skeletal muscles and to the erythrocyte membranes, the data on enzyme-enzyme interactions and the data on the regulation of activity of glycolytic enzymes by cellular metabolites, the structure of the glycolytic enzymes complex adsorbed to a biological support has been proposed. The key role in the formation of multienzyme complex belongs to 6-phosphofructokinase. The enzyme molecule has two association sites, one of which provides the fixation of 6-phosphofructokinase on the support and another is saturated by fructose-1,6-bisphosphate aldolase. The multienzyme complex contains one tetrameric molecule of 6-phosphofructokinase and two molecules of each of other glycolytic enzymes. Hexokinase is not a part of the complex. The molecular mass of the multienzyme complex is about 2.6 X 10(6) daltons. The multienzyme complex has symmetry axis of second order. The formation of the multienzyme complex leads to the compartmentation of glycolytic process. The problem of integration of physico-chemical mechanisms of enzyme activity regulation (allosteric, dissociative and adsorptive mechanisms) is discussed.     

Adsorption of peripheral enzymes to membrane anchor proteins

The character of the isotherms of specific adsorption of peripheral enzymes to dimeric anchor proteins embedded in the membrane has been analysed. The situations are discussed when adsorption corresponds to the stoichiometry of one or two molecules of peripheral enzyme per dimeric binding site. The corresponding expressions describing the competitive interrelationships between peripheral enzymes adsorbed to the same binding sites have been derived. The experimental data on the adsorption of glycolytic enzymes to erythrocyte membranes are used for the illustration of the theoretical predictions. The physiological role of enzyme self-association which leads to the formation of enzyme oligomers of unlimited length is discussed. It is assumed that under in vivo conditions the association sites of such enzymes are saturated through interactions with anchor proteins of subcellular structures and with the enzymes of the corresponding metabolic pathways. Therefore the linearly associating enzymes play the key role in the formation of multienzyme complexes attached to subcellular structures. The significance of 6-phosphofructokinase adsorption to erythrocyte membranes in the formation of the complex of glycolytic enzymes is discussed.     

Principles of integration of cell metabolism

The notion of the "primary blocks" of cellular metabolism (designated as "metabolic system") has been introduced. Metabolic system is defined as a metabolic pathway which corresponds to the really existing multienzyme complex. The complex of glycolytic enzymes which catalyzes the anaerobic reduction of glucose-6-phosphate with production of ATP may serve as an example of metabolic system (this complex does not contain hexokinase). The complex is formed on thin filaments of I-band of the muscle fibers or on dimers of band 3 protein embedded in the erythrocyte membranes. The fixation of the multienzyme complex to the support of biological nature provides the material basis for regulation of the metabolic system by chemical signals produced by the higher levels of metabolic control. Owing to interaction with anchor protein of the support the chemical signals exert the general control of functioning the multienzyme complex (switching on--switching-off of the metabolic system). It is assumed that the glycolytic system in skeletal muscles is stimulated by Ca2+ ions which interact with the anchor protein of the support (troponin C).     

The role of multienzyme complexes in integration of cellular metabolism

The notion of the "primary block" of cellular metabolism designated as "metabolic system" is introduced. Metabolic system is defined as a metabolic pathway which corresponds to the structurally ordered multienzyme complex. The complex of glycolytic enzymes which catalyzes the anaerobic reduction of glucose-6-phosphate with production of ATP may serve as an example of metabolic system (this complex does not contain hexokinase). The complex is formed on thin filaments of I-band of the muscle fibres or on the dimers of band 3 protein embedded in the erythrocyte membrane. The fixation of the multienzyme complex to the support of the biological nature provides the material basis for regulation of the metabolic system by chemical signals produced by the higher levels of metabolic control. Owing to interaction with anchor protein of the support the chemical signals exert the general control of functioning of the multienzyme complex (switching on-switching off the metabolic system). It is assumed that glycolytic system in skeletal muscles is stimulated by Ca2+ ions which interact with the anchor protein of the support (troponin C).     

The role of multi-enzyme complexes in the integration of cell metabolism

General properties of enzymes and structurally ordered multienzyme complexes as controllable systems are discussed: the spatial isolation of working sites and sites of control and the realization of control mechanisms with the participation of "external" factors which provide the optimal functioning of the controllable system in the biological system of higher level of complexity. The basic mechanisms of the control of soluble enzymes are isosteric and allosteric mechanisms which directed to the maintenance of cellular homeostasis. The mechanism of functioning of a multienzyme complex as a whole which is realized with the participation of second messengers is classified as a mechanism for tracing of the signals from higher levels of the control of metabolism (from nervous, hormonal and immune systems). When discussing the control of functioning of the multienzyme complexes, special attention was paid to the complex of glycolytic enzymes formed on the structural proteins of skeletal muscles and on the membranes. An order of assembly of the complex of glycolytic enzymes is proposed. The possible localization of this complex in myofibrils is discussed.     

Equilibrium partition studies of the myofibrillar interactions of glycolytic enzymes

The interactions of several glycolytic enzymes with muscle myofibrils in imidazole-chloride buffer (pH 6.8, I 0.158) have been investigated by equilibrium partition studies. Results for aldolase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and phosphofructokinase are interpreted in terms of a myofibrillar capacity of 76 nmol/g protein and a single intrinsic association constant for each tetravalent enzyme with matrix sites. The existence of separate myofibrillar sites for aldolase and glyceraldehyde-3-phosphate dehydrogenase is established by demonstrating independence of the binding of each enzyme upon the presence of the other. Although this investigation provides further physicochemical support for myofibrillar adsorption of glycolytic enzymes in the cellular environment, its findings are incompatible with the proposition (B. I. Kurganov, N. P. Sugrobova, and L. S. Mil'man (1985) J. Theor. Biol. 116, 509-526) that the phenomenon reflects the formation of a specific multienzyme complex attached to the myofibril.     

Regulation of skeletal muscle metabolism by enzyme binding.

The random diffusion mechanism is usually assumed in analyzing the energetics of specific pathways despite the findings that enzymes associate with each other and (or) with various membranous and contractile elements of the cell. Successive glycolytic enzymes have been shown to associate in the cytosol as enzyme complexes or bind to the thin filaments. Furthermore, the degree of glycolytic enzyme interactions have been shown to change with altered rates of carbon flux through the pathway. In particular, the proportions of aldolase, phosphofructokinase, and glyceraldehyde phosphate dehydrogenase bound to the contractile proteins have been found to increase with increased rates of glycolysis. In addition, decreasing pH and ionic strength are also associated with an increase in glycolytic enzyme interactions. The kinetics displayed by interacting enzymes generally serve to enhance their catalytic efficiencies. The associations of the glycolytic enzymes serve to enhance metabolite transfer rates, increase the local concentrations of intermediates, and provide for regulation of activity via effectors. Therefore these interactions provide an additional mechanism for regulating glycolytic flux in skeletal muscle.     

A reappraisal of the binding of cytosolic enzymes to erythrocyte membranes

Several cytosolic proteins have been shown to be associated with hypotonic erythrocyte ghosts via electrostatic interactions with the anion transport band 3 protein. This article considers the problems of demonstrating binding under physiological conditions and reviews the evidence for the relevance of enzyme binding to the membrane for the regulation of glycolysis. The hypotheses for the existence of topological and sequential multienzyme complexes of the glycolytic enzymes in erythrocytes are also discussed.     

Clustering of sequential enzymes in the glycolytic pathway and the citric acid cycle

In recent years, evidence has been accumulating that metabolic pathways are organized in vivo as multienzyme clusters. Affinity electrophoresis proves to be an attractive in vitro method to further evidence specific associations between purified consecutive enzymes from the glycolytic pathway on the one hand, and from the citric acid cycle on the other hand. Our results support the hypothesis of cluster formation between the glycolytic enzymes aldolase, glyceraldehydephosphate dehydrogenase, and triosephosphate isomerase, and between the cycle enzymes fumarase, malate dehydrogenase, and citrate synthase. A model is presented to explain the possibility of regulation of the citric acid cycle by varying enzyme-enzyme associations between the latter three enzymes, in response to changing local intramitochondrial ATP/ADP ratios.     

The reversible binding of glycolytic enzymes in ovine skeletal muscle in response to tetanic stimulation

The extent of binding of glycolytic enzymes to the particulate fraction of homogenates was measured in sheep hind muscles after electrical stimulation. As compared to the control muscles, stimulation led to significant increases in the amount of phosphofructokinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase bound to the particulate fraction. The bindng of other glycolytic enzymes was not significantly altered. A servey of different hind limb muscles at variable rates of stimulation revealed that each muscle exhibited its own characteristic response pattern in terms of the level of increased enzyme binding. Generally, an increased stimulation rate led to greater enzyme adsorption. The increase in enzyme binding was rapidly reversible for it was shown that the amount of enzyme bound quickly returned to control values when the muscles were allowed to recover in the live anaesthetised animal following cessation of stimulation. Those muscles which exhibited increased enzyme binding were characterised by a marked loss of glycogen and accumulation of lactate suggesting that accelerated glycolytic flux was a necessary condition for the observation of increased enzyme binding. In support of this, enzyme adsorption was observed to be greatest on stimulation of ischemic muscles, whereas in trained muscles, or muscles with depleted glycogen stores induced by prior adrenalin treatment, the increased enzyme binding response was greatly diminished. It is concluded that the variable binding of key glycolytic enzymes has a role to play in the regulation of glycolytic behaviour in skeletal muscle.     

Studies of protein-protein interaction using countercurrent distribution in aqueous two-phase systems: partition behavior of five glycolytic enzymes from crude baker's yeast extract

The partition behavior of five glycolytic enzymes, in extracts from baker's yeast (Saccharomyces cerevisiae), between two aqueous phases has been studied by countercurrent distribution. All enzymes showed distribution patterns which indicated homogeneity and a similar partition behavior. In purified form, three of the enzymes (glyceraldehyde-phosphate dehydrogenase, 3-phosphoglycerate kinase, and enolase) showed the same partition behavior as in the extracts. Pure 6-phosphofructokinase, on the other hand, changed its partition distinctively relative to what was found in the extracts. These results indicate interactions between this enzyme and macromolecular compounds in the extracts and support a model suggested by Kurganov et al. (1985, J. Theor. Biol. 116, 509-526) describing a "glycolytic particle."     

Regulation of concentrations of glycolytic enzymes and creatine-phosphate kinase in “fast-twitch” and “slow-twitch” skeletal muscles of the chicken

The distinctive contractile and metabolic characteristics of different skeletal muscle fiber types are associated with different protein populations in these cells. In the present work, we investigate the regulation of concentrations of three glycolytic enzymes (aldolase, enolase, glyceraldehyde-3-phosphate dehydrogenase) and creatine-phosphate kinase in “fast-twitch” (breast) and “slow-twitch” (lateral adductor) muscles of the chicken. Results of short-term amino acid incorporation experiments conducted both in vivo and with muscle explants in vitro showed that these enzymes turnover at different rates and that aldolase turns over 2 to 3 times faster than the other three enzymes. However, these differences in turnover rates were difficult to detect in long-term double-isotope incorporation experiments, presumably because extensive reutilization of labeled amino acids occurred during these long-term experiments. Mature muscle fibers synthesize these four cytosolic enzymes at very high rates. For example, 11 to 14% of the total labeled leucine incorporated into protein by breast muscle fibers was found in the enzyme aldolase. Results of short-term amino acid incorporation experiments also showed that the relative rates of synthesis of the three glycolytic enzymes were about fourfold higher in mature “fast-twitch” muscle fibers than in mature “slow-twitch” ones while the relative rates of synthesis of creatine-phosphate kinase were similar in the two fiber types. The relative rates of synthesis of these four enzymes and cytosolic proteins in general were found to be very similar in immature muscles of both types. More profound changes in the relative rates of synthesis of major cytosolic proteins, including the glycolytic enzymes, occurred during postembryonic maturation of fast-twitch fibers than occurred during maturation of slow-twitch fibers. Our work demonstrates that (1) the synthesis of creatine-phosphate is independently regulated with respect to the synthesis of the glycolytic enzymes in muscle fibers; and (2) the approximate fourfold higher steady-state concentrations of glycolytic enzymes in fast-twitch muscle fibers as compared with slow-twitch fibers are determined predominantly by regulatory mechanisms operating at the level of protein synthesis rather than protein degradation. Our demonstration that more profound changes in the relative rates of synthesis of major cytosolic proteins occur during maturation of fast-twitch fibers as compared with slow-twitch fibers is discussed in terms of the mode(s) of fiber-type differentiation proposed by others.     

Small-scale hypoxic cultures for monitoring the spatial reorganization of glycolytic enzymes in Saccharomyces cerevisiae

At normal oxygen concentration, glycolytic enzymes are scattered in the cytoplasm of Saccharomyces cerevisiae. Under hypoxia, however, most of these enzymes, including enolase, pyruvate kinase, and phosphoglycerate mutase, spatially reorganize to form cytoplasmic foci. We tested various small-scale hypoxic culture systems and showed that enolase foci formation occurs in all the systems tested, including in liquid and on solid media. Notably, a small-scale hypoxic culture in a bench-top multi-gas incubator enabled the regulation of oxygen concentration in the media and faster foci formation. Here, we demonstrate that the foci formation of enolase starts within few hours after changing the oxygen concentration to 1% in a small-scale cultivation system. The order of foci formation by each enzyme is tightly regulated, and of the three enzymes, enolase was the fastest to respond to hypoxia. We further tested the use of the small-scale cultivation method to screen reagents that can control the spatial reorganization of enzymes under hypoxia. An AMPK inhibitor, dorsomorphin, was found to delay formation of the foci in all three glycolytic enzymes tested. These methods and results provide efficient ways to investigate the spatial reorganization of proteins under hypoxia to form a multienzyme assembly, the META body, thereby contributing to understanding and utilizing natural systems to control cellular metabolism via the spatial reorganization of enzymes.     

Glycolytic and Related Enzymes in Clostridial Classification

The activities of 15 glycolytic and related enzymes were determined in clostridia. All contained 1-phosphofructokinase; three of them lacked 6-phosphofructokinase and mannitol 1-phosphate dehydrogenase. Glucose 6-phosphate dehydrogenase was found in six clostridia, thus demonstrating the presence of hexose monophosphate shunt. Only parts of the citric acid cycle were found to be present in most clostridia with an indication of the full cycle in Clostridium septicum. The intermediary enzyme activities were used to differentiate between the different clostridia.     

The tentative identification in Escherichia coli of a multienzyme complex with glycolytic activity.

Penicillin spheroplasts of Escherichia coli were ruptured osmotically, by freezing and thawing, or mechanically. Differential centrifugation sedimented 20-30% of the glycolytic enzymes without increasing their specific activities. There was, however, evidence of distinct groups of sedimenting enzymes; growth on different carbon sources could influence the distribution. Sucrose gradient studies gave no evidence of enzyme association but provided estimations of the molecular weight of each enzyme which were close to those subsequently observed on gel filtration. Using the determined molecular weight and a literature value for specific activity, the measured activity ratio of the enzymes was compared with that expected from an equimolar mixture. All values agreed within a factor of five, except for hexokinase. The relative roles of hexokinase and phosphotransferase in E. coli are briefly considered. An equimolar multienzyme aggregate of all the enzymes of glycolysis would have a molecular weight of about 1.6 X 10(6). Chromatography on a Biogel column yielded one fraction, corresponding to a molecular weight of 1.6 X 10(6), which contained a proportion of all the glycolytic enzyme studied; the remaining portion of each enzyme activity was eluted from the column at the position expected from its individual molecular weight. The fraction of mol. wt 1 600 000 was tested for complete glycolysis pathway activity and found not to be different from a reconcentrated mixture of the separated enzymes. Both the eluted and the reconstructed systems showed unexpected activity changes at different protein concentrations. The specific radioactivity of pyruvate formed by these systems from [14C]glucose 6-phosphate was reduced by the presence of unlabelled 3-phosphoglycerate, but by less than would have been expected had the latter been able to participate fully in glycolytic activity. This result indicates that these preparations were capable of selectivity compartmenting glycolytic intermediates. Electron microscope investigation of both systems showed large numbers of regular 30 nm diameter particles which, on disruption, appeared to be composed of smaller units: it is possible that these particles may have been aggregates containing glycolytic enzymes. The possible advantages of a glycolytic multienzyme complex are briefly discussed.     

Mouse skeletal muscle fiber-type-specific macroautophagy and muscle wasting are regulated by a Fyn/STAT3/Vps34 signaling pathway

Skeletal muscle atrophy induced by aging (sarcopenia), inactivity, and prolonged fasting states (starvation) is predominantly restricted to glycolytic type II muscle fibers and typical spares oxidative type I fibers. However, the mechanisms accounting for muscle fiber-type specificity of atrophy have remained enigmatic. In the current study, although the Fyn tyrosine kinase activated the mTORC1 signaling complex, it also induced marked atrophy of glycolytic fibers with relatively less effect on oxidative muscle fibers. This was due to inhibition of macroautophagy via an mTORC1-independent but STAT3-dependent reduction in Vps34 protein levels and decreased Vps34/p150/Beclin1/Atg14 complex 1. Physiologically, in the fed state endogenous Fyn kinase activity was increased in glycolytic but not oxidative skeletal muscle. In parallel, Y705-STAT3 phosphorylation increased with decreased Vps34 protein levels. Moreover, fed/starved regulation of Y705-STAT3 phosphorylation and Vps34 protein levels was prevented in skeletal muscle of Fyn null mice. These data demonstrate a Fyn/STAT3/Vps34 pathway that is responsible for fiber-type-specific regulation of macroautophagy and skeletal muscle atrophy.     

A broad view of scaffolding suggests that scaffolding proteins can actively control regulation and signaling of multienzyme complexes through allostery

Enzymes often work sequentially in pathways; and consecutive reaction steps are typically carried out by molecules associated in the same multienzyme complex. Localization confines the enzymes; anchors them; increases the effective concentration of substrates and products; and shortens pathway timescales; however, it does not explain enzyme coordination or pathway branching. Here, we distinguish between metabolic and signaling multienzyme complexes. We argue for a central role of scaffolding proteins in regulating multienzyme complexes signaling and suggest that metabolic multienzyme complexes are less dependent on scaffolding because they undergo conformational control through direct subunit–subunit contacts. In particular, we propose that scaffolding proteins have an essential function in controlling branching in signaling pathways. This new broadened definition of scaffolding proteins goes beyond cases such as the classic yeast mitogen-activated protein kinase Ste5 and encompasses proteins such as E3 ligases which lack active sites and work via allostery. With this definition, we classify the mechanisms of multienzyme complexes based on whether the substrates are transferred through the involvement of scaffolding proteins, and outline the functional merits to metabolic or signaling pathways. Overall, while co-localization topography helps multistep pathways non-specifically, allosteric regulation requires precise multienzyme organization and interactions and works via population shift, either through direct enzyme subunit–subunit interactions or through active involvement of scaffolding proteins. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.     

The effect of endurance-training on the maximum activities of hexokinase, 6-phosphofructokinase,citrate synthase,and oxoglutarate dehydrogenase in red and white muscles of the rat

Adult female rats were subjected to an eleven-week endurance-training programme, and, for the first time, the maximum activities of enzymes that can indicate the quantitative capacities of both anaerobic glycolysis and the Krebs cycle in muscle (viz. 6-phosphofructokinase and oxoglutarate dehydrogenase respectively) were measured in heart plus white and fast-oxidative skeletal muscle. No changes were observed in heart muscle. In fast-oxidative skeletal muscle, activities of hexokinase, citrate synthase, and oxoglutarate dehydrogenase were increased by 51, 26, and 33% respectively but there was no effect on 6-phosphofructokinase. These results demonstrate that in red muscle there is no effect of this training programme on the anaerobic capacity but that of the aerobic system is increased by one third. In white skeletal muscle, only the activity of citrate synthase was increased, which indicates that this activity may not provide even qualitative information about changes in capacity of the Krebs cycle.     

Direct visualization of phosphorylase-phosphorylase kinase complexes by scanning tunneling and atomic force microscopy.

In skeletal muscle the activation of phosphorylase b is catalyzed by phosphorylase kinase. Both enzymes occur in vivo as part of a multienzyme complex. The two enzymes have been imaged by atomic force microscopy and the results compared to those previously found by scanning tunneling microscopy. Scanning tunneling microscopy and atomic force microscopy have been used to view complexes between the activating enzyme phosphorylase kinase and its substrate phosphorylase b. Changes in the size and shape of phosphorylase kinase were observed when it bound phosphorylase b.     

Supramolecular organization of enzymes of the tricarboxylic acid cycle

In virtue of analysis of data on the interaction of tricarboxylic acid cycle enzymes with the mitochondrial inner membrane and data on the enzyme-enzyme interactions, the spatial structure for the tricarboxylic acid cycle enzyme complex (tricarboxylic acid cycle metabolon) is proposed. The alpha-ketoglutarate dehydrogenase complex, adsorbed on the mitochondrial inner membrane along one of its 3-fold symmetry axes, plays the key role in the formation of metabolon. Two association sites of the alpha-ketoglutarate dehydrogenase complex located on opposite sides of the complex participate in the interaction with the membrane. The tricarboxylic acid cycle enzyme complex contains one molecule of the alpha-ketoglutarate dehydrogenase complex and six molecules of each of the other enzymes of the tricarboxylic acid cycle, as well as aspartate aminotransferase and nucleosidediphosphate kinase. Succinate dehydrogenase, the integral protein of the mitochondrial inner membrane, is a component of the anchor site responsible for the assembly of metabolon on the membrane. The molecular mass of the complex (ignoring succinate dehydrogenase) is of 8.10(6) daltons. The metabolon symmetry corresponds to the D3 point symmetry group. It is supposed, that the tricarboxylic acid cycle enzyme complex interacts with other multienzyme complexes of the matrix and the electron transfer chain.