Supramolecular organization of glycolytic enzymes

On the basis of the analysis of the data on adsorption of glycolytic enzymes to structural proteins of skeletal muscle and to 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 glycolytic enzyme complex adsorbed to a biological support has been proposed. The key role in the formation of the 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 fixed on structural proteins of skeletal muscle contains one tetrameric molecule of 6-phosphofructokinase and at two molecules of other glycolytic enzymes. Hexokinase is not involved in the complex composition. The molecular mass of the multienzyme complex is about 2,6 X 10(6) Da. The formation of the multienzyme complex leads to the compartmentation of the glycolytic process. The problem of integration of physico-chemical mechanisms of enzyme activity regulation (allosteric, dissociative and adsorptive mechanisms) is discussed.     

The association of glycolytic enzymes with cellular and model membranes

This article deals with the binding of glycolytic enzymes with membranous or protein subcellular structures. The representative papers of the last three decades dealing with this matter are reviewed. The studies evidencing the binding of some glycolytic enzymes to insoluble subcellular proteins and membranous structures are presented. It is currently generally accepted that the glycolytic enzymes work in some organisation. Such organisation undoubtedly plays a marked role, although still poorly known, in the regulation processes of glycolysis. From this review, the conclusion emerges that the regulatory ability of the binding of glycolytic enzymes to cellular membranes should be added to the list of well-known mechanisms of post-translational regulation of the glycolytic enzymes. Some of the results presented are the background for the hypothesis that planar phospholipid domains in/on the membrane surface are capable of functioning as binding sites for these enzymes. Such binding can modify the conformation state of the enzymes, which results in changes in their kinetic properties; thus, it may function as a regulator of catalytic activity     

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.     

Where is the glycolytic complex? A critical evaluation of present data from muscle tissue

Associations between glycolytic enzymes and subcellular structures have been interpreted as presenting a novel mechanism of glycolytic control; reversible enzyme binding to subcellular structural components is believed to regulate enzyme activity in vivo through the formation of a multi-enzyme complex. However, three lines of evidence suggest that enzyme binding to cellular structures is not involved in the control of glycolysis. (i) Calculations of the distribution of glycolytic enzymes under the physiological cellular conditions of higher ionic strength and higher enzyme concentrations indicate that a large multi-enzyme complex would not exist. (ii) In many cases, binding to subcellular structures is accompanied by changes in enzyme kinetic parameters brought about by allosteric modification, but these changes often inhibit enzyme activity. (iii) In the case where formation of binary enzyme/enzyme complexes activates enzymes, the overall increase in flux through the enzyme reaction is negligible.     

Interactions of glycolytic enzymes with erythrocyte membranes

Partition equilibrium experiments have been used to characterize the interactions of erythrocyte ghosts with four glycolytic enzymes, namely aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase and lactate dehydrogenase, in 5 mM sodium phosphate buffer (pH 7.4). For each of these tetrameric enzymes a single intrinsic association constant sufficed to describe its interaction with erythrocyte matrix sites, the membrane capacity for the first three enzymes coinciding with the band 3 protein content. For lactate dehydrogenase the erythrocyte membrane capacity was twice as great. The membrane interactions of aldolase and glyceraldehyde-3-phosphate dehydrogenase were mutually inhibitory, as were those involving either of these enzymes and lactate dehydrogenase. Although the binding of phosphofructokinase to erythrocyte membranes was inhibited by aldolase, there was a transient concentration range of aldolase for which its interaction with matrix sites was enhanced by the presence of phosphofructokinase. In the presence of a moderate concentration of bovine serum albumin (15 mg/ml) the binding of aldolase to erythrocyte ghosts was enhanced in accordance with the prediction of thermodynamic nonideality based on excluded volume. At higher concentrations of albumin, however, the measured association constant decreased due to very weak binding of the space-filling protein to either the enzyme or the erythrocyte membrane. The implications of these findings are discussed in relation to the likely subcellular distribution of glycolytic enzymes in the red blood cell.     

Organization of enzymes on subcellular structures: relay at the surface

There is a large body of evidence that soluble cytoplasmic enzymes of eukaryotic cells, e.g., glycolytic enzymes and proteins of the translational machinery, are organized in some way in space and in time. The following features of such organization emerge from the experimental data: (1) metabolites are transferred between enzymes directly "from hand to hand" in short-living enzyme-enzyme complexes rather than by diffusion in aqueous media; (2) enzymes show a tendency to be absorbed on surfaces of subcellular structures, such as membranes, cytoskeleton and polyribosomes; (3) enzymes are desorbed from a surface of a subcellular structure after binding specific metabolites, i.e., substrates and/or products of the reactions catalyzed by these enzymes. These features are suggestive of a relay mechanism for the enzyme systems functioning in a cell; an enzyme adsorbed on a surface of a subcellular structure is desorbed after binding its substrate or in the course of the catalytic act. Within a complex with its product the enzyme diffuses into the environment, until it reaches the next enzyme adsorbed on the same surface; then a short-living enzyme-enzyme complex is formed, and a direct "from hand to hand" transfer of the metabolite takes place. As a result, the overall metabolic process appears to be localized near the surface. We termed this mechanism as a "relay at the surface".     

Hypothetical structure of the glycolytic enzyme complex (glycolytic metabolon) formed on erythrocyte membranes

A hypothetical structure of the glycolytic enzyme complex (glycolytic metabolon) adsorbed on the inner surface of the erythrocyte membrane has been proposed. Oligomers of integral membrane protein, band 3 protein (anion-transport system), are the anchor site for the complex. The complex is supposed to have a three-fold symmetry axis, perpendicular to the membrane plane, and contains a triple set of the glycolytic enzymes. The complex is in equilibrium with free enzymes; the equilibrium state depends on the physiological state of the erythrocyte.     

Filamentous actin and its associated binding proteins are the stimulatory site for 6-phosphofructo-1-kinase association within the membrane of human erythrocytes

Glycolytic enzymes reversibly associate with the human erythrocyte membrane (EM) as part of their regulatory mechanism. The site for this association has been described as the amino terminus of band 3, a transmembrane anion transporter. Binding of glycolytic enzymes to this site is recognized to inhibit glycolysis, since binding inhibits the catalytic activity of these enzymes, including the rate-limiting enzyme 6-phosphofructo-1-kinase (PFK). However, the existence of a putative stimulatory site for glycolytic enzymes within the EM has been proposed. PFK has been described as able to reversibly associate with other proteins, such as microtubules, which inhibit the enzyme, and filamentous actin, which activates the enzyme. Here, it is demonstrated that PFK also binds to actin filaments and its associated binding proteins in the protein meshwork that forms the erythrocyte cytoskeleton. Through fluorescence resonance energy transfer experiments using either confocal microscopy or fluorescence spectroscopy, we show that, within the EM, PFK and actin filaments containing its associated binding proteins are located close enough to propose binding between them. Moreover, specifically blocking PFK binding to band 3 results in an association of the enzyme with the EM that increases the enzyme's catalytic activity. Conversely, disruption of the association between PFK and actin filaments containing its associated binding proteins potentiates the inhibitory action of the EM on the enzyme. Furthermore, it is shown that insulin signaling increases the association of PFK to actin filaments and its associated binding proteins, revealing that this event may play a role on the stimulatory effects of insulin on erythrocyte glycolysis. In summary, the present work presents evidence that filamentous actin and its associated binding proteins are the stimulatory site for PFK within the EM.     

Functional aspects of cellular microcompartmentation in the development of neurodegeneration: Mutation induced aberrant protein-protein associations

Research in the last 10 years has revealed that the development of neurodegeneration is a multistep process during which one or few specific mutant protein species of altered conformation initiate aberrant protein-protein interactions resulting in aggregates forming plaques. This review focuses on the heteroassociations of the mutant proteins with subcellular structures, such as cytoskeleton, cell membranes or with glycolytic enzymes, which may be crucial in the initiation of neurodegeneration such as in Huntington's disease or Alzheimer's disease. Triosephosphate isomerase enzymopathy is a unique glycolytic enzyme deficiency coupled with neurodegeneration. We present data on the mutation induced misfolding process, which likely plays a crucial role in the enhanced associations of the enzyme with the truncated fragment of the isomerase, with the red cell membrane or with the microtubular network. On the basis of our recent clinical and experimental results obtained with two compound heterozygote Hungarian brothers it became obvious that the mutations alone are not sufficient to explain the development of the neurological sympthomes. This underscores the fact that the mutations alone are not enough for the development of the clinical phenotype of a disease.     

On the differential release of glycolytic enzymes from cellular structure

In an endeavour to extend the available information on the biological significance of the interactions between glycolytic enzymes and cellular ultrastructure, the role of release of enzymes from digitonized fibroblasts has been studied. Lactate dehydrogenase and phosphofructokinase were rapidly and quantitatively eluted under the experimental conditions, while glyceraldehyde-3-phosphate dehydrogenase and aldolase were retained to an appreciably greater extent by the cells. This differential release of glycolytic enzymes has been related to the known binding propensities between those enzymes and subcellular structures, and are interpreted as providing additional confirmatory evidence of the importance of aldolase and glyceraldehyde-3-phosphate dehydrogenase, in particular, to these associations. The data also shed light on the order of binding of these glycolytic components - phosphofructokinase being indicated as binding subsequently (and probably separately) to aldolase and glyceraldehyde-3-phosphate dehydrogenase. These results have been discussed in relation to the available data on the associations between glycolytic enzymes and cellular structure, the possible physiological significance of this phenomenon, and the access to these problems provided by the present technique.     

The effect of enzyme-enzyme complexes on the overall glycolytic rate in vivo.

Recent studies have demonstrated that most glycolytic enzymes can reversibly associate to form heterogeneous enzyme-enzyme (binary) complexes in vitro. However, kinetic analysis of these complexes has shown that the individual enzymes have a varied response to complex formation: some enzymes are inhibited, some are activated and some are unaffected. In order to determine the potential role of binary complexes in regulating glycolytic flux, we have mathematically calculated enzyme distributions and activities using data from in vitro binding and kinetic studies. These calculations suggest that, overall, formation of binary complexes would lower flux through phosphofructokinase and aldolase, would increase flux through glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase, and would not affect flux through triosephosphate isomerase, phosphoglycerate kinase and pyruvate kinase. The implications of these results are discussed with respect to the effect of complex formation on overall glycolytic flux and on the flux through individual enzyme loci.     

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.     

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.     

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.     

Outer-membrane phospholipase A: known structure, unknown biological function

Outer-membrane phospholipase A (OMPLA) is one of the few enzymes present in the outer membrane of Gram-negative bacteria. The enzymatic activity of OMPLA is strictly regulated to prevent uncontrolled breakdown of the surrounding phospholipids. The activity of OMPLA can be induced by membrane perturbation and concurs with dimerization of the enzyme. The recently elucidated crystal structures of the inactive, monomeric and an inhibited dimeric form of the enzyme provide detailed structural insight into the functional properties of the enzyme. OMPLA is a serine hydrolase with a unique Asn-156-His-142-Ser-144 catalytic triad. Only in the dimeric state, complete substrate binding pockets and functional oxyanion holes are formed. A model is proposed for the activation of OMPLA in which membrane perturbation causes the formation of non-bilayer structures, resulting in the presentation of phospholipids to the active site of OMPLA and leading to the formation of the active dimeric species. Possible roles for OMPLA in maintaining the cell envelope integrity and in pathogenicity are discussed.     

Proteins of diverse function and subcellular location are lysine acetylated in Arabidopsis

Acetylation of the ε-amino group of lysine (Lys) is a reversible posttranslational modification recently discovered to be widespread, occurring on proteins outside the nucleus, in most subcellular locations in mammalian cells. Almost nothing is known about this modification in plants beyond the well-studied acetylation of histone proteins in the nucleus. Here, we report that Lys acetylation in plants also occurs on organellar and cytosolic proteins. We identified 91 Lys-acetylated sites on 74 proteins of diverse functional classes. Furthermore, our study suggests that Lys acetylation may be an important posttranslational modification in the chloroplast, since four Calvin cycle enzymes were acetylated. The plastid-encoded large subunit of Rubisco stands out because of the large number of acetylated sites occurring at important Lys residues that are involved in Rubisco tertiary structure formation and catalytic function. Using the human recombinant deacetylase sirtuin 3, it was demonstrated that Lys deacetylation significantly affects Rubisco activity as well as the activities of other central metabolic enzymes, such as the Calvin cycle enzyme phosphoglycerate kinase, the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase, and the tricarboxylic acid cycle enzyme malate dehydrogenase. Our results demonstrate that Lys acetylation also occurs on proteins outside the nucleus in Arabidopsis (Arabidopsis thaliana) and that Lys acetylation could be important in the regulation of key metabolic enzymes.     

Comparative study of free and bound glycolytic enzymes from sea scorpion brain.

Free and bound forms of hexokinase, pyruvate kinase, and lactate dehydrogenase were prepared from the brain of the sea scorpion (Scorpaena porcus) in a low ionic strength medium. Properties of the free and bound forms were compared to determine whether binding to particulate matter could influence enzyme function or stability in vivo. Changes in pH differently affected the activity of the free and bound forms of all three enzymes. Furthermore, bound forms of hexokinase and pyruvate kinase were more stable than the free enzymes to heating at 45 degrees C. Bound hexokinase showed higher affinity for substrates (ATP, glucose) than the free form and bound lactate dehydrogenase had greater affinity for pyruvate and NADH. Although the affinities of the two forms of pyruvate kinase for substrates were similar, Hill coefficients for phosphoenolpyruvate as well as inhibition by ATP differed between the two enzyme forms. Free and bound lactate dehydrogenase also showed differences in Hill coefficients and bound lactate dehydrogenase was less sensitive to substrate inhibition by high pyruvate concentrations. The possible physiological role of the binding of these glycolytic enzymes to subcellular structures is discussed.     

Evidence for glycolytic enzyme binding during anaerobiosis of the foot muscle ofPatella caerulea (L.)

Summary The effect of anaerobiosis and aerobic recovery on the degree of binding of glycolytic enzymes to the particulate fraction of the cell was studied in the foot muscle of the marine molluscP. caerulea, in order to assess the role of glycolytic enzyme binding in the metabolic transition between aerobic and anoxic states. Short periods of anoxia (2 h, 4 h) resulted in an increase in enzyme binding in association with the increased glycolytic rate observed; this was particularly pronounced for phosphorylase, phosphofructokinase, aldolase, pyruvate kinase and lactate dehydrogenase. Decreased enzyme binding was observed after prolonged periods of anoxia. These effects were reversed and control values re-established when animals were returned to aerobic conditions. The results suggest that glycolytic rate could be regulated by changes in the distribution of glycolytic enzymes between free and bound forms inP. caerulea foot muscle. This reversible interaction of glycolytic enzymes with structural proteins may constitute an additional mechanism for metabolic control.     

Chemical modification of the actin binding site of rabbit muscle aldolase by diethylpyrocarbonate

Summary To extend the available information on the significance of the interactions between glycolytic enzymes and the actin component of the cellular ultrastructure, investigations into the compositional characteristics of the actin binding site on one of the major glycolytic enzymes, aldolase, have been undertaken. As the electrostatic nature of the association has been previously reported indicative of a cationic region on the enzyme involved in the binding, these studies have investigated the possibility of the involvement of histidine residues in this binding region. By the use of the histidine specific reagent, diethylpyrocarbonate, we have been able to establish a difference in nature of an actin binding domain and the active site domain which does contain an essential histidine. The results have been discussed in relation to the significance of this finding with respect to the binding of aldolase to subcellular structure.     

Further evidence for the importance of lipid bilayers in the interaction between lactate dehydrogenase and phosphatidylserine

Lactate dehydrogenase (LDH) is one of the glycolytic enzymes, which have been proved to have the capability to reverse non-specific adsorption on cellular membranous structures in vitro, as well as on the structural proteins of the contractile system of muscle cells. It has been suggested that this binding may play a physiological role, as it alters the enzyme's kinetic properties. Our previous studies on this enzyme showed that its interaction with some anionic phospholipids reveals similar characteristics and similar effect on the activity of the enzyme to those which had been observed for the interaction with membranous structures. Disruption of the lipid bilayers by nonionic detergent (Tween 20) restored the enzyme activity inhibited by the presence of phosphatidylserine (PS) liposomes. In this study, we used the measurement of enzyme tryptophanyl fluorescence spectra to monitor the interaction and possible changes in the enzyme conformation. The investigation provided further evidence of the importance of the bilayer structure in this interaction. Similarly to the effect on the activity of the enzyme, the addition of Tween 20 diminishes the quenching of the LDH tryptophanyl fluorescence, and finally completely restores the fluorescence.