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.     

The binding of glycolytic enzymes to the cytoskeleton--influence of pH

In a continuing study of the interactions between glycolytic enzymes and cytoskeletal structure, the influence of a variation of the pH of the eluting medium has been investigated. This treatment resulted in an increased degree of binding of most of the glycolytic enzymes with a decrease in pH, with the most marked increases in binding occurring with phosphofructokinase, glyceraldehydephosphate dehydrogenase, enolase and pyruvate kinase. The significance of this data has been discussed with reference to the relative affinities of interaction of the individual glycolytic components and the physiological correlations of these phenomena.     

Association of C-terminal region of phosphoglycerate mutase with glycolytic complex regulates energy production in cancer cells

Cancer cells prefer anaerobic ATP synthesis, regardless of the availability of oxygen. It has been hypothesized that in these cells, glycolytic enzymes associate into a large complex, which results in an increased efficiency of glycolytic flux. However, there is no convincing in vivo evidence supporting this hypothesis. Here, we show that all the enzymes of triose phosphate metabolism, from aldolase to pyruvate kinase consecutively, form a macromolecular complex in vivo and that disruption of such complex significantly inhibits lactate release and ATP synthesis in the glycolytic pathway. Composition of the complex and the effectiveness of the glycolytic flux depends on lactate and glucose concentration. High concentrations of exogenous lactate reduces association of the C-terminal region phosphoglycerate mutase (PGAM) with the complex which results in its disruption and inhibition of ATP synthesis. Additionally, high lactate affects nuclear localization of PGAM and ceases cell proliferation. Our findings might provide new prospects for cancer treatment using low-molecular weight competitors to destabilize the glycolytic complex and reduce proliferative potential of cancer cells.     

Interactions between glycolytic enzymes of Mycoplasma pneumoniae

With only 688 protein-coding genes, Mycoplasma pneumoniae is one of the smallest self-replicating organisms. These bacteria use glycolysis as the major pathway for ATP production by substrate-level phosphorylation, suggesting that this pathway must be optimized to high efficiency. In this study, we have investigated the interactions between glycolytic enzymes using the bacterial adenylate cyclase-based two-hybrid system. We demonstrate that most of the glycolytic enzymes perform self-interactions, suggesting that they form dimers or other oligomeric forms. In addition, enolase was identified as the central glycolytic enzyme of M. pneumoniae due to its ability to directly interact with all other glycolytic enzymes. Our results support the idea of the formation of a glycolytic complex in M. pneumoniae and we suggest that the formation of this complex might ensure higher fluxes through the glycolytic pathway than would be possible with isolated non-interacting enzymes.     

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.     

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.     

Long-term control on renal carbohydrate metabolism--II. Effect of starve-feed cycles on renal tubule glycolysis

1. The effects of different and alternative starve-feed cycles on glycolysis from isolated renal tubules as well as the glycolytic enzymes phosphofructokinase and pyruvate kinase have been studied. Adaptive responses of renal glycolysis under the nutritional conditions mentioned are reported. 2. Renal glucose utilization increased in a linear fashion during the feeding state of the nutritional cycles, becoming twice as much in both feeding and fasting cycles. Conversely, a decrease in this metabolic pathway took place during the starve periods of the cycles. During the feed-starve cycle the decrease reached 70% in 48 hr of fasting after being fed with a high carbohydrate diet. Whereas in the opposite cycle it was almost 35%. 3. The activities of renal glycolytic enzymes, phosphofructokinase and pyruvate kinase are parallel to the glycolytic capacity of renal tubules in different nutritional conditions. These changes only occur at cellular substrate concentration. 4. The behaviour of the kinetic parameters of these enzymes throughout these experimental conditions is reported. In general, variations in Km values without changes in Vmax values take place which reflect an increase in the catalytic efficiency of the glycolytic enzymes during the feeding state and conversely a decrease during the starvation state.     

On the existence of a complex of glycolytic enzymes

It has been suggested in the literature that the glycolytic enzymes are organized into a multi-enzymic complex. We have evaluated this hypothesis for the phosphotriose-glycerate phosphate group of glycolytic enzymes of muscle using sucrose density gradient centrifugation, gel filtration, and ultrafiltration. Attempts were made to avoid dilution and changes in pH. The ratio of activities of the phosphotriose-glycerate phosphate group of enzymes was similar to that found in several other tissues that has led to their designation as a constant proportion group of enzymes. However, no evidence was obtained that they exist as a multi-enzymic complex in chicken breast muscle. As the pH of the press juice is raised to 7.0 and the temperature to 25°C, association occurs between some components in the muscle press juice as evidenced by a blocking of the pores of an ultrafiltration membrane. This association, however, does not involve the enzymes of the phosphotriose-glycerate phosphate group.     

A histochemical study about the influence of lytic enzymes on plasma membrane enzyme activities in rat liver and kidney.

1. The effect of lipolytic, glycolytic and proteolytic enzymes on the activities of plasma membrane enzyme activities in rat liver and kidney has been investigated by a pretreatment of tissue sections with the lytic enzymes. 2. The action of the proteolytic enzymes causes a very strong decrease of leucyl-beta-naphthylamidase activity, whereas the activities of ATP-ase, 5'-nucleotidase and alkaline phosphatase show a lesser decrease. This indicates a different membrane anchorage of leucyl-beta-naphthylamidase as compared to that of the phosphatases. 3. Treatment with glycolytic enzymes results in a decrease of 5'-nucleotidase and ATP-ase activity, whereas liver alkaline phosphatase and leucyl-beta-naphthylamidase show an increase in activity. 4. Treatment with phospholipase C gives about the same results. The very strong decrease of 5'-nucleotidase activity indicates a great dependence on phospholipids.     

Dissection of the network of interactions that links RNA processing with glycolysis in the Bacillus subtilis degradosome

The RNA degradosome is a multiprotein macromolecular complex that is involved in the degradation of messenger RNA in bacteria. The composition of this complex has been found to display a high degree of evolutionary divergence, which may reflect the adaptation of species to different environments. Recently, a degradosome-like complex identified in Bacillus subtilis was found to be distinct from those found in proteobacteria, the degradosomes of which are assembled around the unstructured C-terminus of ribonuclease E, a protein not present in B. subtilis. In this report, we have investigated in vitro the binary interactions between degradosome components and have characterized interactions between glycolytic enzymes, RNA-degrading enzymes, and those that appear to link these two cellular processes. The crystal structures of the glycolytic enzymes phosphofructokinase and enolase are presented and discussed in relation to their roles in the mediation of complex protein assemblies. Taken together, these data provide valuable insights into the structure and dynamics of the RNA degradosome, a fascinating and complex macromolecular assembly that links RNA degradation with central carbon metabolism.     

Interactions between glycolytic enzymes and cytoskeletal structure--the influence of ionic strength and molecular crowding

In a study of the interactions between glycolytic enzymes and cytoskeletal structure, the effect of increasing the degree of molecular crowding by the addition of physiological concentrations of saline and protein was studied. Increasing the ionic strength to physiological levels resulted in only a slight decrease in the retention of most of enzymes, whereas the establishment of physiological concentrations of both saline and protein, caused a markedly increased degree of binding of all the glycolytic enzymes. The implications of this data have been discussed in relation to the relative affinities of interaction of the individual components, the influence of molecular crowding and the physiological significance of this phenomenon.     

Ouabain-sensitive interaction between human red cell membrane and glycolytic enzyme complex in cytosol

Binding of 2,3-diphosphoglycerate to monophosphoglycerate mutase, of which it is an obligatory cofactor, causes changes in the resonance positions of the 31P nuclear magnetic resonance spectra of both phosphate groups. It has previously been shown that these resonances shift when other glycolytic enzymes, such as phosphoglycerate kinase, are added to form the 2,3-diphosphoglycerate . monophosphoglycerate mutase . phosphoglycerate kinase complex. In view of this association, we have examined the set of glycolytic enzymes from aldolase to pyruvate kinase and found evidence of direct communication between all of these enzymes. A multi-enzyme complex of 1--2 . 10(6) daltons has been separated from broken cell ghosts by Biogel column filtration and evidence has been presented to show that this complex exhibits aldolase, glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase activity. The glycolytic multi-enzyme complex interacts with the outer face of inside-out vesicles prepared from human red cells and the interaction is suppressed by application of 10(-6) M ouabain to the inner face of these vesicles. These studies show that the conformation of the enzymes comprising the megadalton complex are responsive to the application of ouabain to the outer red cell membrane surface.     

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.     

Enolase takes part in a macromolecular complex associated to mitochondria in yeast

In eucaryotes, glycolytic enzymes are classically regarded as being localised in the cytosol. Recently, we have shown that part of the cellular pool of the glycolytic enzyme, enolase, is tightly associated with the mitochondrial surface in the yeast Saccharomyces cerevisiae (N. Entelis, I. Brandina, P. Kamenski, I.A. Krasheninnikov, R.P. Martin and I. Tarassov, A glycolytic enzyme, enolase, is recruited as a cofactor of tRNA targeting toward mitochondria in Saccharomyces cerevisiae, Genes Dev. 20 (2006) 1609-1620). Here, using enzymatic assays, we show that all glycolytic enzymes are associated with mitochondria in yeast, to extents similar to those previously reported for Arabidopsis cells. Using separation of mitochondrial complexes by blue-native/SDS-PAGE and coimmunoprecipitation of mitochondrial proteins with anti-enolase antibodies, we found that enolase takes part in a large macromolecular complex associated to mitochondria. The identified components included additional glycolytic enzymes, mitochondrial membrane carriers, and enzymes of the TCA cycle. We suggest a possible role of the enolase complex in the channeling of pyruvate, the terminal product of glycolysis, towards the TCA cycle within mitochondria. Moreover, we show that the mitochondrial enolase-containing complex also contains the cytosolic tRNA(CUU)Lys, which is mitochondrially-imported, and its presumed import carrier, the precursor of the mitochondrial lysyl-tRNA synthetase. This suggests an unsuspected novel function for this complex in tRNA mitochondrial import.     

Glycolytic enzymes associate dynamically with mitochondria in response to respiratory demand and support substrate channeling

In Arabidopsis thaliana, enzymes of glycolysis are present on the surface of mitochondria and free in the cytosol. The functional significance of this dual localization has now been established by demonstrating that the extent of mitochondrial association is dependent on respiration rate in both Arabidopsis cells and potato (Solanum tuberosum) tubers. Thus, inhibition of respiration with KCN led to a proportional decrease in the degree of association, whereas stimulation of respiration by uncoupling, tissue ageing, or overexpression of invertase led to increased mitochondrial association. In all treatments, the total activity of the glycolytic enzymes in the cell was unaltered, indicating that the existing pools of each enzyme repartitioned between the cytosol and the mitochondria. Isotope dilution experiments on isolated mitochondria, using (13)C nuclear magnetic resonance spectroscopy to monitor the impact of unlabeled glycolytic intermediates on the production of downstream intermediates derived from (13)C-labeled precursors, provided direct evidence for the occurrence of variable levels of substrate channeling. Pull-down experiments suggest that interaction with the outer mitochondrial membrane protein, VDAC, anchors glycolytic enzymes to the mitochondrial surface. It appears that glycolytic enzymes associate dynamically with mitochondria to support respiration and that substrate channeling restricts the use of intermediates by competing metabolic pathways.     

A quantitative evaluation of the effect of enzyme complexes on the glycolytic rate in vivo: mathematical modeling of the glycolytic complex

The cellular distribution of free and bound glycolytic enzymes in vivo was estimated by means of a model based on previously determined association constants for individual binding interactions and in vivo protein concentrations. The calculations revealed that a significant proportion of the enzymes would be either associated with F-actin, or bound in binary enzyme-enzyme complexes in vivo. An analysis of the relative concentration, and relative activity, of F-actin-bound enzymes suggested that a complete glycolytic complex, composed of all enzymatic steps from phosphofructokinase (PFK) to lactate dehydrogenase (LDH) does not exist. This was indicated by a very low concentration of F-actin-associated phosphoglycerate kinase (PGK) and by a very low activity of F-actin bound aldolase and PGK; this model showed that aldolase and PGK would be absent from any F-actin bound complex. An analysis of soluble enzyme-enzyme associations indicated that formation of binary enzyme complexes may lead to an increased overall flux through glyceraldehyde 3-phosphate dehydrogenase and LDH, but would serve to decrease flux through PFK and aldolase. A 1.4-fold activation of PFK, which occurs when the soluble enzyme binds to F-actin, suggested that reversible binding of PFK to F-actin may represent a novel cellular mechanism for controlling glycolytic flux during periods of increased metabolic demand by controlling the key regulatory enzyme of glycolysis.     

Decrease in phosphofructokinase activity during blood preservation and the effect of intracellular ATP

The activities of 15 enzymes, including all the glycolytic enzymes, were observed during the preservation of human red cells at 4°C. After 8 weeks, phosphofructokinase (PFK) activity had decreased most. The decrease was prevented by the addition of adenine and inosine to the preservation medium, but not at all by inosine alone and only slightly by adenine alone. This decrease paralleled that of the decrease of intracellular ATP. PFK in the hemolysate was inactivated rapidly, but the inactivation was effectively prevented by ATP. The decrease in glycolytic activity during preservation was concluded to be a result of the loss of PFK activity, and this in turn was due to the decrease of ATP.     

Studies on associations of glycolytic and glutaminolytic enzymes in MCF-7 cells: Role of P36

Isoelectric focusing of MCF-7 cell extracts revealed an association of the glycolytic enzymes glyceraldehyde 3-phosphate-dehydrogenase, phosphoglycerate kinase, enolase, and pyruvate kinase. This complex between the glycolytic enzymes is sensitive to RNase. p36 could not be detected within this association of glycolytic enzymes; however an association of p36 with a specific form of malate dehydrogenase was found. In MCF-7 cells three forms of malate dehydrogenase can be detected by isoelectric focusing: the mitochondrial form with an isoelectric point between 8.9 and 9.5, the cytosolic form with pl 5.0, and a p36-associated form with pl 7.8. The mitochondrial form comprises the mature mitochondrial isoenzyme (pl 9.5) and its precursor form (pl 8.9). Refocusing of the pl 7.8 form of malate dehydrogenase also gave rise to the mitochondrial isoenzyme. Thus, the pl 7.8 form of malate dehydrogenase is actually the mitochondrial isoenzyme retained in the cytosol by the association with p36. Addition of fructose 1,6-bisphosphate to the initial focusing column induced a quantitative shift of the pl 7.8 form of malate dehydrogenase to the mitochondrial forms (pl 8.9 and 9.5). In MCF-7 cells p36 is not phosphorylated in tyrosine. Kinetic measurements revealed that the pl 7.8 form of malate dehydrogenase has the lowest affinity for NADH. Compared to both mitochondrial forms the cytosolic isoenzyme has a high capacity when measured in the NAD → NADH direction (malate → oxaloacetate direction). The association of p36 with the mitochondrial isoenzyme may favor the flow of hydrogen from the cytosol into the mitochondria. Inhibition of cell proliferation by AMP which leads to an inhibition of glycolysis has no effect on complex formation by glycolytic and glutaminolytic enzymes in MCF-7 cells. AMP treatment leads to an activation of malate dehydrogenase, which correlates with the increase of pyruvate and the decrease of lactate levels, but has no effect on the distribution of the various malate dehydrogenase forms. © 1996 Wiley-Liss, Inc.     

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.     

Therapeutic Potential of Riboflavin,Niacin and Ascorbic Acid on Carbohydrate Metabolizing Enzymes in Secondary Endometrial Carcinoma Bearing Rats

Curative potential of riboflavin, niacin and ascorbic acid against tamoxifen mediated endometrial carcinoma was established by studies on carbohydrate metabolizing enzymes. The enzymes investigated were glycolytic enzymes namely, hexokinase; aldolase; phosphoglucoisomerase and the gluconeogenic enzymes namely, glucose-6-phosphatase and fructose-1, 6-biphosphatase in endometrial carcinoma bearing rats. A significant increase in glycolytic enzymes and a subsequent decrease in gluconeogenic enzymes were observed in plasma, liver and kidney of endometrial carcinoma animals. The administration of riboflavin (45 mg/kg bw/day), niacin (100 mg/kg bw/day) and ascorbic acid (200 mg/kg bw/day) along with tamoxifen (45 mg/kg bw/day) caused a significant decrease in the activity of glycolytic enzymes and a significant increase in the activities of gluconeogenic enzymes to near normal levels in experimental animals. Our results suggest that riboflavin, niacin and ascorbic acid have potential combination therapy against tamoxifen mediated secondary endometrial carcinoma in experimental rats. However, there were no deleterious side effects observed in combinants alone treated animals.