Mechanism of glyceraldehyde-3-phosphate transfer from aldolase to glyceraldehyde-3-phosphate dehydrogenase

The catalytic interaction of glyceraldehyde-3-phosphate dehydrogenase with glyceraldehyde 3-phosphate has been examined by transient-state kinetic methods. The results confirm previous reports that the apparent Km for oxidative phosphorylation of glyceraldehyde 3-phosphate decreases at least 50-fold when the substrate is generated in a coupled reaction system through the action of aldolase on fructose 1,6-bisphosphate, but lend no support to the proposal that glyceraldehyde 3-phosphate is directly transferred between the two enzymes without prior release to the reaction medium. A theoretical analysis is presented which shows that the kinetic behaviour of the coupled two-enzyme system is compatible in all respects tested with a free-diffusion mechanism for the transfer of glyceraldehyde 3-phosphate from the producing enzyme to the consuming one.     

A new chemical mechanism catalyzed by a mutated aldehyde dehydrogenase.

NAD(P) aldehyde dehydrogenases (EC 1.2.1.3) are a family of enzymes that oxidize a wide variety of aldehydes into acid or activated acid compounds. Using site-directed mutagenesis, the essential nucleophilic Cys 149 in the NAD-dependent phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Escherichia coli has been replaced by alanine. Not unexpectedly, the resulting mutant no longer shows any oxidoreduction phosphorylating activity. The same mutation, however, endows the enzyme with a novel oxidoreduction nonphosphorylating activity, converting glyceraldehyde 3-phosphate into 3-phosphoglycerate. Our study further provides evidence for an alternative mechanism in which the true substrate is the gem-diol entity instead of the aldehyde form. This implies that no acylenzyme intermediate is formed during the catalytic event. Therefore, the mutant C149A is a new enzyme which catalyzes a distinct reaction with a chemical mechanism different from that of its parent phosphorylating glyceraldehyde-3-phosphate dehydrogenase. This finding demonstrates the possibility of an alternative route for the chemical reaction catalyzed by classical nonphosphorylating aldehyde dehydrogenases.     

Compartmentalized ATP synthesis in skeletal muscle triads.

Isolated skeletal muscle triads contain a compartmentalized glycolytic reaction sequence catalyzed by aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase. These enzymes express activity in the structure-associated state leading to synthesis of ATP in the triadic junction upon supply of glyceraldehyde 3-phosphate or fructose 1,6-bisphosphate. ATP formation occurs transiently and appears to be kinetically compartmentalized, i.e., the synthesized ATP is not in equilibrium with the bulk ATP. The apparent rate constants of the aldolase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase reaction are significantly increased when fructose 1,6-bisphosphate instead of glyceraldehyde 3-phosphate is employed as substrate. The observations suggest that fructose 1,6-bisphosphate is especially effectively channelled into the junctional gap. The amplitude of the ATP transient is decreasing with increasing free [Ca2+] in the range of 1 nM to 30 microM. In the presence of fluoride, the ATP transient is significantly enhanced and its declining phase is substantially retarded. This observation suggests utilization of endogenously synthesized ATP in part by structure associated protein kinases and phosphatases which is confirmed by the detection of phosphorylated triadic proteins after gel electrophoresis and autoradiography. Endogenous protein kinases phosphorylate proteins of apparent Mr 450,000, 180,000, 160,000, 145,000, 135,000, 90,000, 54,000, 51,000, and 20,000, respectively. Some of these phosphorylated polypeptides are in the Mr range of known phosphoproteins involved in excitation-contraction coupling of skeletal muscle, which might give a first hint at the functional importance of the sequential glycolytic reactions compartmentalized in triads.     

Dynamic interactions of enzymes involved in triosephosphate metabolism

A steady-state kinetic analysis of the coupled reactions catalysed by the three-enzyme system, aldolase, glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase, was performed. The kinetic parameters of the progress curves of end-product formation calculated for noninteracting enzymes were compared with those measured in the two-enzyme and three-enzyme systems. Changes in the fluorescence anisotropy of labelled dehydrogenase upon addition of aldolase and/or isomerase were also measured. Glyceraldehyde-3-phosphate oxidation catalysed by glyceraldehyde-3-phosphate dehydrogenase in the presence of isomerase (which ensures rapid equilibration of the triosephosphates) follows single first-order kinetics. The rate constant depends simply on the concentration of the dehydrogenase, indicating no kinetically significant isomerase-dehydrogenase interaction. Fluorescence anisotropy measurements also fail to reveal complex formation between the two enzymes. The steady-state velocity of 3-phosphoglycerate formation from fructose 1, 6-bisphosphate in the reactions catalysed by aldolase and dehydrogenase is not increased twofold on addition of the isomerase, even though a 1:2 stoichiometry of fructose 1,6-bisphosphate/glyceraldehyde 3-phosphate is expected. In fact, by increasing the concentration of the isomerase, the steady-state velocity actually decreases. This effect of the isomerase may be a kinetic consequence of an aldolase-isomerase interaction, which results in a decrease of aldolase activity. Furthermore, the fluorescence anisotropy of labelled dehydrogenase, measured at different aldolase concentrations, is significantly lower when the sample contains isomerase. The decrease in the steady-state velocity of the consecutive reactions caused by the elevation of isomerase concentration could be negated by increasing the dehydrogenase concentrations in the three-enzyme system. All of these observations fit the assumption that the amount of aldolase-dehydrogenase complex is reduced due to competition of isomerase with dehydrogenase. The alternate binding of dehydrogenase and isomerase to aldolase may regulate the flux rate of glycolysis.     

Quantitative cytochemical measurement of glyceraldehyde 3-phosphate dehydrogenase activity

Summary A system has been developed for the quantitative measurement of glyceraldehyde 3-phosphate dehydrogenase activity in tissue sections. An obstacle to the histochemical study of this enzyme has been the fact that the substrate, glyceraldehyde 3-phosphate, is very unstable. In the present system a stable compound, fructose 1, 6-diphosphate, is used as the primary substrate and the demonstration of the glyceraldehyde 3-phosphate dehydrogenase activity depends on the conversion of this compound into the specific substrate by the aldolase present in the tissue. The characteristics of the dehydrogenase activity resulting from the addition of fructose 1, 6-diphosphate, resemble closely the known properties of purified glyceraldehyde 3-phosphate dehydrogenase. Use of polyvinyl alcohol in the reaction medium prevents release of enzymes from the sections, as occurs in aqueous media. Although in this study intrinsic aldolase activity was found to be adequate for the rapid conversion of fructose 1, 6-diphosphate into the specific substrate for the dehydrogenase, the use of exogenous aldolase may be of particular advantage in assessing the integrity of the Embden-Meyerhof pathway.     

Quantitative cytochemical measurement of glyceraldehyde 3-phosphate dehydrogenase activity.

A system has been developed for the quantitative measurment of glyceraldehyde 3-phosphate dehydrogenase activity in tissue sections. An obstacle to the histochemical study of this enzyme has been the fact that the substrate, gylceraldehyde 3-phosphate, is very unstable. In the present system a stable compound, fructose 1, 6-diphosphate, is used as the primary substrate and the demonsatration of the glyceraldehyde 3-phosphate dehydrogenase activity depends on the conversion of this compound into the specific substrate by the aldolase present in the tissue. The characteristics of the dehydrogenase activity resulting from the addition of fructose 1, 6-diphosphate, resemble closely the known properties of purified glyceraldehyde 3-phosphate dehydrogenase. Use of polyvinyl alcohol in the reaction medium prevents release of enzymes from the sections, as occurs in aqueous media. Although in this study intrinsic aldolase activity was found to be adequate for the rapid conversion of fructose 1, 6-diphosphate into the specific substrate for the dehydrogenase, the use of exogenous aldolase may be of particular advantage in assessing the intergrity of the Embden-Meyerhof pathway.     

Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT), an Enzyme Essential for NAD+ Biosynthesis,Leads to Altered Carbohydrate Metabolism in Cancer Cells

Nicotinamide phosphoribosyltransferase (NAMPT) has been extensively studied due to its essential role in NAD⁺ biosynthesis in cancer cells and the prospect of developing novel therapeutics. To understand how NAMPT regulates cellular metabolism, we have shown that the treatment with FK866, a specific NAMPT inhibitor, leads to attenuation of glycolysis by blocking the glyceraldehyde 3-phosphate dehydrogenase step (Tan, B., Young, D. A., Lu, Z. H., Wang, T., Meier, T. I., Shepard, R. L., Roth, K., Zhai, Y., Huss, K., Kuo, M. S., Gillig, J., Parthasarathy, S., Burkholder, T. P., Smith, M. C., Geeganage, S., and Zhao, G. (2013) Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), an enzyme essential for NAD⁺ biosynthesis, in human cancer cells: metabolic basis and potential clinical implications. J. Biol. Chem. 288, 3500–3511). Due to technical limitations, we failed to separate isotopomers of phosphorylated sugars. In this study, we developed an enabling LC-MS methodology. Using this, we confirmed the previous findings and also showed that NAMPT inhibition led to accumulation of fructose 1-phosphate and sedoheptulose 1-phosphate but not glucose 6-phosphate, fructose 6-phosphate, and sedoheptulose 7-phosphate as previously thought. To investigate the metabolic basis of the metabolite formation, we carried out biochemical and cellular studies and established the following. First, glucose-labeling studies indicated that fructose 1-phosphate was derived from dihydroxyacetone phosphate and glyceraldehyde, and sedoheptulose 1-phosphate was derived from dihydroxyacetone phosphate and erythrose via an aldolase reaction. Second, biochemical studies showed that aldolase indeed catalyzed these reactions. Third, glyceraldehyde- and erythrose-labeling studies showed increased incorporation of corresponding labels into fructose 1-phosphate and sedoheptulose 1-phosphate in FK866-treated cells. Fourth, NAMPT inhibition led to increased glyceraldehyde and erythrose levels in the cell. Finally, glucose-labeling studies showed accumulated fructose 1,6-bisphosphate in FK866-treated cells mainly derived from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Taken together, this study shows that NAMPT inhibition leads to attenuation of glycolysis, resulting in further perturbation of carbohydrate metabolism in cancer cells. The potential clinical implications of these findings are also discussed.     

A simple approach to identify the mechanism of intermediate transfer: enzyme system related to triose phosphate metabolism

A new approach is described to identify the mechanism of transfer of intermediates of consecutive reactions catalysed by two functionally related enzymes. Interactions resulting in conformational changes of the individual enzymes and/or channelling of the intermediate can be identified by comparing the rate constants of the coupled and individual reactions. Using these kinetic parameters, the relative specific radioactivity of the end product can be calculated according to the different mechanisms. The comparison of these values with the experimentally determined relative specific radioactivity enhances the sensitivity of the determination. The interaction between aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) and glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) was analysed. The data agree with the model in which channeling of the intermediate was assumed. The results suggest that glyceraldehyde 3-phosphate is functionally compartmentalised within the reconstituted enzyme system, which may be relevant under physiological conditions.     

Substrate Specificity, Substrate Channeling, and Allostery in BphJ: An Acylating Aldehyde Dehydrogenase Associated with the Pyruvate Aldolase BphI

BphJ, a nonphosphorylating acylating aldehyde dehydrogenase, catalyzes the conversion of aldehydes to form acyl-coenzyme A in the presence of NAD(+) and coenzyme A (CoA). The enzyme is structurally related to the nonacylating aldehyde dehydrogenases, aspartate-β-semialdehyde dehydrogenase and phosphorylating glyceraldehyde-3-phosphate dehydrogenase. Cys-131 was identified as the catalytic thiol in BphJ, and pH profiles together with site-specific mutagenesis data demonstrated that the catalytic thiol is not activated by an aspartate residue, as previously proposed. In contrast to the wild-type enzyme that had similar specificities for two- or three-carbon aldehydes, an I195A variant was observed to have a 20-fold higher catalytic efficiency for butyraldehyde and pentaldehyde compared to the catalytic efficiency of the wild type toward its natural substrate, acetaldehyde. BphJ forms a heterotetrameric complex with the class II aldolase BphI that channels aldehydes produced in the aldol cleavage reaction to the dehydrogenase via a molecular tunnel. Replacement of Ile-171 and Ile-195 with bulkier amino acid residues resulted in no more than a 35% reduction in acetaldehyde channeling efficiency, showing that these residues are not critical in gating the exit of the channel. Likewise, the replacement of Asn-170 in BphJ with alanine and aspartate did not substantially alter aldehyde channeling efficiencies. Levels of activation of BphI by BphJ N170A, N170D, and I171A were reduced by ≥3-fold in the presence of NADH and ≥4.5-fold when BphJ was undergoing turnover, indicating that allosteric activation of the aldolase has been compromised in these variants. The results demonstrate that the dehydrogenase coordinates the catalytic activity of BphI through allostery rather than through aldehyde channeling.     

Effect of the sodium/potassium ratio on glyceraldehyde 3-phosphate dehydrogenase interaction with red cell vesicles.

Binding of glyceraldehyde 3-phosphate to glyceraldehyde-3-phosphate dehydrogenase, the membrane protein known as Band 6, causes shifts in the 31P nuclear magnetic resonance spectrum of the substrate (Fossel, E.T. and Solomon, A.K (1977) Biochim. Biophys. Acta 464, 82--92). We have studied the resonance shifts produced by varying the sodium/potassium ratio, at constant ionic strength, in order to examine the relationship between the cation transport system and glyceraldehyde-3-phosphate dehydrogenase. Alteration of the potassium concentration at the extracellular face of the vesicle affects the conformation of glyceraldehyde-3-phosphate dehydrogenase at the cytoplasmic face, thus showing that a conformation changed induced by a change in extracellular potassium can be transmitted across the membrane. Alterations of the sodium concentration at the cytoplasmic face also affect the enzyme conformation, whereas sodium changes at the extracellular face are without effect. In contrast, there is no sidedness difference in the effect of potassium concentrations. The half-values for these effects are like those for activation of the red cell (Na4 + K+)-ATPase. We have also produced ionic concentration gradients across the vesicle similar to those Glynn and Lew (1970) J. Physiol. London 207, 393--402) found to be effective in running the cation pump backwards to produce adenosine triphosphate in the human red cell. The sodium/potassium concentration dependence of this process in red cells is mimicked by 31P resonance shifts in the (glyceraldehyde 3-phosphate/glyceraldehyde-3-phosphate dehydrogenase/inside out vesicle) system. These experiments provide strong support for the existence of a functional linkage between the membrane (Na+ + K+)-ATPase and the glyceraldehyde-3-phosphate dehydrogenase at the cytoplasmic face.     

Partial reversible inactivation of enzymes due to binding to the human erythrocyte membrane

Summary Hypotonic human erythrocyte ghosts, devoid of the original glyceraldehyde-3-phosphate dehydrogenase content of the red cell, bind added glyceraldehyde-3-phosphate dehydrogenases, isolated from human erythrocytes, rabbit and pig muscle, as well as rabbit muscle aldolase. There are only slight differences in the affinities towards the various glyceraldehyde-3-phosphate dehydrogenases. On the other hand, glyceraldehyde-3-phosphate dehydrogenases are bound much stronger than aldolase; in an equimolar mixture the former can prevent the binding of the latter, or replace previously bound aldolase at the membrane surface. Binding is always accompanied by the partial inactivation of enzymes, which can be reverted by desorption. Unwashed ghosts rich in hemoglobin seem to have a more pronounced inactivating effect on bound glyceraldehyde-3-phosphate dehydrogenase. In isotonic media ghosts, whether white or unwashed, reseal and do not interact with the enzymes.     

Glycerol 3-phosphate analogues as metabolic inhibitors in Escherichia coli, 3-hydroxy-4-oxobutyl-1-phosphonate, a drug that interferes with normal phosphoglyceride metabolism.

3-Hydroxy-4-oxobutyl-1-phosphonate, the phoshonic acid analogue of glyceraldehyde 3-phosphate, enters Escherichia coli via the glycerol 3-phosphate transport system. There is no differential effect upon the accumulation of deoxyribonucleic acid, ribonucleic acid, or phosphoglycerides, although the accumulation of proteins was less effected. Examination of the phospholipids revealed that phosphatidylglycerol accumulation was most severely inhibited and cardiolipin accumulation was least affected. Concentrations of glyceraldehyde 3-phosphate and its phosphonic acid analogue that markedly inhibit macromolecular and phosphoglyceride biosynthesis have no effect upon the intracellular nucleoside triphosphate pool size. The phosphonate is a competitive inhibitor of sn-glycerol 3-phosphate in reactions catalyzed by acyl coenzyme A:sn-glycerol-3-phosphate acyltransferase and CDP-diacylglycerol:sn-glycerol-3-phosphate phosphatidyltransferase. A Km mutant for the former enzyme was susceptible to the phosphansferase activity. Studies with mutant strains ruled out the aerobic glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate synthase, and fructose-1,6-biphosphate aldolase as the primary sites of action.     

Pcal_0632, a phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Pyrobaculum calidifontis

Genome sequence of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0632, annotated as glyceraldehyde-3-phosphate dehydrogenase, which is partially overlapped with phosphoglycerate kinase. In the phylogenetic tree, Pcal_0632 clustered with phosphorylating glyceraldehyde-3-phosphate dehydrogenases characterized from hyperthermophilic archaea and exhibited highest identity of 54% with glyceraldehyde-3-phosphate dehydrogenase from Sulfolobus tokodaii. To examine biochemical function of the protein, Pcal_0632 gene was expressed in Escherichia coli and the gene product was purified. The recombinant enzyme catalyzed the conversion of glyceraldehyde 3-phosphate and inorganic phosphate into 1,3-bisphosphoglycerate utilizing both NAD and NADP as cofactor with a marked preference for NADP. The enzyme was highly stable against temperature and denaturants. Half-life of the enzyme was 60 min at 100 °C. It retained more than 60% of its activity even after an incubation of 72 h at room temperature in the presence of 6 M urea. High thermostability and resistance against denaturants make Pcal_0632 a novel glyceraldehyde-3-phosphate dehydrogenase.

     

Stereospecificity of C4 nicotinamide hydrogen transfer of the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase

The stereospecificity of the reaction catalysed by the spinach chloroplast enzyme NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NADP+ oxidoreductase (phosphorylating), EC 1.2.1.13) with respect to the C4 nicotinamide hydrogen transfer was investigated. NADPH deuterated at the C4 HA position was synthesized using aldehyde dehydrogenase. 1H-NMR spectroscopy was used to examine the NADP+ product of the GPDH reaction for the presence or absence of the C4 deuterium atom. Chloroplast NADP-dependent glyceraldehyde-3-phosphate dehydrogenase retains the deuterium at the C4 HA position (removing the hydrogen atom), and is therefore a B (pro-S) specific dehydrogenase.     

Cooperative effect of fructose bisphosphate and glyceraldehyde-3-phosphate dehydrogenase on aldolase action

The combination of binding and kinetic approaches is suggested to study (i) the mechanism of substrate-modulated dynamic enzyme associations; (ii) the specificity of enzyme interactions. The effect of complex formation between aldolase and glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) on aldolase catalysis was investigated under pseudo-first-order conditions. No change in kcat but a significant increase in KM of fructose 1,6-bisphosphate for aldolase was found when both enzymes were obtained from muscle. In contrast, kcat rather than KM changed if dehydrogenase was isolated from yeast. Next, the conversion of fructose 1-phosphate was not affected by interactions between enzyme couples isolated from muscle. The influence of fructose phosphates on the enzyme-complex formation was studied by means of covalently attached fluorescent probe. We found that the interaction ws not perturbed by the presence of fructose 1-phosphate; however, fructose 1,6-bisphosphate altered the dissociation constant of the enzyme complex. A molecular model for fructose 1,6-bisphosphate-modulated enzyme interaction has been evaluated which suggests that high levels of fructose bisphosphate would drive the formation of the 'channelling' complex between aldolase and glyceraldehyde-3-phosphate dehydrogenase.     

Aldolase-catalyzed diketone phosphate formation from oxoaldehydes. NMR studies and metabolic significance.

NMR spectroscopy showed fructose-1,6-bisphosphate aldolase from rabbit muscle accepts as substrates, in lieu of glyceraldehyde 3-phosphate, the oxoaldehydes methylglyoxal and phenylglyoxal but not hydroxymethylglyoxal. The enzyme catalyzed an aldol condensation between the oxoaldehyde and dihydroxyacetone phosphate to form a monophosphorylated diketone and was inactivated in the process. Circumvention of this reaction, by metabolism of oxoaldehydes to hydroxy acids, may be a metabolic role for the glyoxalase enzyme system. Transketolase and transaldolase were found not to accept oxoaldehydes as substrates in place of glyceraldehyde 3-phosphate.     

Effect of the sodium/potassium ratio on glyceraldehyde-3-phosphate dehydrogenase interaction with red cell vesicles

Binding of glyceraldehyde 3-phosphate to glyceraldehyde-3-phosphate dehydrogenase, the membrane protein known as Band 6, causes shifts in the ³¹P nuclear magnetic resonance spectrum of the substrate (Fossel, E.T. and Solomon, A.K. (1977) Biochim. Biophys. Acta 464, 82–92). We have studied the resonance shifts produced by varying the sodium/potassium ratio, at constant ionic strength, in order to examine the relationship between the cation transport system and glyceraldehyde-3-phosphate dehydrogenase. Alteration of the potassium concentration at the extracellular face of the vesicle affects the conformation of glyceraldehyde-3-phosphate dehydrogenase at the cytoplasmic face, thus showing that a conformation change induced by a change in extracellular potassium can be transmitted across the membrane. Alterations of the sodium concentration at the cytoplasmic face also affect the enzyme conformation, whereas sodium changes at the extracellular face are without effect. In contrast, there is no sidedness difference in the effect of potassium concentrations. The half-values for these effects are like those for activation of the red cell (Na⁺ + K⁺)-ATPase. We have also produced ionic concentration gradients across the vesicle similar to those Glynn and Lew ((1970) J. Physiol. London 207, 393–402) found to be effective in running the cation pump backwards to produce adenosine triphosphate in the human red cell. The sodium/potassium concentration dependence of this process in red cells is mimicked by ³¹P resonance shifts in the (glyceraldehyde 3-phosphate/glyceraldehyde-3-phosphate dehydrogenase/inside out vesicle) system. These experiments provide strong support for the existence of a functional linkage between the membrane (Na⁺ + K⁺)-ATPase and the glyceraldehyde-3-phosphate dehydrogenase at the cytoplasmic face.     

Enzymes of glycerol and glyceraldehyde metabolism in mouse liver: effects of caloric restriction and age on activities

The influence of caloric restriction on hepatic glyceraldehyde- and glycerol-metabolizing enzyme activities of young and old mice were studied. Glycerol kinase and cytoplasmic glycerol-3-phosphate dehydrogenase activities were increased in both young and old CR (calorie-restricted) mice when compared with controls, whereas triokinase increased only in old CR mice. Aldehyde dehydrogenase and aldehyde reductase activities in both young and old CR mice were unchanged by caloric restriction. Mitochondrial glycerol-3-phosphate dehydrogenase showed a trend towards an increased activity in old CR mice, whereas a trend towards a decreased activity in alcohol dehydrogenase was observed in both young and old CR mice. Serum glycerol levels decreased in young and old CR mice. Therefore increases in glycerol kinase and glycerol-3-phosphate dehydrogenase were associated with a decrease in fasting blood glycerol levels in CR animals. A prominent role for triokinase in glyceraldehyde metabolism with CR was also observed. The results indicate that long-term caloric restriction induces sustained increases in the capacity for gluconeogenesis from glycerol.     

Glycolytic enzyme interactions with tubulin and microtubules

Interactions of the glycolytic enzymes glucose-6-phosphate isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase, enolase, phosphoglycerate mutase, phosphoglycerate kinase, pyruvate kinase, lactate dehydrogenase type-M, and lactate dehydrogenase type-H with tubulin and microtubules were studied. Lactate dehydrogenase type-M, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and aldolase demonstrated the greatest amount of co-pelleting with microtubules. The presence of 7% poly(ethylene glycol) increased co-pelleting of the latter four enzymes and two other enzymes, glucose-6-phosphate isomerase, and phosphoglycerate kinase with microtubules. Interactions also were characterized by fluorescence anisotropy. Since the KD values of glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase for tubulin and microtubules were all found to be between 1 and 4 microM, which is in the range of enzyme concentration in cells, these enzymes are probably bound to microtubules in vivo. These observations indicate that interactions of cytosolic proteins, such as the glycolytic enzymes, with cytoskeletal components, such as microtubules, may play a structural role in the formation of the microtrabecular lattice.     

Covalent binding of 3-pyridinealdehyde nicotinamide adenine dinucleotide and substrate to glyceraldehyde 3-phosphate dehydrogenase.

Glyceraldehyde 3-phosphate dehydrogenase (D-glyceraldehyde-3-phoshate:nicotinamide adenine dinucleotide oxidoreductase (phosphorylating), EC 1.2.1.12) forms a complex with 3-pyridinealdehyde-NAD which survives precipitation with 7% perchloric acid. The molar ratio bound 3-pyridinealdehyde-NAD to the enzyme is 2.5 to 2.9. Lactate, malate, and alcohol dehydrogenases do not form acid-precipitable complexes with 3-pyridinealdehyde-NAD. 3-Pyridinealdehyde-deamino-NAD or glyceraldehyde 3-phosphate also forms an acid-stable complex with glyceraldehyde 3-phosphate dehydrogenase; however, NAD, 3-acetylpyridine-NAD, or thionicotinamide-NAD does not produce an acid-stable complex. Incubation of the glyceraldehyde 3-phosphate dehydrogenase with glyceraldehyde 3-phosphate, acetyl phosphate, iodoacetic acid, or iodosobenzoate inhibits the formation of the acid-stable complex with 3-pyridinealdehyde-NAD. Glyceraldehyde 3-phosphate or 3-pyridinealdehyde-NAD also prevents carboxymethylation of the active site cysteine-149 by[14-C]iodoacetic acid. These studies indicate that the aldehyde group of 3-pyridinealdehyde-NAD forms a thiohemiacetal linkage with cysteine-149 which is the substrate binding site for the dehydrogenase reaction. These findings may account for the fact that 3-pyridinealdehyde-NAD strongly inhibits the dehydrogenase and esterase activities of 3-pyridinealdehyde-NAD forms a thiohemiacetal linkage with cysteine-149 which is the substrate binding site for the dehydrogenase reaction. These findings may account for the fact that 3-pyridinealdehyde-NAD strongly inhibits the dehydrogenase and esterase activities of glyceraldehyde 3-phosphate dehydrogenase which require reduced cysteine-149. However, the analogue does not inhibit the acetyl phosphates activity of the enzyme for which the active site sulfhydryl residues must be oxidized.