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Query: EC:1.2.1.13 (glyceraldehyde-3-phosphate dehydrogenase)
 6,511 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pairwise coupled reactions of fructose-1,6-bisphosphate aldolase and sn-glycerol-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and D-glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase and sn-glycerol-3-phosphate dehydrogenase have been detected by microspectrophotometry in single crystals obtained from myogen A in the presence of polyethylene glycol. Microspectrophotometric measurements with polarized light demonstrate that the protein molecules are oriented and that NADH is bound with a definite orientation to the dehydrogenases within the crystal.
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PMID:Coupled enzymatic reactions measured in a single protein crystal from myogen A. 251 36

Perdeuterated spin label (DSL) analogs of NAD+, with the spin label attached at either the C8 or N6 position of the adenine ring, have been employed in an EPR investigation of models for negative cooperativity binding to tetrameric glyceraldehyde-3-phosphate dehydrogenase and conformational changes of the DSL-NAD+-enzyme complex during the catalytic reaction. C8-DSL-NAD+ and N6-DSL-NAD+ showed 80 and 45% of the activity of the native NAD+, respectively. Therefore, these spin-labeled compounds are very efficacious for investigations of the motional dynamics and catalytic mechanism of this dehydrogenase. Perdeuterated spin labels enhanced spectral sensitivity and resolution thereby enabling the simultaneous detection of spin-labeled NAD+ in three conditions: (1) DSL-NAD+ freely tumbling in the presence of, but not bound to, glyceraldehyde-3-phosphate dehydrogenase, (2) DSL-NAD+ tightly bound to enzyme subunits remote (58 A) from other NAD+ binding sites, and (3) DSL-NAD+ bound to adjacent monomers and exhibiting electron dipolar interactions (8-9 A or 12-13 A, depending on the analog). Determinations of relative amounts of DSL-NAD+ in these three environments and measurements of the binding constants, K1-K4, permitted characterization of the mathematical model describing the negative cooperativity in the binding of four NAD+ to glyceraldehyde-3-phosphate dehydrogenase. For enzyme crystallized from rabbit muscle, EPR results were found to be consistent with the ligand-induced sequential model and inconsistent with the pre-existing asymmetry models. The electron dipolar interaction observed between spin labels bound to two adjacent glyceraldehyde-3-phosphate dehydrogenase monomers (8-9 or 12-13 A) related by the R-axis provided a sensitive probe of conformational changes of the enzyme-DSL-NAD+ complex. When glyceraldehyde-3-phosphate was covalently bound to the active site cysteine-149, an increase in electron dipolar interaction was observed. This increase was consistent with a closer approximation of spin labels produced by steric interactions between the phosphoglyceryl residue and DSL-NAD+. Coenzyme reduction (DSL-NADH) or inactivation of the dehydrogenase by carboxymethylation of the active site cysteine-149 did not produce changes in the dipolar interactions or spatial separation of the spin labels attached to the adenine moiety of the NAD+. However, coenzyme reduction or carboxymethylation did alter the stoichiometry of binding and caused the release of approximately one loosely bound DSL-NAD+ from the enzyme. These findings suggest that ionic charge interactions are important in coenzyme binding at the active site.
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PMID:Catalytic mechanism and interactions of NAD+ with glyceraldehyde-3-phosphate dehydrogenase: correlation of EPR data and enzymatic studies. 254 10

The steady-state kinetics of 1,3-bisphosphoglycerate formation through the action of phosphoglycerate kinase on 3-phosphoglycerate and ATP have been examined. The results show that initial velocities determined by the standard method of coupling bisphosphoglycerate production to NADH reduction in the presence of glyceraldehyde-3-phosphate dehydrogenase do not differ significantly from those determined in the absence of the latter enzyme. This observation invalidates the proposal that bisphosphoglycerate dissociation from phosphoglycerate kinase is much too slow to account for the high rates of phosphoglycerate turnover observed in the coupled two-enzyme system. The capacity for rapid bisphosphoglycerate production and release is an intrinsic catalytic property of phosphoglycerate kinase that does not require the presence of other enzymes or the involvement of a mechanism of channelized (non-diffusional) transfer of bisphosphoglycerate from the producing enzyme to the consuming one.
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PMID:Evidence that 1,3-bisphosphoglycerate dissociation from phosphoglycerate kinase is an intrinsically rapid reaction step. 259 31

1. The kinetics of 1,3-bisphosphoglycerate binding to glyceraldehyde-3-phosphate dehydrogenase have been examined by stopped-flow techniques in the absence and presence of phosphoglycerate kinase, using enzyme concentrations in the range 0.5-40 microM. Rate and equilibrium constant estimates for the interaction of the ligand with the two enzymes are reported. 2. The kinetics of ligand transfer from the binary complex of bisphosphoglycerate and phosphoglycerate kinase to the binary complex of NAD+ and glyceraldehyde-3-phosphate dehydrogenase conform excellently to the predictions of a standard free-diffusion mechanism and exhibit no detectable contributions from a mechanism of direct (channelized) transfer of bisphosphoglycerate between the two enzymes. 3. Previously reported evidence that the binary complex of bisphosphoglycerate and phosphoglycerate kinase may act (in the presence of NADH) as a substrate for glyceraldehyde-3-phosphate dehydrogenase according to Michaelis-Menten kinetics is based on a misinterpretation of the experimental observations that can be attributed to neglect of the autocatalytic effect of NAD+ produced during the reaction. Experiments performed under conditions where the autocatalytic effect of NAD+ is eliminated provide clear evidence that the kinetics of utilization of the kinase-bisphosphoglycerate complex for enzymic NADH reduction are consistent with prior dissociation of the complex according to a free-diffusion mechanism of metabolite transfer and incompatible with a mechanism of direct metabolite transfer. 4. A kinetic argument is presented which renders implausible the very idea that direct metabolite transfer between 'soluble' consecutive enzymes in metabolic pathways may offer any catalytic advantages in comparison to metabolite transfer by free diffusion. A mechanism of direct metabolite transfer seems intuitively attractive only because one tends to disregard the diffusional processes required to bring the consecutive enzymes together and to separate them when the transfer has been completed. Direct metabolite transfer would be expected to be catalytically advantageous only in tightly bound multienzyme complexes showing no kinetically significant tendency to dissociate. 5. It is concluded that mechanisms of direct metabolite transfer have not been convincingly demonstrated to apply, nor are they likely to apply, between 'soluble' consecutive enzymes in metabolic pathways, at least not in the glycolytic sequence of reactions.
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PMID:Mechanism of 1,3-bisphosphoglycerate transfer from phosphoglycerate kinase to glyceraldehyde-3-phosphate dehydrogenase. 259 32

Ultraviolet resonance Raman (UVRR) spectra, with 260-nm excitation, are reported for oxidized and reduced nicotinamide adenine dinucleotides (NAD+ and NADH, respectively). Corresponding spectra are reported for these coenzymes when bound to the enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and liver and yeast alcohol dehydrogenases (LADH and YADH). The observed differences between the coenzyme spectra are interpreted in terms of conformation, hydrogen bonding, and general environment polarity differences between bound and free coenzymes and between coenzymes bound to different enzymes. The possibility of adenine protonation is discussed. UVRR spectra with 220-nm excitation also are reported for holo- and apo-GAPDH (GAPDH-NAD+ and GAPDH alone, respectively). In contrast with the 260-nm spectra, these show only bands due to vibrations of aromatic amino acid residues of the protein. The binding of coenzyme to GAPDH has no significant effect on the aromatic amino acid bands observed. This result is discussed in the light of the known structural change of GAPDH on binding coenzyme. Finally, UVRR spectra with 240-nm excitation are reported for GAPDH and an enzyme-substrate intermediate of GAPDH. Perturbations are reported for tyrosine and tryptophan bands on forming the acyl enzyme.
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PMID:An ultraviolet resonance Raman study of dehydrogenase enzymes and their interactions with coenzymes and substrates. 265 94

Chemical modification of one arginine residue per subunit of tetrameric D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) molecule results in a 85-95% loss of its activity (Nagradova and Asryants (1975) Biochim. Biophys. Acta 386, 365-368; Nagradova, N.K., Asryants, R.A., Benkevich, N.V. and Safronova, M.I. (1976) FEBS Lett. 69, 246-248). Transient kinetic experiments performed in the present work with modified rabbit muscle and Baker's yeast enzymes showed that the first-order rate constant of acyl-enzyme.NADH formation was diminished 30-fold with the rabbit muscle enzyme and 60-fold with the Baker's yeast enzyme. Modification of arginine residues was shown also to affect the second step of the catalytic reaction, the phosphorolysis of the acyl-enzyme (the second-order rate constant of phosphorolysis decreased 9-fold in the case of the rabbit muscle enzyme and 40-fold in the case of the Baker's yeast enzyme). The native and modified enzymes exhibited similar inhibitory constant values with respect to NADH, suggesting no contribution of arginine residues to the acyl-enzyme.NADH complex destabilization. By and large, the experimental data are consistent with the hypothetical scheme proposed on the basis of X-ray crystallography studies to describe a participation of Arg-231 in the catalytic mechanism of D-glyceraldehyde-3-phosphate dehydrogenase (Grau (1982) in the Pyridine Nucleotide Coenzymes, p. 135-187).
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PMID:An examination of the role of arginine residues in the functioning of D-glyceraldehyde-3-phosphate dehydrogenase. 266 76

The effect of ATP on the formation, spectral properties, and reactions of [beta-(2-furyl)acryloyl]glyceraldehyde-3-phosphate dehydrogenase (FA-GPDH) has been investigated. The chromophoric FA-GPDH has the advantage of providing spectrophotometric signals of the interaction of acyl enzyme with nucleotides and dinucleotides. The results are consistent with the exclusive existence of two acyl-enzyme conformations previously inferred from the interaction of the acyl enzyme with NAD+ and NADH. ATP interaction stabilizes a conformation different from that stabilized by NAD+. The inhibitory effects of ATP on these reactions are consistent with the reported inhibitory effect of ATP on the steady-state reaction with the true substrate. The physiological significance of these results to the regulation of glycolysis, via the ligand-dependent fate of 3-phosphoglycerol-GPDH, is discussed.
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PMID:Noncovalent modulation by ATP of the acyl transfer from acyl-glyceraldehyde-3-phosphate dehydrogenase to phosphate. 270 38

In renal tubules isolated from fed rabbits glycerol is not utilized as a glucose precursor, probably due to the rate-limiting transfer of reducing equivalents from cytosol to mitochondria. Pyruvate and glutamate stimulated an incorporation of [14C]glycerol to glucose by 50- and 10-fold, respectively, indicating that glycerol is utilized as a gluconeogenic substrate under these conditions. Glycerol at concentration of 1.5 mM resulted in an acceleration of both glucose formation and incorporation of [14C]pyruvate and [14C]glutamate into glucose by 2- and 9-fold, respectively, while it decreased the rates of these processes from lactate as a substrate. In the presence of fructose, glycerol decreased the ATP level, limiting the rate of fructose phosphorylation and glucose synthesis. As concluded from the 'cross-over' plots, the ratios of both 3-hydroxybutyrate/acetoacetate and glycerol 3-phosphate/dihydroxyacetone phosphate, as well as from experiments performed with methylene blue and acetoacetate, the stimulatory effect of glycerol on glucose formation from pyruvate and glutamate may result from an acceleration of fluxes through the first steps of gluconeogenesis as well as glyceraldehyde-3-phosphate dehydrogenase. As inhibition by glycerol of gluconeogenesis from lactate is probably due to a marked elevation of the cytosolic NADH/NAD+ ratio resulting in a decline of flux through lactate dehydrogenase.
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PMID:Effect of glycerol on gluconeogenesis in isolated rabbit kidney cortex tubules. 290 26

The proliferation of in vitro grown Ehrlich ascites tumor cells is inhibited by pyruvate concentrations greater than 2 mM. In the presence of 4-5 mM pyruvate the growth is reduced to about 50%, in the presence of 20 mM to about 5-10%. Viability of the cells is not severely affected. Increase of DNA corresponds to the cell growth. On recultivation in pyruvate free standard medium, growth is nearly normal. Flow cytometric analyses of the proliferation kinetics of the cells in the presence of 20 mM pyruvate revealed a retardation of the passage of all phases of the cell cycle. No phase specific effects could be detected though the S- and G2M-phase are more afflicted than G1. The growth inhibition of EAT cells by pyruvate seems to depend on the presence of glucose. Exogenous pyruvate (greater than 1-2 mM) causes an activation of pyruvate dehydrogenase, a reduction of lactate production from glucose and a stimulation of lipid biosynthesis; the NAD/NADH ratio of the cells is reduced and a rise of glycolytic intermediates beyond glyceraldehyde-3-phosphate dehydrogenase is observed. Maximal activation of pyruvate dehydrogenase by non toxic concentrations of dichloroacetate is also accompanied by an inhibition of cell growth. It is suggested that an increase of glyceraldehyde-3-phosphate level and the changes in the redox state of the cells are of relevance for the inhibition of cell growth by pyruvate. 100-500 microM exogenous glyceraldehyde-3-phosphate strongly inhibited cell growth.
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PMID:Proliferation kinetics and metabolic features of in vitro grown Ehrlich ascites tumor cells in the presence of exogenous pyruvate. 294 14

A homogeneous multimeric protein isolated from the green alga, Scenedesmus obliquus, has both latent phosphoribulokinase activity and glyceraldehyde-3-phosphate dehydrogenase activity. The glyceraldehyde-3-phosphate dehydrogenase was active with both NADPH and NADH, but predominantly with NADH. Incubation with 20 mM dithiothreitol and 1 mM NADPH promoted the coactivation of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase, accompanied by a decrease in the glyceraldehyde-3-phosphate dehydrogenase activity linked to NADH. The multimeric enzyme had a Mr of 560,000 and was of apparent subunit composition 8G6R. R represents a subunit of Mr 42,000 conferring phosphoribulokinase activity and G a subunit of 39,000 responsible for the glyceraldehyde-3-phosphate dehydrogenase activity. On SDS-PAGE the Mr-42,000 subunit comigrates with the subunit of the active form of phosphoribulokinase whereas that of Mr-39,000 corresponds to that of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. The multimeric enzyme had a S20,W of 14.2 S. Following activation with dithiothreitol and NADPH, sedimenting boundaries of 7.4 S and 4.4 S were formed due to the depolymerization of the multimeric protein to NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (4G) and active phosphoribulokinase (2R). It has been possible to isolate these two enzymes from the activated preparation by DEAE-cellulose chromatography. Prolonged activation of the multimeric protein by dithiothreitol in the absence of nucleotide produced a single sedimenting boundary of 4.6 S, representing a mixture of the active form of phosphoribulokinase and an inactive dimeric form of glyceraldehyde-3-phosphate dehydrogenase. Algal thioredoxin, in the presence of 1 mM dithiothreitol and 1 mM NADPH, stimulated the depolymerization of the multimeric protein with resulting coactivation of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. Light-induced depolymerization of the multimeric protein, mediated by reduced thioredoxin, is postulated as the mechanism of light activation in vivo. Consistent with such a postulate is the presence of high concentrations of the active forms of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase in extracts from photoheterotrophically grown algae. By contrast, in extracts from the dark-grown algae the multimeric enzyme predominates.
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PMID:Properties of a multimeric protein complex from chloroplasts possessing potential activities of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase. 302 12


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