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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)

A mutant of Bacillus stearothermophilus D-glyceraldehyde-3-phosphate dehydrogenase, Ser148----Ala, was produced by oligonucleotide-directed mutagenesis. The study of the catalytic properties of this mutant has shown that this mutation significantly affects the Michaelis constant of inorganic phosphate and to a lesser extent that of 1,3-diphosphoglycerate and D-glyceraldehyde-3-phosphate. This result is consistent with model-building studies which show that, for the phosphorylation step of catalysis, inorganic phosphate must bind to the anion recognition site designated Pi with the C(3) phosphate of the acyl-enzyme intermediate in the alternative anion site Ps. Studies of the enantiomeric specificity using D- and L-glyceraldehyde as substrates show that the hydroxyl group of Ser148, combined with the presence of the C(3) phosphate of the substrate, enhances stereospecificity as well as catalysis. However, the stereospecific effect cannot be a consequence of the direct interaction of Ser148 with the C(2)-hydroxyl of the substrate. The changed Km for glyceraldehyde-3-phosphate suggests that the initial step of hemithioacetal formation may take place with its C(3) phosphate bound in the Pi site. This supports the molecular mechanism proposed by Moody (1984). Therefore, catalysis could be enhanced through interactions of the serine hydroxyl group not only with inorganic phosphate but also with the C(3) phosphate of glyceraldehyde-3-phosphate.
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PMID:Site-directed mutagenesis of glyceraldehyde-3-phosphate dehydrogenase reveals the role of residue Ser148. 250 80

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.
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PMID:Stereospecificity of C4 nicotinamide hydrogen transfer of the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase. 252 66

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 binding of a spin-labeled AMP analog to tetrameric glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle is described. The spin label, perdeuterated and 15N-substituted 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, was attached to C-8 of AMP (C8-SL-AMP). Up to 8 equivalents of C8-SL-AMP bind per enzyme tetramer, i.e., 2 per monomer. Combining sites are the adenine subsite of the coenzyme-binding domain and the phosphate site. Glyceraldehyde 3-phosphate causes a conformational change in the enzyme that brings C8-SL-AMP molecules bound to adjacent R-axis-related subunits closer to one another by 0.2-0.3 nm and allows for spin-spin interaction between the nitroxide radicals. Similar, but less pronounced structural changes take place upon lowering the pH from 8 to 7. Addition of a single equivalent of NAD+ to a complex of the enzyme with 7.6 equivalents of C8-SL-AMP leads to the release of almost 4 C8-SL-AMP molecules. This supports our previous findings that binding of just one NAD+ molecule induces conformational changes in all four subunits.
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PMID:Interaction of glyceraldehyde-3-phosphate dehydrogenase with AMP as studied by means of a spin-labeled analog. 255 43

Ornithine uptake by rat kidney mitochondria is here first shown by monitoring the reduction of the intramitochondrial pyridine nucleotides which occurs as a result of metabolism of imported ornithine via ornithine aminotransferase and 1-pyrroline-carboxylate dehydrogenase. Ornithine uptake shows saturation features (Km and Vmax values, measured at 20 degrees C and at pH 7.20, were found to be about 0.85 mM and 23 nmoles/min x mg protein, respectively) and proves to be inhibited by D-ornithine, inorganic phosphate, praseodimium chloride and mersalyl. Neither malate nor glutamate, but phosphate was found to exchange with ornithine. Phosphate efflux caused by externally added ornithine was shown both as revealed by a c colorimetric assay and as continuously monitored by measuring extramitochondrial reduction of NAD+ in the presence of glyceraldehyde-3-phosphate, glyceraldehyde-3-phosphate dehydrogenase, ADP and 3-phosphoglycerate kinase. The role of ornithine carrier in kidney metabolism will also be discussed.
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PMID:Metabolite transport in rat kidney mitochondria: ornithine/phosphate translocator. 256 42

Glycolytic enzymes have been observed to associate in vitro with membranes and cytoplasmic filaments in a variety of systems, but their distribution in vivo is contested. We have therefore examined the distribution of glyceraldehyde-3-phosphate dehydrogenase (G3PD) in the intact human erythrocyte using indirect immunofluorescence and affinity-purified rabbit antibodies to G3PD. Antibody specificity was demonstrated by immunoblotting as well as immunofluorescence experiments with ghosts specifically depleted of and reconstituted with G3PD. Anti-G3PD immunolabeling experiments utilized both fixed whole cells and fixed cell suspensions infused with 2.3 M sucrose, frozen and thick-sectioned. In all experiments a two-step fixation protocol was employed which ensured that cytoplasmic hemoglobin was retained when cells were subjected to Triton X-100 permeabilization, the anti-genicity of G3PD was preserved, and antibody penetration was complete. We used mixtures of biotinylated affinity-purified antibodies to G3PD and dichlorotriazinylaminofluorescein-labeled, affinity-purified antibodies to hemoglobin, followed by rhodamine-streptavidin, in double-label experiments. In both whole and sectioned human erythrocytes, G3PD staining was predominantly membrane associated while hemoglobin staining was diffusely distributed throughout the cytoplasm. In isolated ghosts, some G3PD was tightly bound to the membrane and was resistant to elution with phosphate-buffered saline and NAD+/arsenate. However, in immunolabeled rat reticulocytes and erythrocytes G3PD was cytoplasmic. Nucleated human blood cells and platelets also exhibited cytoplasmic G3PD. In approximately 10% of the human erythrocyte population G3PD was also cytoplasmic. These cells were flatter in shape and exhibited strong cytoplasmic immunolabeling for hemoglobin which was sometimes concentrated along the cell membrane; possibly, these cells were late reticulocytes or early erythrocytes. We conclude that G3PD is preferentially associated with the plasma membrane of human erythrocytes in a specific fashion.
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PMID:Association of glyceraldehyde-3-phosphate dehydrogenase with the plasma membrane of the intact human red blood cell. 264 1

The catalytically essential amino acid, histidine 176, in the active site of Escherichia coli glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been replaced with an asparagine residue by site-directed mutagenesis. The role of histidine 176 as a chemical activator, enhancing the reactivity of the thiol group of cysteine 149, has been demonstrated, with iodoacetamide as a probe. The esterolytic properties of GAPDH, illustrated by the hydrolysis of p-nitrophenyl acetate, have been also studied. The kinetic results favor a role for histidine 176 not only as a chemical activator of cysteine 149 but also as a hydrogen donor facilitating the formation of tetrahedral intermediates. These results support the hypothesis that histidine 176 plays a similar role during the oxidative phosphorylation of glyceraldehyde 3-phosphate.
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PMID:Role of the histidine 176 residue in glyceraldehyde-3-phosphate dehydrogenase as probed by site-directed mutagenesis. 265 73

Several enzymes active in the presence of NAD with acetaldehyde and propionaldehyde have been purified from human brain and characterized. The enzymes have been identified as aldehyde dehydrogenase (EC 1.2.1.3), NAD-linked succinic semialdehyde dehydrogenase (EC 1.2.1.24), and glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12). Glyceraldehyde-3-phosphate dehydrogenase is extremely heterogeneous with some isozymes active with acetaldehyde, others inactive. The cytoplasmic enzyme, which is the classical glyceraldehyde-3-phosphate dehydrogenase, is inactive with acetaldehyde as substrate; the isozymes that are active with short chain aliphatic aldehydes are localized in the mitochondrial fraction. Properties of glyceraldehyde-3-phosphate dehydrogenase isozymes with respect to short chain aliphatic aldehydes and inhibition by disulfiram are described. Their Km values for acetaldehyde range from 300 to 2000 microM. All glyceraldehyde-3-phosphate dehydrogenases that are active with acetaldehyde are easily inactivated by low concentrations of disulfiram. In all cases activity regain can be obtained with 2-mercaptoethanol; in the case of two glyceraldehyde-3-phosphate isozymes (E8.5 and 9.0), activity can also be regained with cysteine and with glutathione; activity of E6.6 and E6.8 glyceraldehyde-3-phosphate dehydrogenases could not be regained with 33 microM cysteine or glutathione. Succinic semialdehyde dehydrogenase and aldehyde dehydrogenase (EC 1.2.1.3) were also inhibited by disulfiram; their activity could be regained with 2-mercaptoethanol but not with 33 microM cysteine or glutathione. Comparison of human brain succinic semialdehyde dehydrogenase and aldehyde dehydrogenase with glyceraldehyde-3-phosphate dehydrogenase shows that the activity with short chain aldehydes is not unique to aldehyde dehydrogenase; neither is sensitivity to disulfiram; activity with 3,4-dihydroxyphenylacetaldehyde appears to be a unique property of aldehyde dehydrogenase (EC 1.2.1.3).
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PMID:Human brain glyceraldehyde-3-phosphate dehydrogenase, succinic semialdehyde dehydrogenase and aldehyde dehydrogenase isozymes: substrate specificity and sensitivity to disulfiram. 269 Jun 58

1. Human erythrocytes were incubated in autologous plasma containing [32P]Pi, and sampled by a method which avoids washing the cells. 2. In experiments of up to 3 h duration, the specific radioactivity of cellular Pi stabilized at a value below that of extracellular Pi. This can be explained on the basis of a single cellular Pi pool exchanging with a large unlabelled pool of cellular organic phosphates. 3. However, a rapid initial phase of labelling, occurring within 30 s, was inconsistent with the situation described in point 2. A possible explanation is that about 1/4 of cellular Pi occurs in a separate, fast-labelling pool. 4. When the extracellular Pi concentration was doubled, most of the corresponding increase in the steady-state cellular Pi concentration was accounted for by the apparent fast-labelling Pi pool, which also doubled. 5. The observed initial rate of labelling of cellular organic phosphates [which probably occurs through the reaction catalysed by glyceraldehyde-3-phosphate dehydrogenase (E.C. 1.2.1.12)] was considerably lower than that predicted from the flux through the Embden-Meyerhof pathway. This implies that the enzyme is exposed to Pi whose specific radioactivity is lower than the mean specific radioactivity of cellular Pi, and fails to support earlier suggestions that this enzyme uses extracellular Pi. 6. In 3 h incubations, the rate of organic phosphate labelling was roughly constant throughout, even though the specific radioactivity of cellular Pi had risen slowly to a plateau. Viewed in conjunction with point 5, this again suggests some inhomogeneity in cellular Pi. 7. Cellular Pi and extracellular Pi only reached isotopic steady state after 2 days. At this stage some organic phosphates were probably still incompletely labelled. 8. We conclude that, whatever their physical or technical reasons, such labelling inhomogeneities and slow attainment of isotopic steady state may cause serious misinterpretation of results if ignored during 32P-labelling of intact cells.
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PMID:32P-labelling anomalies in human erythrocytes. Is there more than one pool of cellular Pi? 269 64

The analogue of NAD+, 4-chloroacetylpyridine-adenine dinucleotide (clac4PdAD+), inactivated the glyceraldehyde-3-phosphate dehydrogenase from sturgeon at a high rate. An affinity labeling was shown to occur with clac4PdAD+. The mononucleotide 4-chloroacetylpyridine 1-beta-D-ribose 5'-phosphate (clac4PdMN+) reacted with the enzyme in a second-order reaction whose rate was much smaller than that calculated for clac4PdAD+ taken as a second-order rate reagent. The rate of the reaction of clac4PdAD+ with the enzyme was determined by stopped flow, using as a probe the long-wavelength absorption maximum (430 nm) formed concomitantly with inactivation of the enzyme. Computer-assisted graphic simulation showed that the clac4PdAD+ analogue could bind to the active site of the enzyme from Bacillus stearothermophilus in a similar manner to that of NAD+, and that the reactive carbon and the reactive thiolate of Cys-149 were within bonding distance. The absorption at 430 nm was linearly proportional to the substoichiometric concentration of clac4PdAD+/mole subunit. Thiol titration suggested the modification of one thiol residue per subunit. The modified thiol was identified by degradation as Cys-149. In contrast to the absorption band generated during the reaction of the 3-chloroacetylpyridine-adenine dinucleotide (clac3PdAD+) with the same enzyme [Eur. J. Biochem. (1982) 127, 519-524; 129, 437-446], enzyme inactivated with clac4PdAD+ and clac4PdMN+ exhibited an absorption maximum at long wavelength which was still present after denaturation. The chromophore is proposed to be the enol form of the alpha-thioether ketone produced by alkylation of the thiolate of Cys-149 by the chloroacetyl group.
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PMID:4-Chloroacetylpyridine adenine dinucleotide. A highly reactive and chromophoric affinity label of glyceraldehyde-3-phosphate dehydrogenase from sturgeon. 271 79


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