Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.4.1.2 (glutamate dehydrogenase)
 4,380 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In rat pancreatic islets, D-glucose in concentrations exceeding 5.6 mM caused a concentration-related decrease of the mitochondrial NADH/NAD+ ratio, as judged from the changes in the islet content of glutamate, NH4+, and 2-ketoglutarate, and assuming that the glutamate dehydrogenase reaction is near equilibrium with the mitochondrial NAD system. The concentration dependency of the response to D-glucose was vastly different in islet and parotid cells, respectively. L-Leucine, 2-ketoisocaproate, BCH (a nonmetabolized but insulinotropic analog of L-leucine) and 3-phenylpyruvate also lowered the mitochondrial NADH/NAD+ ratio. In the presence of D-glucose, the latter ratio was also decreased by NH4+ or the absence of extracellular Ca2+, but dramatically increased by aminooxyacetate. Taking into account prior metabolic findings, the nutrient-induced fall in the mitochondrial redox state is thought to reflect an increased clearance of mitochondrial NADH through both the respiratory chain and malate-aspartate shuttle. The nutrient-induced decrease in the mitochondrial NADH/NAD+ ratio might favor both the circulation of metabolites in the Krebs cycle and the exit of Ca2+ from the mitochondria.
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PMID:Hexose metabolism in pancreatic islets: regulation of the mitochondrial NADH/NAD+ ratio. 755 20

This review is an exhaustive description of the biochemistry and enzymology of all 17 known NAD(P)(+)-amino acid dehydrogenases. These enzymes catalyze the oxidative deamination of an amino acid to its keto acid and ammonia, with the concomitant reduction of either NAD+ or NADP+. These enzymes have many important applications in industrial and medical settings and have been the object of prodigious enzymological research. This article describes all that is known about the poorly characterized members of the family and contains detailed information on the better characterized enzymes, including valine, phenylalanine, leucine, alanine, and glutamate dehydrogenases. The latter three enzymes have been the subject of extensive enzymological experimentation, and, consequently, their chemical mechanisms are discussed. The three-dimensional structure of the Clostridium symbiosum glutamate dehydrogenase has been determined recently and remains the only structure known of any amino acid dehydrogenase. The three-dimensional structure and its implications to the chemical mechanisms and rate-limiting steps of the amino acid dehydrogenase family are discussed.
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PMID:The biochemistry and enzymology of amino acid dehydrogenases. 770 1

125I-N6-(N-[6-N-(5-iodo-4-azidosalicyl)-aminohexyl]- aminocarbamoylmethyl)-nicotinamide adenine dinucleotide (125I-N6-I-ASA-AH-NAD+) was synthesized by coupling N6-([6-aminohexyl]-carbamoylmethyl)-NAD+ with 4-azidosalicylic acid N-hydroxysuccinimide ester followed by radioiodination. The utility of 125I-N6-I-ASA-AH-NAD+ as an effective site-directed photoprobe was demonstrated by the photolabeling of both glutamate dehydrogenase and 15-hydroxyprostaglandin dehydrogenase. Both enzymes can be saturated with labeled probe with apparent dissociation constants comparable to those reported for NAD+. Photoincorporation of the probe into both enzymes was found to be protected specifically by NAD+. These results indicate that 125I-N6-I-ASA-AH-NAD+ can be a specific photoprobe for NAD(+)-linked enzymes.
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PMID:An 125I-labeled N6-substituted azido analog of NAD+ for the photoaffinity labeling of NAD(+)-linked enzymes. 780 Jul 17

The time-course of reaction between Ellman's reagent (DTNB) and clostridial glutamate dehydrogenase has been investigated over a wide range of reagent concentrations (50-5000 microM) and showed pseudo-first-order kinetics throughout. The reaction was followed both by monitoring loss of enzyme activity and by detection of released thionitrobenzoate through its absorbance at 412 nm, and, when both methods were used for the same DTNB concentration, the pseudo-first-order rate constants were identical within experimental error, suggesting that the two methods detect the same process. The dependence of the rate constants on DTNB concentration clearly shows saturation, with a limiting value of 1.62 x 10(-3) s-1 and a dissociation constant of 1.0 mM governing the formation of the implied non-covalent enzyme-DTNB complex. This information has allowed a detailed analysis of the protection of the enzyme by NAD+, yielding a value of 334 microM for the dissociation constant for the enzyme-coenzyme binary complex. In view of the convenience of protection studies as a means of determining dissociation constants, this study emphasizes the importance of establishing whether a chemical modification reaction follows simple first-order kinetics with respect to the chemical reagent.
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PMID:Initial formation of a non-covalent enzyme-reagent complex during the inactivation of clostridial glutamate dehydrogenase by Ellman's reagent: determination of the enzyme's dissociation constant for the binary complex with NAD+ from protection studies. 781 94

The effect of polyamines on glutamate dehydrogenase [L-glutamate: NAD(P) oxidoreductase (deaminating) [EC 1.4.1.3]) activity has been studied in both permeabilized kidney-cortex mitochondria and isolated renal tubules of rabbit. Spermidine was the most potent inhibitor of glutamate synthesis in permeabilized mitochondria resulting in about 80% decrease of the enzyme activity at 5 mM concentration. Putrescine, alpha-monofluoromethylputrescine (MFMP) and (R,R)-delta-methyl-alpha-acetylenic-putrescine (MAP) were more efficient than spermine. The inhibitory action of polyamines was potentiated by an elevated NADH content in the reaction mixture. Increasing concentrations of either NH4Cl, KCl or NaCl in the incubation medium resulted in a decrease of polyamine-induced inhibition of the enzyme activity, indicating that monovalent cations can compete with polyamines for the binding site at glutamate dehydrogenase. The inhibitory action of spermidine on glutamate synthesis was abolished by 2 mM ADP or 10 mM L-leucine, allosteric activators of the enzyme, as well as on the addition of either oxalate or sulphate at 20 mM concentrations. Spermidine did not affect glutamate formation when NADH was substituted by NADPH, suggesting an importance of the NADH binding to the inhibitory site of the enzyme for a decrease of reductive amination of 2-oxoglutarate by polyamine. Although spermidine did not influence glutamate deamination in the presence of NAD+, it stimulated this process by about 70% when NAD+ was substituted by NADP+. In the presence of ADP the stimulatory effect of polyamine was not significant. The data indicate that in permeabilized rabbit kidney-cortex mitochondria the effect of polyamines on both glutamate formation and glutamate deamination via the reaction catalysed by glutamate dehydrogenase is dependent upon the coenzyme utilized by the enzyme. In the presence of NADH their inhibitory effect on the glutamate formation may be alleviated by allosteric activators of the enzyme, and concentrations of potassium, sodium, sulphate and oxalate. In isolated rabbit renal tubules incubated with 5 mM methionine sulfoximine and aminooxyacetate, in order to inhibit glutamine synthetase and aminotransferases, respectively, 5 mM spermidine decreased glutamate formation by about 30%, while putrescine and spermine did not significantly diminish the enzyme activity. In the presence of octanoate glutamate formation was reduced by about 30% by naturally occurring polyamines as well as MFMP and MAP, indicating that under these conditions NADH rather than NADPH is utilized as the coenzyme. In view of these data it is possible to suggest that polyamines may be of importance to control glutamate dehydrogenase activity under physiological conditions.
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PMID:Effect of polyamines on glutamate dehydrogenase within permeabilized kidney-cortex mitochondria and isolated renal tubules of rabbit. 791 Apr 59

A putative catalytic aspartyl residue, Asp-165, in the active site of clostridial glutamate dehydrogenase has been replaced with serine by site-directed mutagenesis. The mutant enzyme is efficiently overexpressed in Escherichia coli as a soluble protein and can be successfully purified by the dye-ligand chromatographic procedure normally employed for the wild-type enzyme. By several criteria, including circular dichroism spectrum, sulphydryl reactivity with Ellman's reagent, crystallization and mobility in non-denaturing electrophoresis, the enzyme appears to be correctly folded. NAD+ protects the D165S mutant against modification by Ellman's reagent, suggesting unimpaired binding of coenzyme. In standard assays the specific activity is decreased 10(3)-fold in the reductive amination reaction and 10(5)-fold for oxidative deamination. Kinetic studies show that apparent Km values for NADH and 2-oxoglutarate are almost unchanged. The large reduction in the reaction rate coincides with a weakening of the affinity for ammonium ion (Km > 300 mM, compared with 60 mM for the wild-type). The data are entirely consistent with the direct involvement of D165 in catalysis rather than in the binding of coenzyme or 2-oxoglutarate.
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PMID:The catalytic role of aspartate in the active site of glutamate dehydrogenase. 803 59

The binding of NAD+ and L-Glutamate to glutamate dehydrogenase (GDH) from Clostridium symbiosum has been investigated by stopped-flow fluorescence spectroscopy. The formation of the binary complexes produces little change in the protein fluorescence but formation of the ternary complex results in quenching of its fluorescence with a maximum value of 40%. This finding, coupled with the finding that a step prior to hydride transfer but subsequent to ternary complex formation is rate limiting, has enabled us to monitor the kinetics of ternary complex formation in detail. The ternary complex can be formed via the GDH-NAD+ or the GDH-L-Glu binary complexes, but the route via the GDH-NAD+ binary complex is the preferred pathway. The equilibrium and rate constants for the formation of the two binary complexes and the ternary complex formed via the two possible pathways have been determined. These studies have revealed an interaction between the coenzyme-binding site and the substrate-binding site, which lead to a decrease in the binding constant for the second substrate binding to the enzyme. The free energy coupling between the binary and ternary complexes is about 2.4-2.8 kJ.mol-1. We propose that there is a further isomerisation of the ternary complex, which is rate limiting for the steady-state turnover of the enzyme. Formation of this complex is characterised by an increased negative interaction, with a free energy coupling between these complexes of 6.3-11.6 kJ.mol-1.
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PMID:The mechanism of substrate and coenzyme binding to clostridial glutamate dehydrogenase during oxidative deamination. 809 28

Protein chemical studies of NAD(+)-dependent glutamate dehydrogenase (GDH; EC 1.4.1.2) from Clostridium symbiosum indicate only two cysteine residues/subunit, in good agreement with the gene sequence. Experiments with various thiol-modifying reagents reveal that in native clostridial GDH only one of these two cysteines is accessible for reaction. This residue does not react with iodoacetate, iodoacetamide, N-ethylmaleimide or N-phenylmaleimide, but reaction with either p-chloromercuribenzene sulphonate or 5,5'-dithiobis(2-nitrobenzoic acid) causes complete inactivation, preventable by NAD+ or NADH but not by glutamate or 2-oxoglutarate. Protection studies with combinations of substrates show that glutamate enhances protection by NADH, whereas 2-oxoglutarate diminishes it. These studies were also used to determine a dissociation constant (0.69 mM) for the enzyme-NAD+ complex. Similar data for NADH indicated mildly cooperative binding with a Hill coefficient of 1.32. The significance of these results is discussed in the light of the high-resolution crystallographic structure for clostridial GDH and in relation to information for GDH from other sources.
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PMID:Site and significance of chemically modifiable cysteine residues in glutamate dehydrogenase of Clostridium symbiosum and the use of protection studies to measure coenzyme binding. 812 8

Covalent adducts of NAD+ with pyruvate and 2-oxoglutarate have been reported to inhibit differentially the activities of bovine glutamate dehydrogenase (GDH) towards these two oxoacid substrates, implying separate active sites. Thorough reinvestigation fails to confirm this finding, with the pyruvate adduct uniformly the more potent inhibitor of both substrate activities under several assay conditions. This suggests that bovine GDH provides amino acid dehydrogenation sites of one structural type only. Clostridial GDH, with a strong preference for oxoglutarate over pyruvate as substrate, is also more strongly inhibited by the pyruvate adduct in the oxoglutarate assay. These findings challenge the generality of the view that carbonyl substrates used in forming such adducts confer specificity for the corresponding substrate binding pocket in enzyme active sites.
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PMID:Inhibition of glutamate dehydrogenase by covalent coenzyme-substrate adducts: a re-examination. 836 10

An investigation on the relative presence of some protein metabolic enzymes, namely aspartate aminotransferase (AST), alanine aminotransferase (ALT), NAD+ and NADP+ dependent glutamate dehydrogenase (GLDH) and arginase in cyst wall (CW), cyst fluid (CF) and zoite (ZT) fractions of the sarcocysts of Sarcocystis fusiformis in the oesophageal muscles of Indian water buffalo was carried out. Both the transaminases were present in all the fractions of the cyst, although in variable amounts. There was a higher level of AST activity than of ALT activity. AST activity was the highest in ZT, whereas ALT activity was at a maximum in the CF fraction. The levels of activity of NAD+ and NADP+ dependent GLDH and arginase remained beyond detectable limits. The study revealed that the intermediates of carbohydrate metabolism are linked to protein metabolism by transaminases. The possibility of concomitant removal of ammonia and its subsequent incorporation into the urea cycle is ruled out in this parasitic protozoan.
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PMID:Sarcocystis fusiformis: some protein metabolic enzymes in various fractions of sarcocysts of buffalo (Bubalus bubalis). 844 61


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