Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.4.3.11 (glutamate dehydrogenase)
4,437 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An oxidized nicotinamide adenine dinucleotide phosphate/oxidized nicotinamide adenine dinucleotide (NADP+/NAD+) nonspecific L-glutamate dehydrogenase from Bacteroides thetaiotaomicron was purified 40-fold (NADP+ or NAD+ activity) over crude cell extract by heat treatment, (NH4)2SO2 fractionation, diethylaminoethyl-cellulose, Bio-Gel A 1.5m, and hydroxylapatite chromatography. Both NADP+- and NAD+-dependent activities coeluted from all chromatographic treatments. Moreover, a constant ratio of NADP+/NAD+ specific activities was demonstrated at each purification step. Both activities also comigrated in 6% nondenaturing polyacrylamide gels. Affinity chromatography of the 40-fold-purified enzyme using Procion RED HE-3B gave a preparation containing both NADP+- and NAD+-linked activities which showed a single protein band of 48,5000 molecular weight after sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The dual pyridine nucleotide nature of the enzyme was most readily apparent in the oxidative direction. Reductively, the enzyme was 30-fold more active with reduced NADP than with reduced NAD. Nonlinear concave 1/V versus 1/S plots were observed for reduced NADP and NH4Cl. Salts (0.1 M) stimulated the NADP+-linked reaction, inhibited the NAD+-linked reaction, and had little effect on the reduced NADP-dependent reaction. The stimulatory effect of salts (NADP+) was nonspecific, regardless of the anion or cation, whereas the degree of NAD+-linked inhibition decreased in the order to I- greater than Br- greater than Cl- greater than F-. Both NADP+ and NAD+ glutamate dehydrogenase activities were also detected in cell extracts from representative strains of other bacteroides deoxyribonucleic acid homology groups.
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PMID:Characterization of a pyridine nucleotide-nonspecific glutamate dehydrogenase from Bacteroides thetaiotaomicron. 736 28

To investigate the mechanisms of the antiammoniagenic effect of ketone bodies, acidotic dogs (NH4Cl) were infused with either beta-hydroxybutyrate or acetoacetate. Total blood ketones ranged from 2 to 4 mM. Renal ammoniagenesis fell by a mean of 53%, with a proportional decrease in glutamine extraction. Glutamate release in the renal vein rose, renal extraction of lactate fell, and aspartate and alanine production decreased. Study of the metabolite profile of the renal cortex by the freeze-clamp technique before and after ketone infusion showed that tissue glutamine concentration was unchanged, whereas glutamate, alpha-ketoglutarate, malate, and citrate rose. The intermediates of the gluconeogenic pathway, such as phosphoenolypyruvate, 2-phosphoglycerate, 3-phosphoglycerate, and glucose-6-phosphate, fell significantly. The redox state as calculated from the free NAD+/NADH ratios in the cytosolic (lactate dehydrogenase) and the mitochondrial (glutamate dehydrogenase and beta-hydroxybutyrate dehydrogenase) compartments was reduced. The present study suggests that ketone bodies inhibit renal ammoniagenesis through increased generation of alpha-ketoglutarate (metabolic or bicarbonate effect) and a decrease in the mitochondrial and cytosolic redox potentials in the kidney.
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PMID:Cellular mechanisms of the antiammoniagenic effect of ketone bodies in the dog. 743 17

1. The binding of NAD+ to glutamate dehydrogenase may be followed quantitatively by titration, using high-sensitivity circular dichroism (CD) difference spectroscopy. 2. The CD of the bound coenzyme in the binary complex E . NAD closely resembles that of bound ADP, although the affinity is much lower, being 350-fold less for NAD+ at 20 degrees C in 0.1 M phosphate, pH 7. 3. A family of CD spectra may be analysed by unconstrained linear regression assuming only three components: free enzyme, free coenzyme, and a single binary complex, E . NAD. 4. Taking the molar CD of bound ADP as representing the molar CD of the adenine chromophore of bound NAD+, the linear regression shows the formation of a simple 1 : 1 complex E . NAD with Kd = 0.72 mM in a simple binding process without positive or negative cooperativity. 5. NADP+ binding is more than 10-fold weaker than NAD+ binding. 6. From the similarity of the CD of bound ADP and bound NAD+ it is probable that NAD+, in forming a simple binary complex, binds preferentially at the regulatory (adenine nucleotide) binding site (site II). 7. Direct evidence has been obtained for the binding of a second molecule of NAD+ to the ternary complex E . NAD . glutarate. This process occurs with low affinity and is probably also located at the adenine regulatory site. 8. This second-site binding of NAD+ may contribute to the phenomena of non-Michaelis-Menten kinetics and apparent negative homotropic interactions in the binding of NAD+, previously attributed to subunit-subunit cooperative interactions.
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PMID:The binding of oxidised coenzyme to bovine-liver glutamate dehydrogenase studied by circular-difference spectroscopy. 746 Sep 36

Glutamate dehydrogenases from many sources display nonclassical kinetic behavior suggestive of allosteric interaction among the six subunits of the hexamer. A three-dimensional structure now potentially offers a framework for explaining the basis of such behavior in clostridial glutamate dehydrogenase, and this paper offers evidence of extreme, all-or-none cooperativity in the binding of glutamate by this enzyme. A site-directed mutant of clostridial glutamate dehydrogenase in which Ala163 in the glutamate binding site is replaced by glycine displays a markedly sigmoid dependence of reaction rate on glutamate concentration (S0.5 = 200 mM), with a Hill coefficient of 3.4 when assayed at pH 10.5 with 1 mM NAD+. Under the same conditions the wild-type enzyme gave no measurable rate with glutamate concentrations in the range normally used for kinetics (0-100 mM) but gave a steep rise in reaction rate from 600 to 1200 mM glutamate. At pH 9.0, where the wild-type enzyme has previously been shown to be "inactive" in a standard assay, a study extending to much higher glutamate concentrations again revealed a sigmoid dependence, with a Hill coefficient of 5.4 and an S0.5 at 150 mM glutamate. With the mutant A163G the apparent cooperativity was less, with a Hill coefficient of 2.3, and the affinity for glutamate was higher, with S0.5 of 7 mM. Both proteins gave normal hyperbolic dependence on glutamate concentration at pH 7 and pH 8. At pH 9 and with saturating glutamate, both enzymes showed a hyperbolic dependence of the rate on NAD+ concentration. The NAD+ concentration, however, affected the observed degree of cooperativity with varied glutamate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Positive cooperativity with Hill coefficients of up to 6 in the glutamate concentration dependence of steady-state reaction rates measured with clostridial glutamate dehydrogenase and the mutant A163G at high pH. 754 69

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


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