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)

The dinucleotide binding beta alpha beta motif in the crystal structures of seven different enzymes has been analysed in terms of their three-dimensional structures and primary sequences. We have identified that the hydrogen bonding of the adenine ribose to the glycine-rich turn containing the fingerprint sequence GXGXXG/A occurs via a direct or indirect mechanism, depending on the nature of the fingerprint sequence but independent of coenzyme specificity. The major determinant of the type of interaction is the nature of the residue occupying the last position of the above fingerprint. In the NAD(+)-linked dehydrogenases, an acidic residue is commonly used to form important hydrogen bonds to the adenine ribose hydroxyls and, hitherto, this residue has been thought to be an indicator of NAD+ specificity. However, on the basis of the three-dimensional structure of the NAD(+)-linked glutamate dehydrogenase (GDH) from Clostridium symbiosum we have demonstrated that this residue is not a universal requirement for the construction of an NAD+ binding site. Furthermore, considerations of sequence homology unambiguously identify an equivalent acidic residue in both NADP+ and dual specificity glutamate dehydrogenases. The conservation of this residue in these enzymes, coupled to its close proximity to the 2' phosphate implied by the necessary similarity in three-dimensional structure to C. symbiosum GDH, implicates this residue in the recognition of the 2' phosphate either via water-mediated or direct hydrogen-bonding schemes. Analysis of the latter has led us to suggest that two patterns of recognition for the 2' phosphate group of NADP(+)-binding enzymes may exist, which are distinguished by the ionization state of the 2' phosphate.
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PMID:Structural consequences of sequence patterns in the fingerprint region of the nucleotide binding fold. Implications for nucleotide specificity. 145 69

The reaction mechanism of Azospirillum brasilense glutamate synthase has been investigated by several approaches. 15N nuclear magnetic resonance studies demonstrate that the amide nitrogen of glutamine is reductively transferred to 2-oxoglutarate in an irreversible manner with no release of the transferred ammonia group into the medium. Identical results were obtained using thio-NADPH and acetylpyridine-NADPH, which are shown to be less efficient substrates of the enzyme than NADPH. Similarly, no exchange of the ammonia group being transferred with exogenous ammonium ion was observed during catalysis. The glutamate formed as the product of the iminoglutarate reduction was determined to be in the L configuration. The enzyme was also found to catalyze, under anaerobic conditions, the exchange of the 4proS H of NADPH with solvent both in the absence and in the presence of 2-oxoglutarate and glutamine. The reductive half-reaction is therefore a reversible segment of the overall irreversible amidotransferase reaction. 15N NMR studies also showed that the enzyme does not catalyze glutamate dehydrogenase/oxidase reactions or any observable glutaminase activity under neutral (pH 7.5) conditions. Glutaminase activity was also not observable with the reduced enzyme alone or in the presence of D-glutamate (a competitive inhibitor of glutamate synthase with respect to 2-oxoglutarate, with a Ki of about 11 microM) or with the oxidized enzyme in the presence of 2-oxoglutarate, D-glutamate, or NADP+. These data confirm species-dependent differences of A. brasilense glutamate synthase with respect to the enzyme from other sources.
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PMID:Mechanistic studies on Azospirillum brasilense glutamate synthase. 168 91

Two pathways serve for assimilation of ammonia in Paracoccus denitrificans. Glutamate dehydrogenase (NADP+) catalyzes the assimilation at a high NH4+ concentration. If nitrate serves as the nitrogen source, glutamate is synthesized by glutamate-ammonia ligase and glutamate synthase (NADPH). At a very low NH4+ concentration, all three enzymes are synthesized simultaneously. No direct relationship exists between glutamate dehydrogenase (NADP+) and glutamate-ammonia ligase in P. denitrificans, while the glutamate synthase (NADPH) activity changes in parallel with that of the latter enzyme. Ammonia does not influence the induction or repression of glutamate dehydrogenase (NADP+). The inner concentration of metabolites indicates a possible repression of glutamate dehydrogenase (NADP+) by the high concentration of glutamine or its metabolic products as in the case when NH4+ is formed by assimilative nitrate reduction. No direct effect of the intermediates of nitrate assimilation on the synthesis of glutamate dehydrogenase (NADP+) was observed.
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PMID:Assimilation of ammonia in Paracoccus denitrificans. 168 63

We established a simple and rapid enzymatic method for measuring potassium ion in serum by using tryptophanase (EC 4.1.99.1) purified from Escherichia coli K12 strain (E. coli K12 IFO 3301). The presence of pyridoxal 5-phosphate promotes this enzymatic reaction, and potassium and (or) ammonium ions further accelerate it, with ammonium and potassium ions providing equivalent acceleration. We eliminated endogenous ammonium ion by using glutamate dehydrogenase (GLDH; EC 1.4.1.4), then produced ammonium ion in the presence of tryptophanase, tryptophan, and pyridoxal 5-phosphate. The concentration of formed ammonium ion, which was proportional to that of potassium ion in sample, was determined by adding GLDH to produce NADP+ in the presence of 2-oxoglutarate and NADPH; we then read the change of absorbance at 340 nm. The standard curve was linear for potassium ion concentrations up to 7.00 mmol/L. The within-assay variation (CV) was 0.89% at 5.51 mmol/L and 1.32% at 3.37 mmol/L. The day-to-day CVs were 0.99% at 6.85 mmol/L and 1.71% at 3.52 mmol/L. Analytical recoveries ranged from 98.7% to 108.9%. The correlation coefficient between values obtained with this enzymatic assay (y) and by flame photometry (x) was 0.995: y = 0.984x + 0.091 mmol/L (Sy.x = 0.105, n = 100). The presence of hemoglobin, bilirubin, or other cations little affects this system.
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PMID:New enzymatic method with tryptophanase for determining potassium in serum. 173 5

In adult male and female rat liver, the activity of NAD(+)-and NADP(+)-dependent glutamate dehydrogenase (GDH) was microquantitatively measured in tissue samples of 50-150 ng, microdissected continuously along the sinusoidal length. Total activity of GDH with NAD+ as co-factor was found to be higher by a ratio of about 1:2.3 than with NADP+. All intra-acinar enzyme profiles, irrespective of sex, showed an increasing gradient of GDH activity from the periportal beginning to the perivenous end. These findings are at variance with the immunohistochemical localization of GDH in rat liver. The microquantitative GDH profiles with higher perivenous values could indicate a more pronounced glutamine synthesis in Zone 3 of the liver acinus.
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PMID:Microquantitative analysis of the intra-acinar profiles of glutamate dehydrogenase in rat liver. 185 59

The amino acid sequence is reported for CNBr and tryptic peptide fragments of the NAD(+)-dependent glutamate dehydrogenase of Clostridium symbiosum. Together with the N-terminal sequence, these make up about 75% of the total sequence. The sequence shows extensive similarity with that of the NADP(+)-dependent glutamate dehydrogenase of Escherichia coli (52% identical residues out of the 332 compared) allowing confident placing of the peptide fragments within the overall sequence. This demonstrated sequence similarity with the E. coli enzyme, despite different coenzyme specificity, is much greater than the similarity (31% identities) between the GDH's of C. symbiosum and Peptostreptococcus asaccharolyticus, both NAD(+)-linked. The evolutionary implications are discussed. In the 'fingerprint' region of the nucleotide binding fold the sequence Gly X Gly X X Ala is found, rather than Gly X Gly X X Gly. The sequence found here has previously been associated with NADP+ specificity and its finding in a strictly NAD(+)-dependent enzyme requires closer examination of the function of this structural motif.
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PMID:The partial amino acid sequence of the NAD(+)-dependent glutamate dehydrogenase of Clostridium symbiosum: implications for the evolution and structural basis of coenzyme specificity. 195 26

The amination of 2-oxoglutarate catalyzed by NADP-specific glutamate dehydrogenase (EC 1.4.1.4, L-glutamate:NADP+ oxidoreductase (deaminating)) from Halobacterium halobium has been analyzed by initial rate, graphical analysis, and product and competitive inhibition studies. Initial rate and graphical analysis reveal that a B term (representing 2-oxoglutarate) is not statistically necessary for an initial rate equation. However, the absence of a B term does not distinguish between ordered and random binding of NADPH and ammonia. The patterns of product inhibition by NADP+ and L-glutamate, and competitive inhibition by hydroxylamine and succinate permit deduction of the kinetic mechanism as ordered, with NADPH, 2-oxoglutarate and ammonia added in that order, and L-glutamate release preceding NADP+ release.
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PMID:Analysis of the kinetic mechanism of halophilic NADP-dependent glutamate dehydrogenase. 198 84

1. On transferring Clostridium symbiosum glutamate dehydrogenase from pH 7 to assay mixtures at pH 8.8, reaction time courses showed a marked deceleration that was not attributable to the approach to equilibrium of the catalysed reaction. The rate became approximately constant after declining to 4-5% of the initial value. Enzyme, stored at pH 8.8 and assayed in the same mixture, gave an accelerating time course with the same final linear rate. The enzyme appears to be reversibly converted from a high-activity form at low pH to a low-activity form at high pH. 2. Re-activation at 31 degrees C upon dilution from pH 8.8 to pH 7 was followed by periodic assay of the diluted enzyme solution. At low ionic strength (5 mM-Tris/HCl), no re-activation occurred, but various salts promoted re-activation to a limiting rate, with full re-activation in 40 min. 3. Re-activation was very temperature-dependent and extremely slow at 4 degrees C, suggesting a large activation energy. 4. 2-Oxoglutarate, glutarate or succinate (10 mM) accelerated re-activation; L-glutamate and L-aspartate were much less effective. 5. The monocarboxylic amino acids alanine and norvaline appear to stabilize the inactive enzyme: 60 mM-alanine does not promote re-activation, and, as substrates at pH 8.8 for enzyme stored at pH 7, alanine and norvaline give progress curves showing rapid complete inactivation. 6. Mono- and di-nucleotides (AMP, ADP, ATP, NAD+, NADH, NADP+, CoA, acetyl-CoA) at low concentrations (10(-4)-10(-3) M) enhance re-activation at pH 7 and also retard inactivation at pH 8.8. 7. The re-activation rate is independent of enzyme concentration: ultracentrifuge experiments show no changes in molecular mass with or without substrates. 8. The activation-inactivation appears to be due to a slow pH-dependent conformational change that is sensitively responsive to the reactants and their analogues.
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PMID:A pH-dependent activation-inactivation equilibrium in glutamate dehydrogenase of Clostridium symbiosum. 224 20

Pathways of ammonia assimilation into glutamic acid were investigated in ammonia-grown and N2-fixing Clostridium kluyverii and Clostridium butyricum by measuring the specific activities of glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. C. kluyverii had NADPH-glutamate dehydrogenase with a Km of 12.0 mM for NH4+. The glutamate dehydrogenase pathway played an important role in ammonia assimilation in ammonia-grown cells but was found to play a minor role relative to that of the glutamine synthetase/NADPH-glutamate synthase pathway in nitrogen-fixing cells when the intracellular NH4+ concentration and the low affinity of the enzyme for NH4+ were taken into account. In C. butyricum grown on glucose-salt medium with ammonia or N2 as the nitrogen source, glutamate dehydrogenase activity was undetectable, and the glutamine synthetase/NADH-glutamate synthase pathway was the predominant pathway of ammonia assimilation. Under these growth conditions, C. butyricum also lacked the activity of glucose-6-phosphate dehydrogenase, which catalyzes the regeneration of NADPH from NADP+. However, high activities of glucose-6-phosphate dehydrogenase as well as of NADPH-glutamate dehydrogenase with a Km of 2.8 mM for NH4+ were present in C. butyricum after growth on complex nitrogen and carbon sources. The ammonia-assimilating pathway of N2-fixing C. butyricum, which differs from that of the previously studied Bacillus polymyxa and Bacillus macerans, is discussed in relation to possible effects of the availability of ATP and of NADPH on ammonia-assimilating pathways.
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PMID:Ammonia assimilation pathways in nitrogen-fixing Clostridium kluyverii and Clostridium butyricum. 256 48

Activities and properties of the ammonium assimilation enzymes NADP+-dependent glutamate dehydrogenase (GDH), glutamate synthase (GOGAT) and glutamine synthetase (GS) were determined in batch and continuous cultures of Candida albicans. NADP+-dependent GDH activity showed allosteric kinetics, with an S0.5 for 2-oxoglutarate of 7.5 mM and an apparent Km for ammonium of 5.0 mM. GOGAT activity was affected by the buffer used for extraction and assay, but in phosphate buffer, kinetics were hyperbolic, yielding Km values for glutamine of 750 microM and for 2-oxoglutarate of 65 microM. The enzymes GOGAT and NADP+-dependent GDH were also assayed in batch cultures of Saccharomyces cerevisiae and three other pathogenic Candida spp.: Candida tropicalis, Candida pseudotropicalis and Candida parapsilosis. Evidence is presented that GS/GOGAT is a major pathway for ammonium assimilation in Candida albicans and that this pathway is also significant in other Candida species.
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PMID:Ammonium assimilation by Candida albicans and other yeasts: evidence for activity of glutamate synthase. 257 53


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