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)

The glutamate dehydrogenase-NADPH-alpha-ketoglutarate complex, an active intermediate on the reaction pathway has a number of unusual properties: 1) it is the only blue-shifted natural complex of this enzyme; 2) it has an anomalously slow rate of dissociation; 3) its off-rate shows a substantial pH-independent D2O solvent isotope effect not exhibited by any other ternary complex of this enzyme; and 4) it has an unusually large enthalpy of interaction parameter. These properties must be ascribable to at least one of the two possibilities conferred on the complex by the presence of the alpha-carbonyl group of alpha-ketoglutarate; the ability to engage in carbonyl addition reactions; and/or the ability to form a specific hydrogen bond. Oxalylglycine, a competitive inhibitor of alpha-ketoglutarate in this enzyme-catalyzed reaction, provides a means of discriminating between these two modes of action. The structure of oxalylglycine provides a dicarboxylic compound which has the same intercarboxylate proton distance and has a carbonyl group in a position spatially analogous to that of alpha-ketoglutarate. Its carbonyl group, however, is that of an amide group and cannot, therefore, engage in carbonyl addition reactions, but can hydrogen bond. Therefore, any effects observed with both oxalylglycine and alpha-ketoglutarate must be ascribed to formation of specific alpha-carbonyl hydrogen bonding, whereas any effects observed with alpha-ketoglutarate alone must be due to an alpha-carbonyl addition reaction. We have used this logic to test the source of the four phenomena listed above. In each case, oxalylglycine and alpha-ketoglutarate showed the same effect. Therefore, we conclude that all four phenomena are in fact due to the formation of a specific alpha-carbonyl hydrogen bond and that the specific carbonyl addition reaction between alpha-ketoglutarate and an enzyme lysine group, postulated in one proposed catalytic mechanism, does not occur.
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PMID:The anomalous properties of the glutamate dehydrogenase-NADPH-alpha-ketoglutarate complex are not ascribable to a carbonyl addition reaction. 304 Jul 12

Lysine-ketoglutarate reductase was purified 675-fold from bovine liver mitochondria. Product inhibition studies gave results similar to those reported for this enzyme extracted from other sources. Inhibition studies with L-citrulline exhibited mixed inhibition patterns. No inhibition of the partially-purified enzyme by ammonium salts was detected; in contrast, marked inhibition of the enzyme by ammonium was apparently observed in crude liver homogenates. This was probably due to depletion of NADPH and/or 2-oxoglutarate in the assay mixture as a result of conversion of ammonium to glutamate by glutamate dehydrogenase. A similar explanation could account for the high levels of lysine observed in humans with urea cycle disorders.
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PMID:Inhibition of bovine liver lysine-ketoglutarate reductase by urea cycle metabolites and saccharopine. 313 24

A highly conserved lysine at position 128 of Escherichia coli glutamate dehydrogenase (GDH) has been altered by site-directed mutagenesis of the gdhA gene. Chemical modification studies have previously shown the importance of this residue for catalytic activity. We report the properties of mutants in which lysine-128 has been changed to histidine (K128H) or arginine (K128R). Both mutants have substantially reduced catalytic centre activities and raised pH optima for activity. K128H also has increased relative activity with amino acid substrates other than glutamate, especially L-norvaline. These differences, together with alterations in Km values, Kd values for NADPH and Ki values for D-glutamate, imply that lysine-128 is intimately involved in either direct or indirect interactions with all the substrates and also in catalysis. These multiple interactions of lysine-128 explain the diverse effects of chemical modifications of the corresponding lysine in homologous GHDs. In contrast, lysine-27, another highly reactive residue in bovine GDH, is not conserved in all of the sequenced NADP-specific GDHs and is therefore not likely to be involved in catalysis.
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PMID:Multiple interactions of lysine-128 of Escherichia coli glutamate dehydrogenase revealed by site-directed mutagenesis studies. 314 42

Modification of glutamate dehydrogenase with 3,4,5,6-tetrahydrophthalic anhydride at pH 8.0 results in the progressive loss of enzymatic activity and a concomitant increase in the negative charge of the protein. Although the rate of inactivation at room temperature is too rapid to allow accurate rate constant determination, modification at 4 degrees C shows that the pseudo-first-order rate constant for inactivation appears to show a saturation effect with increasing reagent concentration, with a maximum of approximately 1 min-1. Control experiments showed that tetrahydrophthalic anhydride was hydrolyzed at a much slower rate, with a pseudo-first-order rate constant of 0.041 min-1. Protection studies indicated that inactivation was decreased by the active site ligands, NADP and 2-oxoglutarate. The extents of inactivation, whether assayed with glutamate at pH 7.0 or norvaline at pH 8.0, were the same. Changes in mobility on native gels and isoelectric point were used to follow the incorporated negative charge resulting from modification. Enzyme modified in the presence of protecting ligands (where activity is maintained) showed mobility changes which suggested that a single site of modification was protected. Modified enzyme incorporated 0.78 mol pyridoxal 5-phosphate less than native enzyme, consistent with modification of lysine-126. Enzyme modified under limiting conditions was shown to have a quaternary structure similar to that of the native enzyme, as judged by crosslinking patterns obtained with dimethylpimelimidate. The modified protein is readily resolved from unmodified protein using an NaCl double gradient elution from DEAE-Sephacel. The modification is reversed with regain of activity by incubation of the modified enzyme at low pH. We have made use of the recently demonstrated ability of guanidine hydrochloride to dissociate the hexamer of glutamate dehydrogenase into trimers that can then be reassociated to construct heterohexamers of glutamate dehydrogenase, in which one trimer of the heterohexamer contains native subunits while the other has been inactivated by the 3,4,5,6-tetrahydrophthalic anhydride modification. The heterohexamer is separated from either native or fully modified hexamers by DEAE-Sephacel chromatography. Significantly, the heterohexamer has little detectable catalytic activity, although activity is regained by reversal of the modification of the one modified trimer in the hexamer. This demonstrates that catalytic site cooperation between trimers in the hexamer of glutamate dehydrogenase is an essential component of the enzymatic activity of this enzyme.
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PMID:3,4,5,6-Tetrahydrophthalic anhydride modification of glutamate dehydrogenase: the construction and activity of heterohexamers. 337 6

6-[(4-Bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate (6-BDB-TADP) has been shown to react at the reduced diphosphopyridine nucleotide (DPNH) inhibitory site of bovine liver glutamate dehydrogenase with incorporation of 1 mol of reagent/mol of enzyme subunit [Batra, S. P., & Colman, R. F. (1984) Biochemistry 23, 4940-4946]. The modified enzyme had lost one of the six free sulfhydryl groups per enzyme subunit as detected by 5,5'-dithiobis(2-nitrobenzoate). In the unmodified enzyme digested with trypsin, six cysteinyl peptides labeled with [14C]iodoacetic acid were detected by high-performance liquid chromatography (HPLC), whereas only five were observed in the 6-BDB-TADP-modified enzyme. A cysteinyl peptide has been isolated from modified enzyme digested with trypsin and chymotrypsin. Purification of the nucleotidyl peptide was accomplished by chromatography on phenyl boronate-agarose, followed by gel filtration on Sephadex G-25 and Bio-Gel P-4 in 50 mM ammonium bicarbonate, pH 8.0. The modified peptides were finally purified by HPLC on a C18 column using 0.1% trifluoroacetic acid with an acetonitrile gradient. By comparison of the amino acid composition and N-terminal residue of the isolated peptide with the known amino acid sequence of the enzyme, the peptide in the DPNH inhibitory site labeled by 6-BDB-TADP has been identified as the 19-membered fragment from Glu-311 to Lys-329. A unique residue, Cys-319, was identified as the reactive amino acid within the DPNH inhibitory site.
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PMID:Isolation and identification of cysteinyl peptide labeled by 6- [( 4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate in the reduced diphosphopyridine nucleotide inhibitory site of glutamate dehydrogenase. 371 40

The catalytic and regulator properties of glutamate dehydrogenase by modification of Lys-126 residue by puridoxal-5'-phosphate was studied. The phosphopyridoxyl derivative of the enzyme with blocked NADH-induced binding site of GTP not capable of being polymerized was taken as a model. It was shown that: blocking the epsilon-amino group of Lys-126 residue brings to a simultaneous inactivation of the enzyme and desensibilization of its residual activity to GTP action; the modification of Lys-126 residue and resulting inactivation of the enzyme and desensibilization to GTP action were non-cooperative processes, with equal values of pseudofirst order rate constants; modification of Lys-126 residue of any of hexamer's protomer results in the desensibilization to GTP action on one of the contacting, catalytically active protomers. The experimental dependence of the inhibition degree of the enzyme by GTP upon the average number of modified residues of Lys-126 is explained by the model of the hexamer of glutamate dehydrogenase with identical interlocation of any of the protomers in relation to the one in contact.
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PMID:[Modification of glutamate dehydrogenase by pyridoxal-5'-phosphate. Study of the structural organisation of the hexamer and its possible role in the realization of GTP action]. 376 31

Photooxidation of bovine liver glutamate dehydrogenase (GDH, EC 1.4.1.3) in the presence of methylene blue at a low light intensity occurs in two stages. At the first stage, the duration of which depends on temperature and dye concentration, a slight activation is observed simultaneously with the oxidation of two histidine residues. At the second stage, the inactivation is concomitant with the oxidation of three histidine and one tryptophan residues. The inactivation is a first order reaction (k = 3,22 X 10(-2) min-1) and is correlated with changes in the circular dichroism spectra. These data testify to the structural role of histidine residues in the GDH molecule. The kinetic behaviour of GDH during its modification with diethylpyrocarbonate (DEP) depends on pH and the reagent concentration. Four histidine residues undergo carbethoxylation at pH 6.0 and 7.5, but the modification rate is much higher at pH 7.5. At low DEP concentrations, a remarkable activation is observed with a simultaneous modification of one histidine residue, which is independent of pH. At high DEP concentrations, a rapid inactivation takes place at pH 7.5. Treatment of the carbethoxylated inactive enzyme with hydroxylamine results in the deacylation of histidine residues without any noticeable reactivation. The data on the combined effect of DEP and pyridoxal-5'-phosphate suggest that GDH inactivation by DEP at pH 7.5 is a result of modification of an essential epsilon-NH2 group of lysine-126.
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PMID:[Structural role of histidine residues in NAD(P)-glutamate dehydrogenase from the bovine liver]. 407 86

A tentative primary structure of the NADP-specific glutamate dehydrogenase [L-glutamate: NADP oxidoreductase (deaminating), EC 1.4.1.4] from Neurospora crassa has been determined. The proposed sequence contains 452 amino-acid residues in each of the identical subunits of the hexameric enzyme. Comparison of the sequence with that of the bovine liver enzyme reveals considerable homology in the amino-terminal portion of the chain, including the vicinity of the reactive lysine, with only shorter stretches of homology within the carboxyl-terminal regions. The significance of this distribution of homologous regions is discussed.
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PMID:Amino-acid sequence of NADP-specific glutamate dehydrogenase of neurospora crassa. 415 68

1. The reaction of 4-iodoacetamidosalicylate with bovine liver glutamate dehydrogenase is dependent on pH. The pH-activity curve is bell-shaped and can be described by apparent pK values of 7.8+/-0.2 and 9.1+/-0.2. 2. Enzyme in which lysine-126 has been modified by 4-iodoacetamidosalicylate has unaltered sedimentation characteristics except when measured in the presence of GTP and NADH. 3. GTP binding to the inhibited enzyme is unaltered. However, GTP can no longer promote the binding of a second molecule of NADH, since this is already bound to the inhibited enzyme without GTP. 4. The equilibrium binding of ADP, GTP, NAD-sulphite and NADH (when measured at low concentrations) was largely unchanged by modification. 5. The number of binding sites for 2-oxoglutarate to the enzyme-NADH complex were decreased by 60% in an enzyme that has been inhibited by 70%.
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PMID:The effect of modifying lysine-126 on the physical, catalytic and regulatory properties of bovine liver glutamate dehydrogenase. 435 37

A tentative but almost complete amino acid sequence for the subunit peptide chain of bovine liver glutamate dehydrogenase indicates a minimal size of 506 residues with a molecular weight of 56,100, in accord with the physical size of the subunit of 55,900. Inactivation with pyridoxal 5'-phosphate, followed by reduction with sodium borohydride, has permitted identification of the essential lysine as residue 97. Nitration of tyrosine-412 is accompanied by loss of the allosteric inhibitory effect of guanosine triphosphate. Comparison of the sequences of glutamate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase has indicated that only two 12-residue sequences are similar in the two enzymes; this sequence includes reactive lysine-97 of the former enzyme.
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PMID:Bovine liver glutamate dehydrogenase: tentative amino acid sequence; identification of a reactive lysine; nitration of a specific tyrosine and loss of allosteric inhibition by guanosine triphosphate. 528 18


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