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:1.4.1.2 (glutamate dehydrogenase)
4,380 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

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

1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) (14)C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus (14)C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of (14)C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate --> threonine --> glycine right harpoon over left harpoon serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine.
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PMID:Biosynthesis of amino acids in Clostridium pasteurianum. 541 50

1. Inhibition of ox liver glutamate dehydrogenase with N-(N'-acetyl-4[(35)S]-sulphamoylphenyl)maleimide (ASPM) is more specific at pH7.3 than at pH6.9. At pH7.3 inhibition accompanies the incorporation at 1 mole of ASPM residues into about 53000g. of protein. 2. Digestion of the modified protein with chymotrypsin and trypsin yields a unique radioactive peptide. 3. Acid hydrolysis of 1 mole of this peptide yields 1 mole of N(in)-succin-2-yl-lysine. The in-amino group of a lysyl residue is thus the site of modification of the protein. 4. The sequence containing the modified lysyl residue is: [Formula: see text] where Asx respresents either aspartic acid or asparagine.
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PMID:A peptide containing a reactive lysyl group from ox liver glutamate dehydrogenase. 578 69

The fluorescent nucleotide analogue 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine (5'-FSB epsilon A) reacts irreversibly with bovine liver glutamate dehydrogenase and modifies one of the natural inhibitory guanosine 5'-triphosphate (GTP) sites [Jacobson, M.A., & Colman, R.F. (1982) Biochemistry 21, 2177-2186]. Enzyme with 1.28 mol of 5'-(p-sulfonylbenzoyl)-1,N6-ethenoadenosine/mol of subunit incorporated and exhibiting maximum change in sensitivity to GTP inhibition is now shown by amino acid analysis to contain 0.95 mol of O-[(4-carboxyphenyl)sulfonyl]tyrosine (CBS-Tyr) and 0.33 mol of N epsilon-[(4-carboxyphenyl)sulfonyl]-lysine (CBS-Lys), quantitatively accounting for the total incorporation prior to acid hydrolysis. As a function of time of incubation with 5'-FSB epsilon A, the amount of CBS-Tyr formed was directly proportional to the change in GTP inhibition. In contrast, an initial formation of CBS-Lys was observed, followed by relatively little additional CBS-Lys although the percent change in GTP inhibition continued to increase. It was concluded that the tyrosine is an essential residue in the GTP binding site of glutamate dehydrogenase, while the lysine modified is not involved in the inhibitory action of GTP. The nucleotide analogue 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) was evaluated for its ability to occupy the adenosine 5'-diphosphate (ADP) activator site and to function as an energy acceptor conjointly with 5'-SB epsilon A covalently bound at the GTP site as the energy donor. TNP-ADP activates native enzyme 2-fold and competes kinetically with ADP. As determined by fluorometric titration, the maximum number of TNP-ADP binding sites on native enzyme was 0.5 mol/mol of subunit in the absence and 1 mol/mol of subunit in the presence of reduced coenzyme. The 5'-SB epsilon A-modified enzyme also binds TNP-ADP: 0.5 mol/mol of subunit in the absence or presence of reduced coenzyme. TNP-ADP competes for binding with ADP to native and 5'-SB epsilon A-modified enzyme, indicating that this nucleotide analogue is a satisfactory fluorescent probe of the ADP site of glutamate dehydrogenase. An energy-transfer efficiency of 0.77 was determined from the decrease in donor fluorescence upon addition of TNP-ADP in the absence of reduced coenzyme to modified enzyme containing 1.23 mol of 5'-SB epsilon A/mol of subunit. A value of 18 A was calculated as the average distance between the GTP and ADP regulatory sites. This result indicates that the inhibitory GTP and the activatory ADP sites are close but not identical.
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PMID:Resonance energy transfer between the adenosine 5'-diphosphate site of glutamate dehydrogenase and a guanosine 5'-triphosphate site containing a tyrosine labeled with 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine. 641 7

Differential digitonin extraction of rat liver mitochondria and of mitochondria of livers of affected and unaffected male sparse fur mice released a lysine transcarbamylase activity from the mitochondria at a digitonin to protein ratio in between that for myokinase and glutamate dehydrogenase, but at a slightly lower ratio than the ornithine transcarbamylase activity. Homocitrulline formation by isolated rat liver mitochondria is independent of the uptake of lysine by mitochondria as evidenced by the insensitivity of homocitrulline formation to changes in the matrix pH, in contrast to citrulline formation from ornithine. High-performance liquid chromatography separates the lysine transcarbamylase activity from the ornithine transcarbamylase activity. It is concluded that the lysine transcarbamylase activity is localized outside the inner mitochondrial membrane.
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PMID:Further evidence for a separate enzymic entity for the synthesis of homocitrulline, distinct from the regular ornithine transcarbamylase. 643 2


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