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)

An affinity labeling reagent for the estrogenic-binding site of bovine liver L-glutamate dehydrogenase (EC 1.4.1.3) was prepared by conversion of diethylstilbestrol to its alkylating analogue, bromoacetyldiethylstilbestrol. Under standard assay conditions, the analogue acted as a reversible allosteric ligand with regulatory activity much like that of diethylstilbestrol. However, incubation of the enzyme with the alkylating agent in the presence of DPNH resulted in a permanent decrease in glutamate (X form) and an increase in alanine (Y form) activities, and in covalent attachment of diethylstilbestrol in the ratio of 1 mol per subunit (of particle weight 52,000). The brominated analogue behaved as an affinity label that mimicked the allosteric effects of diethylstilbestrol. Diethylstilbestrol protection of the enzyme against alkylation by bromoacetylated sterol suggested competition for the same binding site, while ADP protection indicated a shift of protein equilibrium into the X form. The diethylstilbestrol-enzyme compound was desensitized (relative to the native enzyme) to allosteric reagents such as ADP and GTP. The results were consistent with conformational freezing of the modified protein molecule into the Y form.
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PMID:Covalent attachment of diethylstilbestrol to glutamate dehydrogenase: implications for allosteric regulation. 432 3

1. Glutamate dehydrogenase was inhibited by l-serine O-sulphate, beta-chloro-l-alanine, O-phospho-l-serine and beta-chloro-l-alanine methyl ester. With the exception of beta-chloro-l-alanine methyl ester which was an irreversible inhibitor, it was possible to reverse the inhibitory effects by dialysis. 2. Both NAD(+) and glutamate afford some protection against the inhibition due to the methyl ester. No change in the normal stimulatory effect exhibited by ADP was observed in the presence of beta-chloro-l-alanine methyl ester but the effect due to GTP was modified. 3. Irradiation of glutamate dehydrogenase in the presence of Rose Bengal produced rapid inactivation. Amino acid analysis of the inactivated enzyme showed that eight histidine residues had been destroyed in the process.
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PMID:The inhibition of glutamate dehydrogenase by L-serine O-sulphate and related compounds and by photo-oxidation in the presence of Rose Bengal. 433 Nov 81

1. Glutamate dehydrogenase was subject to rapid inactivation when irradiated in the presence of Rose Bengal or incubated in the presence of ethoxyformic anhydride. 2. Inactivation in the presence of Rose Bengal led to the photo-oxidation of four histidine residues. Oxidation of three histidine residues had little effect on enzyme activity, but oxidation of the fourth residue led to the almost total loss of activity. 3. Acylation of glutamate dehydrogenase with ethoxyformic anhydride at pH6.1 led to the modification of three histidine residues with a corresponding loss of half the original activity. Acylation at pH7.5 led to the modification of two histidine residues and a total loss of enzyme activity. 4. One of the histidine residues undergoing reaction at pH6.1 also undergoes reaction at pH7.5. 5. The presence of either glutamate or NAD(+) in the reaction mixtures at pH6.1 had no appreciable effect. At pH7.5 glutamate caused a marked decrease in both the degree of alkylation and degree of inactivation. NAD(+) had no effect on the degree of inactivation at pH7.5 but did modify the extent of acylation. 6. The normal response of the enzyme towards ADP was unaffected by acylation at pH6.1 or 7.5. 7. The normal response of the enzyme towards GTP was altered by treatment at both pH6.1 and 7.5.
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PMID:The role of histidine residues in glutamate dehydrogenase. 434 75

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

1. One mol of diethyl pyrocarbonate will react with one mol of glutamate dehydrogenase polypeptide chains to form one mol of N(1)-carbethoxyhistidine. Reaction is prevented by NADH. 2. The 1:1 complex has an increased specific activity (1.4-2.0-fold). 3. The reason for the activation is discussed. The results are not consistent with NADH dissociation from the enzyme-glutamate-NADH complex being rate-limiting in the steady state measured. 4. The effects of modification on the properties of the enzyme were investigated. The effects of GTP and NAD(+) on the enzyme activity are unaltered by activation. NADH binding is unaltered and there is no apparent change in the molecular weight. However, the activated enzyme can still be further activated by ADP. K(s) for ADP is decreased fivefold.
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PMID:The reaction of a histidine residue in glutamate dehydrogenase with diethyl pyrocarbonate. 435 38

Ammonia assimilation has been investigated in four strains of Saccharomyces cerevisiae by measuring, at intervals throughout the growth cycle, the activities of several enzymes concerned with inorganic ammonia assimilation. Enzyme activities in extracts of cells were compared after growth in complete and defined media. The effect of shift from growth in a complete to growth in a defined medium (and the reverse) was also determined. The absence of aspartase (EC 4.3.1.1, l-aspartate-ammonia lyase) activity, the low specific activities of alanine dehydrogenase, glutamine synthetase [EC 6.3.1.2, l-glutamate-ammonia ligase (ADP)], and the marked increase in activity of the nicotinamide adenine dinucleotide phosphate-linked glutamate dehydrogenase (NADP-GDH) [EC 1.4.1.4, l-glutamate:NADP-oxidoreductase (deaminating)] during the early stages of growth support the conclusion that yeasts assimilate ammonia primarily via glutamate. The NADP-GDH showed a rapid increase in activity just before the initiation of exponential growth, reached a maximum at the mid-exponential stage, and then gradually declined in activity in the stationary phase. The NADP-GDH reached a higher level of activity when the yeasts were grown on the defined medium as compared with complete medium. The nicotinamide adenine dinucleotide-linked glutamate dehydrogenase (NAD-GDH) [EC 1.4.1.2, l-glutamate:NAD-oxidoreductase (deaminating)] showed only slight increases in activity during the exponential phase of growth. There was an inverse relationship in that the NADP-GDH increased in activity as the NAD-GDH decreased. The NAD-GDH activity was higher after growth on the complete medium. The glutamate-oxaloacetate transaminase (EC 2.6.1.1. l-aspartate:2-oxoglutarate aminotransferase) activity rose and fell in parallel with the NADP-GDH, although its specific activity was somewhat lower. Although other ammonia-assimilatory enzymes were demonstrable, it seems unlikely that their combined activities could account for the remainder of the ammonia-assimilatory capacity not accounted for by the NADP-GDH. The ability of aspartate to serve as effectively as glutamate as the sole source of nitrogen for the growth of yeast apparently resides in their ability to utilize aspartate for amino acid biosynthesis via transamination.
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PMID:Inorganic nitrogen assimilation in yeasts: alteration in enzyme activities associated with changes in cultural conditions and growth phase. 440 Apr 14

1. The binding of NAD(+) and NADP(+) to glutamate dehydrogenase has been studied in sodium phosphate buffer, pH7.0, by equilibrium dialysis. Approximate values for the dissociation constants are 0.47 and 2.5mm respectively. For NAD(+) the value agrees with that estimated from initial-rate results. 2. In the presence of the substrate analogue glutarate both coenzymes are bound more firmly, and there is one active centre per enzyme subunit. The binding results cannot be described in terms of independent and identical active centres, and binding is stronger at low coenzyme concentrations than at high concentrations. Either the six subunits of the oligomer are not identical or there are negative interactions between them in the binding of coenzymes in ternary complexes with glutarate. The latter explanation is favoured. 3. The binding studies support the conclusions drawn from earlier kinetic studies of the glutamate reaction. 4. ADP and GTP respectively decrease and increase the affinity of the enzyme for NAD(+) and NADP(+), in both the presence and absence of glutarate. The negative binding interactions in the presence of glutarate are abolished by ADP, which decreases the affinity for the coenzymes at low concentrations of the latter. 5. In the presence of glutarate, GTP and NAD(+) or NADP(+), the association of enzyme oligomers is prevented, and the solubility of the enzyme is decreased; the complex of enzyme and ligands readily crystallizes. 6. The results are discussed in relation to earlier kinetic studies.
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PMID:The binding of oxidized coenzymes by glutamate dehydrogenase and the effects of glutarate and purine nucleotides. 440 8

A combination of kinetic and isotope effect studies in the presence and absence of the effectors ADP and GTP was used to elucidate the mechanism of regulation of bovine liver glutamate dehydrogenase. ADP at low concentrations of glutamate competes with TPN for free enzyme. GTP exhibits a similar effect at high concentrations (100 microM and above). When ADP binds at its allosteric site, it increases the off rates of both alpha-ketoglutarate and TPNH from their product complexes. This results in a decrease in V/K for both substrates, an increase in V, and an increase in the deuterium isotope effects for all three parameters so that they are all about 1.3. The rate of release of glutamate from E-TPNH-glutamate is also apparently enhanced since no substrate inhibition by glutamate is observed in the presence of ADP. The effect of GTP is in opposition to that of ADP in that GTP decreases the off rates for both TPN and glutamate from E-TPN-glutamate as well as the off rates for alpha-ketoglutarate and TPNH. This results in an increase in the V/K's for both substrates, a decrease in V, and a decrease in the deuterium isotope effects for all three parameters to a value of 1. Substrate inhibition by glutamate is also eliminated by GTP probably by preventing any significant accumulation of E-TPNH to which glutamate binds as an inhibitor.
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PMID:Kinetic studies to determine the mechanism of regulation of bovine liver glutamate dehydrogenase by nucleotide effectors. 612 Jul 19

Studies of isolated rat liver mitochondria were undertaken in order to evaluate the importance of glutamate transport, oxidation reduction state, and product inhibition on the rates of formation of ammonia from glutamate. Uptake and efflux of glutamate across the mitochondrial membrane were measured isotopically in the presence of rotenone. Efflux was stimulated by H+ in the mitochondrial matrix and was found to be first order with respect to matrix glutamate except when the matrix pH was unphysiologically low. The data suggest that the Km of matrix glutamate for efflux is decreased by H+. Matrix H+ also appeared to stimulate glutamate uptake, but the effect was to increase both the Km of medium glutamates and Vmax. Mitochondria were incubated at 15 and 28 degrees C with glutamate and malonate. Under these conditions, glutamate was metabolized only by the deamination pathway. Flux was evaluated by assay of ammonia formation. Oxidation reduction state was varied with ADP and uncoupling agents. Matrix alpha-ketoglutarate was varied either by the omission of malonate from the incubation media or by adding alpha-ketoglutarate to the external media. Influx and efflux of glutamate could be calculated from previously determined transport parameters. The difference between calculated influx and efflux was found to be equal to ammonia formation under all conditions. It was, therefore, possible to evaluate the relative contributions of oxidation reduction state, transport, and product inhibition as effectors of ammonia formation. The contribution of transport was relatively small while oxidation reduction state exerted a large influence. alpha-Ketoglutarate was found to be a potent competitive inhibitor of ammonia production and glutamate dehydrogenase. Inhibition of glutamate dehydrogenase by alpha-ketoglutarate was judged to be a potentially important modulator of metabolic fluxes.
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PMID:Ammonia formation in isolated rat liver mitochondria. 613 94

Experiments were designed to examine the early events in the initiation of glutamate deamination in kidney. Perfused kidneys from methionine sulfoximine-treated rats formed ammonia from [15N]glutamate via the purine nucleotide cycle. The turnover of the 6-amino group of adenine nucleotides to yield ammonia occurred at the rate of 0.30 mumol/g of kidney/min. This rate is 3-4 times larger than in liver and is in agreement with published rates of the purine nucleotide cycle in kidney. The addition of 0.1 mM fluorocitrate to glutamate perfusions stimulated ammonia formation 3 1/2-fold. The turnover of the 6-amino group of adenine nucleotides increased during the first 5 min after adding fluorocitrate to form ammonia predominately from tissue glutamate and aspartate. This turnover correlates with a 3 1/2-fold increase in kidney tissue IMP levels. As the ATP/ADP ratio fell the purine nucleotide cycle was inhibited and glutamate dehydrogenase was stimulated to form ammonia stoichiometric with glutamate taken up from the perfusate. Ammonia formation via glutamate dehydrogenase occurred at a rate of 1.0 mumol/g of kidney/min. Fluorocitrate completely blocked ammonia formation from aspartate in perfusions. The perfused kidney formed ammonia from aspartate via the purine nucleotide cycle at a rate of 1.0 mumol/g of kidney/min. The results indicate a discrete role for aspartate in renal metabolism. Ammonia formation via the purine nucleotide cycle can occur at significant rates and equal to the rate of ammonia formation from glutamate via glutamate dehydrogenase.
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PMID:Early events in the initiation of ammonia formation in kidney. 613 Oct 71


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