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)

The NADP dependent glutamate dehydrogenase from wild type Neurospora crassa is inactivated by exposure to light in the presence of the dye, Methylene Blue. Photo-oxidation appears to disturb the conformational equilibrium which controls the activity of this enzyme. Data obtained suggests that the modified group is the same as that reactive to the histidine reagent, diethylpyrocarbonate.
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PMID:Glutamate dehydrogenase from wild type Neurospora crassa; inactivation by photo-oxidation. 623 Feb 73

5'-p-Fluorosulfonylbenzoyladenosine (5'-FSBA) is a specific affinity label for the inhibitory NADH site of bovine liver glutamate dehydrogenase. Reaction of the enzyme with 5'-FSBA results in the loss of inhibition by high concentrations of NADH with covalent attachment of 0.53 sulfonylbenozyladenosine/subunit, i.e. modification of three subunits of the hexameric enzyme. Equal amounts of N epsilon-(4-carboxybenzenesulfonyl)lysine (Lys-(CBS] and O-(4-carboxybenzenesulfonyl)tyrosine (Tyr-(CBS] are found throughout the course of the reaction (Saradambal, K. V., Bednar, R. A., and Colman, R. F. (1981) J. Biol. Chem. 256, 11866-11872). Modified enzyme, prepared by incubating 2 mg/ml glutamate dehydrogenase with 0.3 mM 3H-labeled 5'-FSBA at pH 8 for 1 h, was carboxymethylated and digested with thermolysin. Two nucleosidyl peptides were isolated by a combination of chromatography on phenyl boronate-agarose, high-performance liquid chromatography in ammonium bicarbonate and high-performance liquid chromatography in trifluoroacetic acid. By comparison of the amino acid analysis and NH2-terminal residue of each isolated peptide with the known amino acid sequence of the enzyme, the peptides were identified as Leu-Gly-Arg-Lys(CBS) and Ile-Gly-His-Tyr(CBS)-Asp. These sequences correspond to residues 417-420 and 187-191, respectively. Lys-420 and Tyr-190 of glutamate dehydrogenase react with 5'-FSBA, and both are apparently located in the NADH inhibitory site.
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PMID:Identification of the lysine and tyrosine peptides labeled by 5'-p-fluorosulfonylbenzoyladenosine in the NADH inhibitory site of glutamate dehydrogenase. 643 99

Initial rate kinetic studies and reduced coenzyme binding studies with bovine glutamate dehydrogenase have shown that an enzyme-NAD(P)H-glutamate abortive complex is a major participant in the overall enzyme-catalyzed reaction. The allosteric regulator ADP is shown to activate the enzyme by destabilizing this abortive complex. Destabilization of this abortive complex with concomitant activation is also achieved by the ethoxyformylation of a single histidine residue per subunit in the hexameric enzyme. ADP, in addition to its activatory effects, is shown to (a) remove the nonlinearity from the Lineweaver-Burk plots which is attributable to negative cooperativity and (b) inhibit the enzyme as a competitive inhibitor with respect to coenzyme under the appropriate conditions. Ethoxyformylation with diethyl pyrocarbonate, which mimics the activatory effects of ADP, has no effect on the negative cooperativity shown by this enzyme. A model for the action of ADP is proposed in which ADP activates glutamate dehydrogenase by binding to a regulatory binding site and blocks the negative cooperativity by mimicking the natural coenzyme at the active site of the enzyme.
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PMID:Effects of adenosine 5'-diphosphate on bovine glutamate dehydrogenase: diethyl pyrocarbonate modification. 747 Apr 50

There is now increasing evidence that surface-associated enzymes, previously considered to be involved in intermediary metabolism or virulence, play a role in physiological reactions such as signal transduction, transport systems, and metabolic processes. Herein we report the molecular aspects of two such enzymes, the cysteine proteinase gingivain and NAD-dependent glutamate dehydrogenase of Porphyromonas gingivalis. The gdh gene comprises an open reading frame of 1,335 base pairs that encodes a 49,000-M(r) protein of 445 amino acids. The gdh gene showed high homology (78.3%) with that of Clostridium symbiosum. Optimal codons accounted for 35.9% of the total codon usage, indicating high expression of this enzyme. These data are currently being used to carry out targeted mutagenesis, which was established here for gingivain. Conditions for targeted mutagenesis within the histidine domain of the catalytic site of gingivain using Tn 4351 was successfully achieved. Consequently, the catalytic functions, such as gingivain's capacity to hydrolyze the synthetic substrate alpha-benzoyl-arginine-4-nitroanilide, were disrupted.
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PMID:Molecular analysis of surface-associated enzymes of Porphyromonas gingivalis. 754 41

The initial velocity, pH and temperature optima, and Km values of Schizosaccharomyces pombe NADP-glutamate dehydrogenase (NADP-GDH:EC 1.4.1.4) have been determined. NADP-GDH was found to be specific for the substrates used in the reaction mixtures. NADP-GDH activity showed a sigmoidal response to changes in alpha-ketoglutarate concentrations, following Hill kinetics with a coefficient nH = 2. A two-fold and a three-fold increase in activity was found in extracts of cells grown on a medium containing cytosine or histidine as a sole nitrogen source, respectively, relative to the activity found in cells grown on other sole nitrogen sources including ammonium, adenine, arginine, aspartate, asparagine, glutamate, glutamine, leucine, lysine, proline, uridine and urea. Five NADP-GDH-defective mutants were isolated on the basis of no growth on ammonium plus allantoin as sole nitrogen sources. The mutants also failed to grow on allantoin alone but, in contrast, they were phenotypically indistinguishable from the wild-type growing on solid minimal medium with ammonium. Additionally, the mutants were found to grow as wild-type on minimal medium with alanine, arginine, asparagine, aspartate, glutamate, glutamine, leucine, ornithine and proline in the absence or presence of allantoin. In liquid minimal medium with ammonium as sole nitrogen source they had a slower growth than the wild-type. Normal growth was observed in cells grown on alanine, arginine, asparagine, aspartate, glutamate, glutamine, leucine, ornithine and proline. The mutants had undetectable levels of NADP-GDH activity, but retained wild-type levels of NAD-GDH, glutame synthase (GOGAT) and glutamine synthetase (GS).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Biochemical and genetical studies of NADP-specific glutamate dehydrogenase in the fission yeast Schizosaccharomyces pombe. 788 25

1. At a physiological concentration of glutamine (0.5 mM), 87% of the total transport across the plasma membrane of liver cells isolated from fed rats involved the Na(+)-dependent system N; this was substantially inhibited by L-histidine. The residual Na(+)-independent component was attributed to system L on the basis of inhibition by 2-amino-2-norbornanecarboxylate and L-tryptophan. 2. Catabolism of glutamine by intact liver cells or by isolated mitochondria was inhibited by glutamate gamma-hydrazide with IC50 values of 13.7 +/- 3.5 microM and 22.6 +/- 3.8 microM respectively and a maximal inhibition of approx. 75%. The site of inhibition was identified as glutaminase; glutamate gamma-hydrazide inhibited this enzyme in cell-free extracts (IC50 37.8 +/- 7.7 microM) but had no activity against glutamate dehydrogenase or transport of glutamine, whether across mitochondrial or plasma membranes. 3. The major control site in cells from fed animals incubated with 0.5 mM L-glutamine was glutaminase (flux control coefficient 0.96). Appreciable control also resided in both plasma membrane transport systems, with coefficients of 0.51 for system N and -0.46 for system L, such that both interacted to provide a fine control of the intracellular concentration of the amino acid. Similar values were obtained by computer simulation based on theoretical determination of elasticities. 4. Previous controversy about the locus of regulation of hepatic glutamine metabolism is resolved by this distribution of control.
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PMID:A quantitative analysis of the control of glutamine catabolism in rat liver cells. Use of selective inhibitors. 824 Feb 66

To date, no attempt has been made to study alterations occurring in the amino acid profile in chronic models of thioacetamide-induced liver cirrhosis. In this work, changes in serum amino acids and proteins in rats with thioacetamide-induced liver cirrhosis are reported, together with changes in enzyme activities in the liver and serum. Seventeen female Wistar rats were used. Eight rats were given 300 mg thioacetamide/l in drinking water for 4 months and nine rats were given water ad libitum during the same time-period. Significant increases in glycine, alanine, serine, methionine, glutamate, ornithine, phenylalanine, tyrosine, histidine and proline were observed in rats with the resulting experimental liver cirrhosis. Threonine, taurine, glutamine, lysine and citrulline tended to increase while isoleucine, leucine, aspartate, arginine and tryptophan tended to decrease. Total and nonessential amino acids increased significantly in cirrhotic animals. Total essential and aromatic amino acids tended to increase in the thioacetamide-treated group, whereas branched chain amino acids tended to decrease in the same group. Regarding serum proteins, a decrease in albumin concentration in the thioacetamide-treated animals was the only change detected. The liver enzyme activities under observation (aspartate and alanine aminotransferases, glutamate dehydrogenase and threonine deaminase) were lower in the thioacetamide group. Decreases were significant for both transaminases and threonine deaminase. Results for serum activities showed that transaminases did not change in thioacetamide-treated rats in comparison with controls. In contrast, alkaline phosphatase rose dramatically in cirrhotic rats. We conclude that the serum amino acid pattern in this chronic model of liver cirrhosis resembles in part that of the corresponding human disease.
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PMID:Serum amino acid changes in rats with thioacetamide-induced liver cirrhosis. 857 92

X-ray crystallographic studies have previously shown that glutamate dehydrogenase from Clostridium symbiosum is a homohexamer. Mutation of the active-site aspartate-165 to histidine causes an alteration in the structural properties of the enzyme. The mutant enzyme, D165H exists predominantly as a single species of lower molecular mass than the wild-type enzyme as indicated by gel filtration and sedimentation velocity analysis. The latter technique gives an S20,w value for D165H of (6.07 +/- 0.01)S which compares with (11.08 +/- 0.01)S for the wild-type, indicative of alteration of the homohexameric quaternary structure of the native enzyme to a dimeric form, a result confirmed by sedimentation equilibrium experiments. Further support for this is provided by chemical modification by Ellman's reagent of cysteine-144 in the mutant, a residue which is buried at the dimer-dimer interface in the wild-type enzyme and is normally inaccessible to modification. The results suggest a possible structural route for communication between the active sites and subunit interfaces which may be important for relaying signals between subunits in allosteric regulation of the enzyme.
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PMID:Alteration of the quaternary structure of glutamate dehydrogenase from Clostridium symbiosum by a single mutation distant from the subunit interfaces. 918 63

The nitrogen assimilation control protein (NAC) from Klebsiella aerogenes or Escherichia coli (NACK or NACE, respectively) is a transcriptional regulator that is both necessary and sufficient to activate transcription of the histidine utilization (hut) operon and to repress transcription of the glutamate dehydrogenase (gdh) operon in K. aerogenes. Truncated NAC polypeptides, generated by the introduction of stop codons within the nac open reading frame, were tested for the ability to activate hut and repress gdh in vivo. Most of the NACK and NACE fragments with 100 or more amino acids (wild-type NACK and NACE both have 305 amino acids) were functional in activating hut and repressing gdh expression in vivo. Full-length NACK and NACE were isolated as chimeric proteins with the maltose-binding protein (MBP). NACK and NACE released from such chimeras were able to activate hut transcription in a purified system in vitro, as were NACK129 and NACE100 (a NACK fragment of 129 amino acids and a NACE fragment of 100 amino acids) released from comparable chimeras. A set of NACE and NACK fragments carrying nickel-binding histidine tags (his6) at their C termini were also generated. All such constructs derived from NACE were insoluble, as was NACE itself. Of the his6-tagged constructs derived from NACK, NACK100 was inactive, but NACK120 was active. Several NAC fragments were tested for dimerization. NACK120-his6 and NACK100-his6 were dimers in solution. MBP-NACK and MBP-NACK129 were monomers in solution but dimerized when the MBP was released by cleavage with factor Xa. MBP-NACE was readily cleaved by factor Xa, but the resulting NACE was also degraded by the protease. However, MBP-NACE-his6 was completely resistant to cleavage by factor Xa, suggesting an interaction between the C and N termini of this protein.
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PMID:The amino-terminal 100 residues of the nitrogen assimilation control protein (NAC) encode all known properties of NAC from Klebsiella aerogenes and Escherichia coli. 992 58

The pH dependence of kinetic parameters for a competitive inhibitor (glutarate) was determined in order to obtain information on the chemical mechanism for NAD-dependent glutamate dehydrogenase from Halobacterium salinarum. The maximum velocity is pH dependent, decreasing at low pHs giving a pK value of 7.19+/-0.13, while the V/K for l-glutamate at 30 degrees C decreases at low and high pHs, yielding pK values of 7.9+/-0.2 and 9.8+/-0.2, respectively. The glutarate pKis profile decreases at high pHs, yielding a pK of 9. 59+/-0.09 at 30 degrees C. The values of ionization heat calculated from the change in pK with temperature are: 1.19 x 10(4), 5.7 x 10(3), 7 x 10(3), 6.6 x 10(3) cal mol-1, for the residues involved. All these data suggest that the groups required for catalysis and/or binding are lysine, histidine and tyrosine. The enzyme shows a time-dependent loss in glutamate oxidation activity when incubated with diethyl pyrocarbonate (DEPC). Inactivation follows pseudo-first-order kinetics with a second-order rate constant of 53 M-1min-1. The pKa of the titratable group was pK1=6.6+/-0.6. Inactivation with ethyl acetimidate also shows pseudo-first-order kinetics as well as inactivation with TNM yielding second-order constants of 1.2 M-1min-1 and 2.8 M-1min-1, and pKas of 8.36 and 9.0, respectively. The proposed mechanism involves hydrogen binding of each of the two carboxylic groups to tyrosyl residues; histidine interacts with one of the N-hydrogens of the l-glutamate amino group. We also corroborate the presence of a conservative lysine that has a remarkable ability to coordinate a water molecule that would act as general base.
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PMID:Amino acid residues involved in the catalytic mechanism of NAD-dependent glutamate dehydrogenase from Halobacterium salinarum. 1007 69


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