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 purified glutamate dehydrogenase (GDH) from Sulfolobus solfataricus showed remarkable thermostability and retained 90-95% of the initial activity after incubation at -20 degrees C, 4 degrees C, and 25 degrees C for up to 6 months. Unlike mammalian GDHs, the activity of GDH from Sulfolobus solfataricus was not significantly affected by the presence of various allosteric effectors such as ADP, GTP, and leucine. Incubation of GDH with increasing concentration of o-phthalaldehyde resulted in a progressive decrease in enzyme activity, suggesting that the o-phthalaldehyde-modified lysine or cysteine is directly involved in catalysis. The inhibition was competitive with respect to both 2-oxoglutarate (Ki = 30 microM) and NADH (Ki = 100 microM), further supporting a possibility that the o-phthalaldehyde-modified residues may be directly involved at the catalytic site. The modification of GDH by the arginine-specific dicarbonyl reagent phenylglyoxal was also examined with the view that arginine residues might play a general role in the binding of coenzyme throughout the family of pyridine nucleotide-dependent dehydrogenases. The purified GDH was inactivated in a dose-dependent manner by phenylglyoxal. Either NADH or 2-oxoglutarate did not gave any protection against the inactivation caused by a phenylglyoxal. This result indicates that GDH saturated with NADH or 2-oxoglutarate is still open to attack by phenylglyoxal. Phenylglyoxal was an uncompetitive inhibitor (Ki = 5 microM) with respect to 2-oxoglutarate and a noncompetitive inhibitor (Ki = 6 microM) with respect to NADH. The above results suggests that the phenylglyoxal-modified arginine residues are not located at the catalytic site and the inactivation of GDH by phenylglyoxal might be due to a steric hindrance or a conformational change affected by the interaction of the enzyme with its inhibitor.
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PMID:Regulatory properties of glutamate dehydrogenase from Sulfolobus solfataricus. 1077 43

The NAD(+)-dependent glutamate dehydrogenase (NAD-GDH) from Pseudomonas aeruginosa PAO1 was purified, and its amino-terminal amino acid sequence was determined. This sequence information was used in identifying and cloning the encoding gdhB gene and its flanking regions. The molecular mass predicted from the derived sequence for the encoded NAD-GDH was 182.6 kDa, in close agreement with that determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme (180 kDa). Cross-linking studies established that the native NAD-GDH is a tetramer of equal subunits. Comparison of the derived amino acid sequence of NAD-GDH from P. aeruginosa with the GenBank database showed the highest homology with hypothetical polypeptides from Pseudomonas putida, Mycobacterium tuberculosis, Rickettsia prowazakii, Legionella pneumophila, Vibrio cholerae, Shewanella putrefaciens, Sinorhizobium meliloti, and Caulobacter crescentus. A moderate degree of homology, primarily in the central domain, was observed with the smaller tetrameric NAD-GDH (protomeric mass of 110 kDa) from Saccharomyces cerevisiae or Neurospora crassa. Comparison with the yet smaller hexameric GDH (protomeric mass of 48 to 55 kDa) of other prokaryotes yielded a low degree of homology that was limited to residues important for binding of substrates and for catalytic function. NAD-GDH was induced 27-fold by exogenous arginine and only 3-fold by exogenous glutamate. Primer extension experiments established that transcription of gdhB is initiated from an arginine-inducible promoter and that this induction is dependent on the arginine regulatory protein, ArgR, a member of the AraC/XyIS family of regulatory proteins. NAD-GDH was purified to homogeneity from a recombinant strain of P. aeruginosa and characterized. The glutamate saturation curve was sigmoid, indicating positive cooperativity in the binding of glutamate. NAD-GDH activity was subject to allosteric control by arginine and citrate, which function as positive and negative effectors, respectively. Both effectors act by influencing the affinity of the enzyme for glutamate. NAD-GDH from this organism differs from previously characterized enzymes with respect to structure, protomer mass, and allosteric properties indicate that this enzyme represents a novel class of microbial glutamate dehydrogenases.
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PMID:The gdhB gene of Pseudomonas aeruginosa encodes an arginine-inducible NAD(+)-dependent glutamate dehydrogenase which is subject to allosteric regulation. 1113 42

An enzymatic end-point method for the quantitative determination of L-arginine was evaluated with samples of synthetic wine and natural grape juice. The enzymes arginase, urease, and glutamate dehydrogenase were used in this simple assay, similar to those described for many metabolites by Boehringer-Mannheim. In synthetic wine, recovery of L-arginine ranged between 98.3 and 104.4% and the precision as coefficient of variation was between 0.4 and 1.47% in the concentration range of the method, 0-100 mg/L L-arginine. The recovery of L-arginine in a grape juice with added L-arginine after clarification with polyvinylpolypyrrolidone ranged between 100 and 101.3%, and the coefficient of variation was 0.6%. The method has low material costs of approximately 0.43 U.S.$ per assay, and the time course of the reaction facilitates measurement of several samples concurrently. The results of this evaluation indicate that the enzymatic assay is a preferred method over colorimetric methods for the manual determination of L-arginine.
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PMID:Quantitative determination of L-arginine by enzymatic end-point analysis. 1126 90

The hyperinsulinism/hyperammonemia (HI/HA) syndrome is a form of congenital hyperinsulinism in which affected children have recurrent symptomatic hypoglycemia together with asymptomatic, persistent elevations of plasma ammonium levels. We have shown that the disorder is caused by dominant mutations of the mitochondrial enzyme, glutamate dehydrogenase (GDH), that impair sensitivity to the allosteric inhibitor, GTP. In 65 HI/HA probands screened for GDH mutations, we identified 19 (29%) who had mutations in a new domain, encoded by exons 6 and 7. Six new mutations were found: Ser(217)Cys, Arg(221)Cys, Arg(265)Thr, Tyr(266)Cys, Arg(269)Cys, and Arg(269)HIS: In all five mutations tested, lymphoblast GDH showed reduced sensitivity to allosteric inhibition by GTP (IC(50), 60--250 vs. 20--50 nmol/L in normal subjects), consistent with a gain of enzyme function. Studies of ATP allosteric effects on GDH showed a triphasic response with a decrease in high affinity inhibition of enzyme activity in HI/HA lymphoblasts. All of the residues altered by exons 6 and 7 HI/HA mutations lie in the GTP-binding domain of the enzyme. These data confirm the importance of allosteric regulation of GDH as a control site for amino acid-stimulated insulin secretion and indicate that the GTP-binding site is essential for regulation of GDH activity by both GTP and ATP.
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PMID:Hyperinsulinism/hyperammonemia syndrome in children with regulatory mutations in the inhibitory guanosine triphosphate-binding domain of glutamate dehydrogenase. 1129 18

By reaction of adenosine 5'-monothiophosphate with benzophenone-4-maleimide, we synthesized adenosine 5'-O-[S-(4-succinimidyl-benzophenone)thiophosphate] (AMPS-Succ-BP) as a photoreactive ADP analogue. Bovine liver glutamate dehydrogenase is known to be allosterically activated by ADP, but the ADP site has not been located in the crystal structure of the hexameric enzyme [Peterson, P. E., and Smith, T. J. (1999) Structure 7, 769-782]. In the dark, AMPS-Succ-BP reversibly activates GDH. Irradiation of the complex of glutamate dehydrogenase and AMPS-Succ-BP at lambda >300 nm causes a time-dependent, irreversible 2-fold activation of the enzyme. The k(obs) for photoactivation shows nonlinear dependence on the concentration of AMPS-Succ-BP, with K(R) = 4.9 microM and k(max) = 0.076 min(-)(1). The k(obs) for photoreaction by 20 microM AMPS-Succ-BP is decreased 10-fold by 200 microM ADP, but is reduced less than 2-fold by NAD, NADH, GTP, or alpha-ketoglutarate. Modified enzyme is no longer activated by ADP, but is still inhibited by GTP and high concentrations of NADH. These results indicate that reaction of AMPS-Succ-BP occurs within the ADP site. The enzyme incorporates up to 0.5 mol of [(3)H]AMPS-Succ-BP/mol of enzyme subunit or 3 mol of reagent/mol of hexamer. The peptide Lys(488)-Glu(495) has been identified as the only reaction target, and the data suggest that Arg(491) is the modified amino acid. Arg(491) (in the C-terminal helix close to the GTP #2 binding domain of GDH) is thus considered to be at or near the enzyme's allosteric ADP site. On the basis of these results, the AMPS-Succ-BP was positioned within the crystal structure of glutamate dehydrogenase, where it should also mark the ADP binding site of the enzyme.
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PMID:Adenosine 5'-0-[S-(4-succinimidyl-benzophenone)thiophosphate]: a new photoaffinity label of the allosteric ADP site of bovine liver glutamate dehydrogenase. 1132 16

Human glutamate dehydrogenase (GDH) exists in two isoforms encoded by the GLUD1 and GLUD2 genes, respectively. Although the two enzymes share in their mature form all but 15 of their 505 amino acids, they differ markedly in their allosteric regulation. To identify the structural basis for these allosteric characteristics, we performed site-directed mutagenesis on the human GLUD1 gene at sites that differ from the GLUD2 gene using a cloned GLUD1 cDNA. Results showed that substitution of Ala for Gly-456, but not substitution of His for Arg-470 or Ser for Asn-498, renders the enzyme markedly resistant to GTP inhibition (IC(50) = 2.80 microm) as compared with the wild type GLUD1-derived GDH (IC(50) = 0.19 microm). The G456A mutation abolished the cooperative behavior of the enzyme, as revealed by the GTP inhibitory curves. The catalytic and kinetic properties of the G456A mutant and its activation by ADP were comparable with those of the wild type GDH. Gly-456 lies in a very tightly packed region of the GDH molecule, and its replacement by Ala may lead to steric clashes with neighboring amino acids. These, in turn, may affect the conformational state of the protein that is essential for the allosteric regulation of the enzyme by GTP.
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PMID:Single amino acid substitution (G456A) in the vicinity of the GTP binding domain of human housekeeping glutamate dehydrogenase markedly attenuates GTP inhibition and abolishes the cooperative behavior of the enzyme. 1195 Aug 37

Although the structure of glutamate dehydrogenase (GDH) has been reported from various sources including mammalian GDH, there are conflicting views regarding the location and mechanism of actions of the coenzyme binding. We have expanded these speculations by photoaffinity labeling and cassette mutagenesis. Photoaffinity labeling with a specific probe, [(32)P]nicotinamide 2-azidoadenosine dinucleotide, was used to identify the NAD(+) binding site within human GDH encoded by the synthetic human GDH gene and expressed in Escherichia coli as a soluble protein. Photolabel-containing peptides generated with trypsin were isolated by immobilized boronate affinity chromatography. Photolabeling of these peptides was most effectively prevented by the presence of NAD(+) during photolysis, demonstrating a selectivity of the photoprobe for the NAD(+) binding site. Amino acid sequencing and compositional analysis identified Glu(279) as the site of photoinsertion into human GDH, suggesting that Glu(279) is located at or near the NAD(+) binding site. The importance of the Glu(279) residue in the binding of NAD(+) was further examined by cassette mutagenesis with mutant enzymes containing Arg, Gly, Leu, Met, or Tyr at position 279. The mutagenesis at Glu(279) has no effects on the expression or stability of the different mutants. The K(m) values for NAD(+) were 10-14-fold greater for the mutant GDHs than for wild-type GDH, whereas the V(max) values were similar for wild-type and mutant GDHs. The efficiency (k(cat)/K(m)) of the mutant GDH was reduced up to 18-fold. The decreased efficiency of the mutants results from the increase in K(m) values for NAD(+). In contrast to the K(m) values for NAD(+), wild-type and mutant GDHs show similar K(m) values for glutamate, indicating that substitution at position 279 had no appreciable effect on the affinity of enzyme for glutamate. There were no differences in sensitivities to ADP activation and GTP inhibition between wild-type and mutant GDH, suggesting that Glu(279) is not directly involved in allosteric regulation. The results with photoaffinity labeling and cassette mutagenesis studies suggest that Glu(279) plays an important role for efficient binding of NAD(+) to human GDH.
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PMID:Importance of glutamate 279 for the coenzyme binding of human glutamate dehydrogenase. 1219 7

Human glutamate dehydrogenase (GDH) exists in GLUD1 (housekeeping) and in GLUD2-specified (brain-specific) isoforms, which differ markedly in their basal activity and allosteric regulation. To determine the structural basis of these functional differences, we mutagenized the GLUD1 GDH at four residues that differ from those of the GLUD2 isoenzyme. Functional analyses revealed that substitution of Ser for Arg-443 (but not substitution of Thr for Ser-331, Leu for Met-370, or Leu for Met-415) virtually abolished basal activity and totally abrogated the activation of the enzyme by l-leucine (1-10 mm) in the absence of other effectors. However, when ADP (0.025-0.1 mm) was present in the reaction mixture, l-leucine (0.3-6.0 mm) activated the mutant enzyme up to >2,000%. The R443S mutant was much less sensitive to ADP (SC(50) = 383.9 +/- 14.6 microm) than the GLUD1 GDH (SC(50) = 31.7 +/- 4.2 microm; p < 0.001); however, at 1 mm ADP the V(max) for the mutant (136.67 micromol min(-1) mg(-1)) was comparable with that of the GLUD1 GDH (152.95 micromol min(-1) mg(-1)). Varying the composition and the pH of the reaction buffer differentially affected the mutant and the wild-type GDH. Arg-443 lies in the "antenna" structure, in a helix that undergoes major conformational changes during catalysis and is involved in intersubunit communication. Its replacement by Ser is sufficient to impair both the catalytic and the allosteric function of human GDH.
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PMID:Substitution of Ser for Arg-443 in the regulatory domain of human housekeeping (GLUD1) glutamate dehydrogenase virtually abolishes basal activity and markedly alters the activation of the enzyme by ADP and L-leucine. 1232 73

In Bacillus subtilis, expression of the rocG gene, encoding glutamate dehydrogenase, and the rocABC operon, involved in arginine catabolism, requires SigL (sigma(54))-containing RNA polymerase as well as RocR, a positive regulator of the NtrC/NifA family. The RocR protein was purified and shown to bind specifically to the intergenic region located between rocG and the rocABC operon. DNaseI footprinting experiments were used to define the RocR-binding site as an 8 bp inverted repeat, separated by one base pair, forming an imperfect palindrome which is present twice within the rocG-rocABC intergenic region, acting as both a downstream activating sequence (DAS) and an upstream activating sequence (UAS). Point mutations in either of these two sequences significantly lowered expression of both rocG and rocABC. This bidirectional enhancer element retained partial activity even when moved 9 kb downstream of the rocA promoter. Electron microscopy experiments indicated that an intrinsically curved region is located between the UAS/DAS region and the promoter of the rocABC operon. This curvature could facilitate interaction of RocR with sigma(54)-RNA polymerase at the rocABC promoter.
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PMID:Specificity of the interaction of RocR with the rocG-rocA intergenic region in Bacillus subtilis. 1263 42

Molecular biological studies confirmed that two glutamate dehydrogenase isozymes (hGDH1 and hGDH2) of distinct genetic origin are expressed in human tissues. hGDH1 is heat-stable and expressed widely, whereas hGDH2 is heat-labile and specific for neural and testicular tissues. A selective deficiency of hGDH2 has been reported in patients with spinocerebellar ataxia. We have identified an amino acid residue involved in the different thermal stability of human GDH isozymes. At 45 degrees C (pH 7.0), heat inactivation proceeded faster for hGDH2 (half life=45 min) than for hGDH1 (half-life=310 min) in the absence of allosteric regulators. Both hGDH1 and hGDH2, however, showed much slower heat inactivation processes in the presence of 1 mM ADP or 3 mM L-Leu. Virtually most of the enzyme activity remained up to 100 min at 45 degrees C after treatment with ADP and L-Leu in combination. In contrast to ADP and L-Leu, the thermal stabilities of the hGDH isozymes were not affected by addition of substrates or coenzymes. In human GDH isozymes, the 443 site is Arg in hGDH1 and Ser in hGDH2. Replacement of Ser by Arg at the 443 site by cassette mutagenesis abolished the heat lability of hGDH2 with a similar half-life of hGDH1. The mutagenesis at several other sites (L415M, A456G, and H470R) having differences in amino acid sequence between the two GDH isozymes did not show any change in the thermal stability. These results suggest that the Ser443 residue plays an important role in the different thermal stability of human GDH isozymes.
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PMID:Important role of Ser443 in different thermal stability of human glutamate dehydrogenase isozymes. 1504 2


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