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)

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

The complete nucleotide sequence of a 39,090 bp segment from the left arm of yeast chromosome IV was determined. Twenty-one open reading frames (ORFs) longer than 100 amino acids and a Gly-tRNA gene were discovered. Nine of the 21 ORFs (D0892, D1022, D1037, D1045, D1057, D1204, D1209, D1214, D1219) correspond to the previously sequenced Saccharomyces cerevisiae genes for the NAD-dependent glutamate dehydrogenase (GDH), the secretory component (SHR3), the GABA transport protein (UGA4), the high mobility group-like protein (NHP2), the hydroxymethylbilane synthase (HEM3), the methylated DNA protein-cysteine S-methyltransferase (MGT1), a putative sugar transport protein, the Shm1 protein (SHM1) and the anti-silencing protein (ASF2). The inferred amino acid sequences of 11 ORFs show significant similarity with known proteins from various organisms, whereas the remaining ORF does not share any similarity with known proteins.
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PMID:The nucleotide sequence of a 39 kb segment of yeast chromosome IV: 12 new open reading frames, nine known genes and one genes for Gly-tRNA. 904 97

It has been suggested that reactive lysine residue(s) may play an important role in the catalytic activities of glutamate dehydrogenase (GDH). There are, however, conflicting views as to whether the lysine residues are involved in Schiff's base formation with catalytic intermediates, stabilization of negatively charged groups or the carbonyl group of 2-oxoglutarate during catalysis, or some other function. We have expanded on these speculations by constructing a series of cassette mutations at Lys130, a residue that has been speculated to be responsible for the activity of GDH and the inactivation of GDH by pyridoxal 5'-phosphate (PLP). For these studies, a 1557-bp gene that encodes human GDH has been synthesized and inserted into Escherichia coli expression vectors. The mutant enzymes containing Glu, Gly, Met, Ser, or Tyr at position 130, as well as the wild-type human GDH encoded by the synthetic gene, were efficiently expressed as a soluble protein and are indistinguishable from that isolated from human and bovine tissues. Despite an approximately 400-fold decrease in the respective apparent Vmax of the Lys130 mutant enzymes, apparent Km values for NADH and 2-oxoglutarate were almost unchanged, suggesting the direct involvement of Lys130 in catalysis rather than in the binding of coenzyme or substrate. Unlike the wild-type GDH, the mutant enzymes were unable to interact with PLP, indicating that Lys130 plays an important role in PLP binding. The results with analogs of PLP suggest that the aldehyde moiety of PLP, but not the phosphate moiety, is required for efficient binding to GDH.
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PMID:Cassette mutagenesis of lysine 130 of human glutamate dehydrogenase. An essential residue in catalysis. 1138 22

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

Proteins in extracts from cotyledons, hypocotyls, and roots of 5-d-old, dark-grown soybean (Glycine max L. Merr. cv Williams) seedlings were separated by polyacrylamide gel electrophoresis. Three isoforms of glutamate dehydrogenase (GDH) were resolved and visualized in gels stained for GDH activity. Two isoforms with high electrophoretic mobility, GDH1 and GDH2, were in protein extracts from cotyledons and a third isoform with the lowest electrophoretic mobility, GDH3, was identified in protein extracts from root and hypocotyls. Subcellular fractionation of dark-grown soybean tissues demonstrated that GDH3 was associated with intact mitochondria. GDH3 was purified to homogeneity, as determined by native and sodium dodecyl sulfate-polyacrylamide gels. The isoenzyme was composed of a single 42-kD subunit. The pH optima for the reductive amination and the oxidative deamination reactions were 8.0 and 9.3, respectively. At any given pH, GDH activity was 12- to 50-fold higher in the direction of reductive amination than in the direction of the oxidative deamination reaction. GDH3 had a cofactor preference for NAD(H) over NADP(H). The apparent Michaelis constant values for [alpha]-ketoglutarate, ammonium, and NADH at pH 8.0 were 3.6, 35.5, and 0.07 mM, respectively. The apparent Michaelis constant values for glutamate and NAD were 15.8 and 0.10 mM at pH 9.3, respectively. To our knowledge, this is the first biochemical and physical characterization of a purified mitochondrial NAD(H)-dependent GDH isoenzyme from soybean.
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PMID:Purification of Mitochondrial Glutamate Dehydrogenase from Dark-Grown Soybean Seedlings. 1222 51

The air-breathing ureogenic walking catfish (Clarias batrachus) faces various environmental constraints throughout the year leading to the problem of accumulation of toxic ammonia. In the present study, the possible role of conversion of accumulated ammonia to various non-essential free amino acids (FAAs) was tested in this fish under hyper-ammonia stress caused by exposing the fish at 25 mM NH(4)Cl for 7 days. Significant accumulation of ammonia of approximately two- to threefold was observed in different tissues (except in the brain), which was accompanied with the significant accumulation of non-essential FAAs in the NH(4)Cl-exposed fish. There was approximately two- to threefold increase of non-essential FAAs in different tissues and in the plasma of the NH(4)Cl-exposed fish compared to the control fish after 7 days of exposure, which was mainly attributable to the increase of Asp, Ala, Gly, Glu, Gln and taurine (Tau) concentrations in general, with certain tissue-specific variations. This was also accompanied with significant increase of activity of certain amino acid metabolism-related enzymes such as the glutamine synthetase (approx. two- to threefold), glutamate dehydrogenase (ammonia utilizing direction) (approx. twofold), aspartate and alanine aminotransaminases (approx. twofold) mainly in the liver, kidney and muscle of the NH(4)Cl-exposed fish. Thus, it appears that the walking catfish has the capacity of active conversion of accumulated ammonia to non-essential FAAs under condition of high concentrations of external ammonia. However, the increase of urea excretion rate due to active conversion of ammonia to urea via the induced urea cycle appears to be quantitatively much more important pathway than the increase of tissue levels of FAAs in dealing with a severe ammonia load.
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PMID:Role of amino acid metabolism in an air-breathing catfish, Clarias batrachus in response to exposure to a high concentration of exogenous ammonia. 1238 86

Nitrogen metabolism is not only one of the basic processes of plant physiology, but also one of the important parts of global chemical cycle. Plant nitrogen assimilation directly takes part in the synthesis and conversion of amino acid through the reduction of nitrate. During this stage, some key enzymes, e.g., nitrate reductase (NR), glutamine synthetase (GS), glutamate dehydrogenase (GDH), glutamine synthase (GOGAT), aspargine synthetase (AS), and asparate aminotransferase (AspAT) participate these processes. The protein is assimilated in plant cell through amino acid, and becomes a part of plant organism through modifying, classifying, transporting and storing processes, etc. The nitrogen metabolism is associated with carbonic metabolism through key enzyme regulations and the conversion of products, which consists of basic life process. Among these amino acids in plant cell, glutamic acid (Glu), glutamine (Gln), aspartic acid (Asp) and asparagines (Asn), etc., play a key role, which regulates their conversion each other and their contents in the plant cell through regulating formation and activity of those key enzymes. Environmental factors also affect the conversion and recycle of the key amino acids through regulating gene expression of the key enzymes and their activities. Nitrate and light intensity positively regulate the gene transcription of NR, but ammonium ions and Glu, Gln do the negative way. Water deficit is a very serious constraint on N2 fixation rate and soybean (Glycine max Merr.) grain yield, in which, ureide accumulation and degradation under water deficit appear to be the key issues of feedback mechanism on nitrogen fixation. Water stress decreases NR activity, but increases proteinase activity, and thus, they regulate plant nitrogen metabolism, although there are some different effects among species and cultivars. Water stress also decreases plant tissue protein content, ratio of protein and amino acid, and reduces the absorption of amino acid by plant. On the contrary, soil flooding decreases the content and accumulation amount of root nitrogen in winter wheat by 11.9% from booting to flowering stages and 39.1% during grain filling stage, and reduces the ratio of carbon and nitrogen by 79.6%. The results misadjust the metabolism between carbon and nitrogen, and result in the end of the root growth. Elevated CO2 level could decrease plant leaf nitrogen content under well-watered condition, but almost maintain stable under water deficit condition. The radiation of UV-B significantly reduces the partitioning coefficient and synthetic rate of Rubisco, which significantly decreases the photosynthetic rate. This paper reviewed the pathway of plant nitrogen assimilation, characteristics of key enzymes and their regulating mechanisms with picturing the regulating mode of NR, and described the signal sensing and conduct of plant nitrogen metabolism and the formation, transportation, storage and degradation of plant cell protein with picturing the schedule of protein transport of membrane system in plant cell. Seven key tasks are emphasized in this paper in terms of the review on the effects and mechanisms of key ecological factors including water stress on plant nitrogen metabolism. They are: 1) the absorption mechanism of plant based on different nitrogen sources and environmental regulations, 2) the localization and compartmentalization of the key enzymes of nitrogen mechanism in plant cell, 3) the gene and environmental regulating model and their relationships in various key enzymes of nitrogen metabolism, 4) the function of main cell organs and their responses to environmental factors in nitrogen metabolism process, 5) physiological and chemical mechanism of nitrogen and the relationship between the mechanism and protein formation during crop grain filling, 6) improving gene structure of special species or cultivars using gene engineering methods to enhance the resistance to environmental factor stress and the efficiency of absorption and transportation of nitrogen, and 7) the mechanism of natural nitrogen cycle and its response to human activity disturbance.
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PMID:[Research advance in nitrogen metabolism of plant and its environmental regulation]. 1522 8

A mutant (D165N) of clostridial glutamate dehydrogenase (GDH) in which the catalytic Asp is replaced by Asn surprisingly showed a residual 2% of wild-type activity when purified after expression in Escherichia coli at 37 degrees C. This low-level activity also displayed Michaelis constants for substrates that were remarkably similar to those of the wild-type enzyme. Expression at 8 degrees C gave a mutant enzyme preparation 1000 times less active than the first preparation, but progressively, over 2 weeks' incubation at 37 degrees C in sealed vials, this enzyme regained 90% of the specific activity of wild type. This suggested that the mutant might undergo spontaneous deamidation. Mass spectrometric analysis of tryptic peptides derived from D165N samples treated in various ways showed (i) that the Asn is in place in D165N GDH freshly prepared at 8 degrees C; (ii) that there is a time-dependent reversion of this Asn to Asp over the 2-week incubation period; (iii) that detectable deamidation of other Asn residues, in Asn-Gly sequences, mainly occurred in sample workup rather than during the 2-week incubation; (iv) that there is no significant deamidation of other randomly chosen Asn residues in this mutant over the same period; and (v) that when the protein is denatured before incubation, no deamidation at Asn-165 is detectable. It appears that this deamidation depends on the residual catalytic machinery of the mutated GDH active site. A literature search indicates that this finding is not unique and that Asn may not be a suitable mutational replacement in the assessment of putative catalytic Asp residues by site-directed mutagenesis.
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PMID:Spontaneous chemical reversion of an active site mutation: deamidation of an asparagine residue replacing the catalytic aspartic acid of glutamate dehydrogenase. 1573 73

The role of residue C323 in catalysis by human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was examined by substituting Arg, Gly, Leu, Met, or Tyr at C323 by cassette mutagenesis using synthetic human GDH isozyme genes. As a result, the Km of the enzyme for NADH and alpha-ketoglutarate increased up to 1.6-fold and 1.1-fold, respectively. It seems likely that C323 is not responsible for substrate-binding or coenzyme-binding. The efficiency (kcat/Km) of the mutant enzymes was only 11-14% of that of the wild-type isozymes, mainly due to a decrease in kcat values. There was a linear relationship between incorporation of [14C]p-chloromercuribenzoic acid and loss of enzyme activity that extrapolated to a stoichiometry of one mol of [14C] incorporated per mol of monomer for wild type hGDHs. No incorporation of [14C]p-chloromer-curibenzoic acid was observed with the C323 mutants. ADP and GTP had no effect on the binding of p-chloromercuribenzoic acid, suggesting that C323 is not directly involved in allosteric regulation. There were no differences between the two hGDH isozymes in sensitivities to mutagenesis at C323. Our results suggest that C323 plays an important role in catalysis by human GDH isozymes.
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PMID:Critical role of the cysteine 323 residue in the catalytic activity of human glutamate dehydrogenase isozymes. 1575 Mar 46


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