Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.4.3.11 (glutamate dehydrogenase)
 4,437 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Genetic manipulation of nitrogenase and key glutamate-forming enzymes can provide mutants that excrete fixed N2 as NH4+. A derepressed N2 fxation mutant (SK-24) has been isolated , which excretes up to 20.2 mumol of fixed N2 as NH4+ per mg of cell protein in 24 hr at room temperature. Biochemical analysis shows that this mutant, which requires glutamate for growth, releases fixed N2 as NH4+ into the environment because of (i) constitutive synthesis of nitrogenase and (ii) genetic blocks resulting in losses of glutamate synthase [L-glutamine:2-oxoglutarate aminotransferase (NADPH oxidizing), EC 2.6.1.53] and glutamate dehydrogenase [L-glutamate:NADP oxidoreductase (deaminating), EC 1.4.1.4] activities, enzymes essential for NH4+ assimilation into cell material. The parent strain (asm-1), missing only glutamate synthase activity, also actively excretes NH4+ during early phases of its growth but eventually reutilizes the NN4+. A miximum yield of 4.0 mumol of NH4+/ml per 24 hr has been noted for asm-1 only during the growth period. Biosynthesis of NH4+ PROCEEDS AT THE EXPENSE OF A Variety of fermentable sugars, such as sucrose or glucose, with a maximum energy conversion efficiency of about 5 glucose degraded per NH4+ formed. The use of microbes for production of NH4+ fertilizer is discussed.
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PMID:Microbial production of ammonium ion from nitrogen. 109 Sep 30

Wild-type glutamate dehydrogenase (EC 1.4.1.4) from Salmonella typhimurium reacts at 25 degrees C in 0.1 M phosphate buffer, pH 7, with the nucleotide analogue 2-[(4-bromo-2,3-dioxobutyl)thio]-adenosine 2',5'-bisphosphate (2-BDB-TA 2',5'-DP) to give 78% inactivation. Protection against inactivation was achieved with NADPH, indicating that modification occurred in the region of the coenzyme binding site. After reaction of the enzyme with 2-BDB-TA 2',5'-DP, the dioxo moiety of the bound reagent was reduced with [3H]NaBH4. The radioactive peptide which corresponds to the sequence Leu282-Cys283-Glu284-Ile285-Lys286 was isolated by HPLC from tryptic digests of inactive modified enzyme but was absent in digests of active enzyme modified in the presence of NADPH. Mutant enzyme E284Q was 64% inactived by 2-BDB-TA 2',5'-DP and modification of the corresponding Leu282-Lys286 peptide was found, while neither mutant enzyme C283I nor C283I:E284Q was inactivated by the nucleotide analogue and no corresponding radioactive peptides were found. These results show that cysteine-283 is the target of the reagent and is located near the coenzyme binding site. The nucleotide analogue 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A 2',5'-DP) has also been shown to react with cysteine-283 (L. Haeffner-Gormley et al., 1991, J. Biol. Chem. 266, 5388-5394). However, the predominant form of the Leu282-Lys286 peptide after reaction with 2-BDB-TA 2',5'-DP contained only 0.17 mol tritium/mol leucine, whereas the 2-BDB-T epsilon A 2',5'-DP-modified peptide contained 1.80 mol tritium/mol leucine; these results indicate that the reaction product of 2-BDB-T epsilon A 2',5'-DP retains two reducible carbonyl groups while these are not available in the product of 2-BDB-TA 2',5'-DP. It is suggested that cysteine-283 reacts primarily at a carbonyl group of 2-BDB-TA 2',5'-DP to form a thiohemiacetal derivative, while it reacts at the methylene group of 2-BDB-T epsilon A 2',5'-DP with displacement of bromide. Both nucleotide analogues also yielded, in small amount, a crosslinked peptide containing the sequences 282-286 and 299-333, indicating proximity between these regions in the native structure.
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PMID:Reaction of the nucleotide analogue 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 2',5'-bisphosphate at the coenzyme site of wild-type and mutant NADP(+)-specific glutamate dehydrogenases from Salmonella typhimurium. 130 91

Glutamate, glutamine, and ammonia pool size have been determined in two S. cerevisiae strains (GOGAT+ and GOGAT-) growing under ammonia excess and limitation at a dilution rate of 0.10/h. The biomass levels and glutamate dehydrogenase NADPH-dependent (NADPH-GDH) activities were also measured for both strains. The strain that lacks GOGAT activity showed lower levels of metabolites under both media and lower levels of biomass under carbon limitation (ammonia excess) compared to the GOGAT+ strain. Under nitrogen limitation, the biomass level was the same for both strains, but GOGAT- changed from rounded to ellipsoidal cells.
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PMID:Ammonia assimilation in S. cerevisiae under chemostatic growth. 132 53

In vitro alterations induced by a 10 micrograms/ml and 50 micrograms/ml dose each of thiophenate and fenbendazole on the absorptive surfaces of Haemonchus contortus (Nematoda: Trichostrongylidae) were studied. The most significant changes were induced in the gut epithelium. Alkaline phosphatase and adenosine triphosphatase activities were decreased, succinic dehydrogenase activity was increased, while acid phosphatase and glucose-6-phosphatase were completely lost from the intestinal epithelium after treatment with either of the drugs. A stimulatory effect of these two anthelmintics was observe on lactic dehydrogenase and reduced nicotinamide adenine dinucleotide diaphorase distribution. Thiophenate caused an increase in the activities of glutamate dehydrogenase (GDH), glucose-6-phosphate dehydrogenase (G-6-PD) and nonspecific esterases and a decrease in reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-D) activity. Fenbendazole treatment led to the inhibition of GDH, while G-6-PD, NADPH-D, cytochrome oxidase, monoamine oxidase and nonspecific esterase activity remained unaltered in the epithelium.
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PMID:Histoenzymic effects of thiophenate and fenbendazole on the absorptive surfaces of Haemonchus contortus. 133 82

The purification and some properties of NADP-dependent glutamate dehydrogenase (GDH) and glutamine synthetase (GS) from the facultatively anaerobic Gram-negative bacterium Paracoccus denitrificans were investigated. The enzymes were purified to homogeneity using a procedure which involved affinity chromatography on Blue Sepharose CL-6B as the major purification step. The recoveries in the purification of GDH and GS were 28% and 64%, respectively. The specific activity of purified GDH was 183 nkat (mg protein)-1 (deaminating reaction). GDH was composed of subunits of molecular mass 47 kDa and the native enzyme was either a tetramer or hexamer. The apparent Km values for L-glutamate, NADP, 2-oxoglutarate, NADPH and ammonia were 1.5 mM, 5.9 microM, 0.47 microM, 12.5 microM and 14 mM, respectively. The specific activity of purified GS was 1125 nkat (mg protein)-1 (transferase reaction). The molecular mass of native GS was 570 kDa; it was composed of 12 subunits of molecular mass 50.1 kDa. The apparent Km values for L-glutamine and hydroxylamine in the transferase reaction were 2.1 and 2.4 mM, respectively; those of ammonia, L-glutamate and ATP in the biosynthetic reaction were 0.03, 1 and 0.17 mM, respectively. After the adenylylation of GS, the Km for L-glutamine and L-glutamate increased and reached the values of 8.0 and 27 mM, respectively. The effects of the changes in GS activity on the ammonia metabolism of Paracoccus denitrificans are discussed.
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PMID:Purification and some properties of glutamate dehydrogenase and glutamine synthetase from Paracoccus denitrificans. 135 41

Characteristics of the three major ammonia assimilatory enzymes, glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GO-GAT) in Corynebacterium callunae (NCIB 10338) were examined. The GDH of C. callunae specifically required NADPH and NADP+ as coenzymes in the amination and deamination reactions, respectively. This enzyme showed a marked specificity for alpha-ketoglutarate and glutamate as substrates. The optimum pH was 7.2 for NADPH-GDH activity (amination) and 9.0 for NADP(+)-GDH activity (deamination). The results showed that NADPH-GDH and NADP(+)-GDH activities were controlled primarily by product inhibition and that the feedback effectors alanine and valine played a minor role in the control of NADPH-GDH activity. The transferase activity of GS was dependent on Mn+2 while the biosynthetic activity of the enzyme was dependent on Mg2+ as essential activators. The pH optima for transferase and biosynthetic activities were 8.0 and 7.0, respectively. In the transfer reaction, the Km values were 15.2 mM for glutamine, 1.46 mM for hydroxylamine, 3.5 x 10(-3) mM for ADP and 1.03 mM for arsenate. Feedback inhibition by alanine, glycine and serine was also found to play an important role in controlling GS activity. In addition, the enzyme activity was sensitive to ATP. The transferase activity of the enzyme was responsive to ionic strength as well as the specific monovalent cation present. GOGAT of C. callunae utilized either NADPH or NADH as coenzymes, although the latter was less effective. The enzyme specifically required alpha-ketoglutarate and glutamine as substrates.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Some properties of glutamate dehydrogenase, glutamine synthetase and glutamate synthase from Corynebacterium callunae. 135 47

Corynebacterium callunae (NCIB 10338) grows faster on glutamate than ammonia when used as sole nitrogen sources. The levels of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (GOGAT; EC 1.4.1.13) of C. callunae were found to be influenced by the nitrogen source. Accordingly, the levels of GS and GOGAT activities were decreased markedly under conditions of ammonia excess and increased under low nitrogen conditions. In contrast, glutamate dehydrogenase (GDH; EC 1.4.1.4) activities were not significantly affected by the type or the concentration of the nitrogen source supplied. The carbon source in the growth medium could also affect GDH, GS and GOGAT levels. Of the carbon sources tested in the presence of 2 mM or 10 mM ammonium chloride as the nitrogen source pyruvate, acetate, fumarate and malate caused a decrease in the levels of all three enzymes as compared with glucose. GDH, GS and GOGAT levels were slightly influenced by aeration. Also, the enzyme levels varied with the growth phase. Methionine sulfoximine, an analogue of glutamine, markedly inhibited both the growth of C. callunae cells and the transferase activity of GS. The apparent Km values of GDH for ammonia and glutamate were 17.2 mM and 69.1 mM, respectively. In the NADPH-dependent reaction of GOGAT, the apparent Km values were 0.1 mM for alpha-ketoglutarate and 0.22 mM for glutamine.
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PMID:The effect of various culture conditions on the levels of ammonia assimilatory enzymes of Corynebacterium callunae. 135 48

The catalytic activity, expressed as Km and Vmax values, of 16 enzymes of practical interest with the macromolecular coenzymes poly(ethylene glycol)-N6-(2-aminoethyl)-NAD+ and poly(ethylene glycol)-N6-(2-aminoethyl)-NADP+ and their low molecular weight precursors N6-(2-aminoethyl)-NAD+ and N6-(2-aminoethyl)-NADP+, was investigated. The enzymes examined are of direct interest for organic synthesis (i.e. alcohol dehydrogenase from yeast, horse liver, or Thermoanaerobium brockii, lactic dehydrogenase, and several hydroxysteroid dehydrogenases) or are used for the regeneration of NAD+, NADP+, NADH, or NADPH (i.e. glutamate dehydrogenase from liver or Proteus, formate dehydrogenase, glucose dehydrogenase, and malic enzyme). The cycling efficiency of poly(ethylene glycol)-N6-(2-aminoethyl)-NADP+ was examined with coupled-enzymes or coupled-substrates systems. Poly(ethylene glycol)-N6-(2-aminoethyl)-NAD+ and, even more so, poly(ethylene glycol)-N6-(2-aminoethyl)-NADP+ were excellent coenzymes with several dehydrogenases. In addition, the coenzymatic properties of N6-(3-sulfonatopropyl)-NAD+, an NAD+ derivative carrying a strong anionic group, were compared with those of the newly synthesized N6-(2-hydroxy-3-trimethylammonium propyl)-NAD+, an NAD+ derivative carrying a strong cationic group. It was expected that the presence of the sulfonic or quaternary ammonium group would enhance the residence time of the coenzyme inside continuous-flow reactors if membranes with anionic or cationic groups, respectively, were used.
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PMID:Coenzymatic properties of low molecular-weight and macromolecular N6-derivatives of NAD+ and NADP+ with dehydrogenases of interest for organic synthesis. 136 82

The determination of ammonia in plasma, using glutamate dehydrogenase, is complicated by non-specific oxidation of the coenzyme, promoted by components of the sample matrix. Measurements performed with appropriate plasma blanks show that 2'-phosphorylated coenzymes (NADPH, deamino-NADPH) are much less oxidized than NADH. By adding lactate dehydrogenase, NADH oxidation by endogenous pyruvate can be completed within a short time. Considerable consumption of coenzyme occurs, however, and endogenous L-alanine aminotransferase also represents a possible source of interference. The results of ammonia determinations using deamino-NADPH (y) or NADPH (x) were identical (a = 0.0 mumol/l, b = 1.00; r = 0.996, n = 62). With NADH as the coenzyme, the method displays adequate specificity only at high sample dilution, e.g. in the measurement of urea after conversion to ammonia.
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PMID:Which is the appropriate coenzyme for the measurement of ammonia with glutamate dehydrogenase? 145 16

Affinity labeling studies of NADP(+)-glutamate dehydrogenase from Salmonella typhimurium have shown that the peptide Leu-282-Lys-286 is located near the coenzyme site [Haeffner-Gormley et al. (1991) J. Biol. Chem. 266, 5388-5394]. The present study was undertaken to evaluate the role of lysine-286. The mutant enzymes K286R, K286Q, and K286E were prepared by site-directed mutagenesis, expressed in Escherichia coli, and purified. The Vmax values (micromoles of NADPH per minute per milligram of protein) were similar for WT (270), K286R (529), K296Q (409), and K286E (382) enzymes. As measured at pH 7.9, the Km value for NADPH was much greater for K286E (280 microM) than for WT (9.8 microM), K286R (30 microM), or K286Q (66 microM) enzymes. The efficiencies (kcat/Km) of the WT and K286R mutant were similar (1.2 x 10(3) min-1 microM-1 and 1.0 x 10(3) min-1 microM-1, respectively) while those of K286Q (0.30 x 10(3) min-1 microM-1) and K286E (0.07 x 10(3) min-1 microM-1) were greatly reduced. The decreased efficiency of the K286E mutant results from the increase in Km-NADPH, consistent with a role for a basic residue at position 286 which enhances the binding of NADPH. Plots of Vmax vs pH showed the pH optima to be 8.1-8.3 for all enzymes at saturating NADPH concentrations. A 40-fold increase in Km-NADPH for K286E was observed as the pH increased from 5.98 to 8.08, from which a unique pKe of 6.5 was calculated.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Importance of lysine-286 at the NADP site of glutamate dehydrogenase from Salmonella typhimurium. 151 Sep 67


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