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)

Reduced pyridine nucleotide dependent glutamate synthase [L-glutamate: NADP+ oxidoreductase (transaminating); EC 1.4.1.13] was purified to homogeneity from Bacillus subtilis PCI 219. The molecular weight of the enzyme was 210,000, and the enzyme was composed of two nonidentical subunits with molecular weights of 160,000 and 56,000. The absorption and CD spectra of the enzyme indicated that the enzyme is an iron-sulfur flavoprotein. The enzyme was found to contain 1:1:7.4:8.7 mol of FMN, FAD, iron atoms, and acid-labile sulfur atoms per mol (MW 210,000). EPR measurements of the NADPH-reduced enzyme at 77K revealed the formation of a stable flavin semiquinone intermediate; however, none of the signals originating from the iron-sulfur cluster was observed. Still at 4.2K the EPR signals in the region of g = 2, which may originate from the paramagnetic iron-sulfur cluster, were clearly observed for both the isolated and dithionite-reduced states of the enzyme. The enzyme exhibited a wide coenzyme specificity, and either NADPH or NADH could be used as electron donor, although the latter was less effective. The enzyme activity was also expressed when ammonium chloride was substituted for L-glutamine. The optimum pHs for NADPH-Gln-, NADH-Gln-, and NADPH-NH3-dependent reactions were 7.8, 6.9, and 9.4, respectively. The apoenzyme exhibited substantial inactivation of the Gln-dependent activities but still retained the NH3-dependent activities. Enzyme reduction-oxidation experiments, initial velocity experiments, and product inhibition patterns revealed that both the NADPH-Gln- and NADH-Gln-dependent reactions coincided with the two-site ping-pong uni-uni bi-bi kinetic mechanism, while the NADPH-NH3-dependent reaction deviated from Michaelis-Menten kinetics. The Gln-dependent activities were inhibited by several TCA cycle members, especially L-malate and fumarate, as well as L-methionine-SR-sulfoximine, pyridoxal-5'-phosphate, and pCMB. The regulation of the glutamate synthase, glutamine synthetase [EC 6.3.1.2], and glutamate dehydrogenase [EC 1.4.1.3] activities was examined with cultures of cells grown with various nitrogen and carbon sources.
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PMID:Glutamate synthase from Bacillus subtilis PCI 219. 301 66

Pyrococcus furiosus is a strictly anaerobic archaeon that grows optimally at 100 degrees C by a fermentative-type metabolism in which complex peptide mixtures such as yeast extract and Tryptone, and also certain sugars, are oxidized to organic acids, H2 and CO2. Enzymes involved in the utilization of peptides such as proteases, aromatic amino transferases, and glutamate dehydrogenase have been previously purified from this organism. It is shown here that P. furiosus also contains significant cytoplasmic concentrations of a new enzyme termed indolepyruvate ferredoxin oxidoreductase (IOR). This catalyzes the oxidative decarboxylation of aryl pyruvates, which are generated by the transamination of aromatic amino acids, to the corresponding aryl acetyl-CoA. IOR is a tetramer (alpha 2 beta 2) of two identical subunits (66,000 and 23,000 Da) with a molecular weight of 180,000. The enzyme contains one molecule of thiamine pyrophosphate and four [4Fe-4S]2+,1+ and one [3Fe-4S]0,1+ cluster, as determined by iron analyses and EPR spectroscopy. Significant amounts of other metals such as copper and zinc were not detected. IOR was virtually inactive at 25 degrees C and exhibited optimal activity above 90 degrees C (at pH 8.0) and at pH 8.5-10.5 (at 80 degrees C). The enzyme was sensitive to inactivation by O2, losing 50% of its activity after exposure to air for 20 min at 23 degrees C, and was quite thermostable, with a half-life of activity at 80 degrees C (under anaerobic conditions) of about 80 min. The Km values (in microM) for indolepyruvate, p-hydroxyphenylpyruvate, phenylpyruvate, CoASH, and P. furiosus ferredoxin, the physiological electron carrier, were 250, 110, 90, 17, and 48, respectively. IOR was inhibited by KCN (apparent Ki = 7.5 mM), but not by CO (1 atm). An enzyme analogous to IOR has not been reported previously. Curiously, it has few properties in common with the pyruvate ferredoxin oxidoreductase of P. furiosus, even though the two enzymes catalyze virtually identical reactions. In fact, of known ketoacid oxidoreductases, the catalytic mechanism of IOR appears to be most similar to that of the pyruvate ferredoxin oxidoreductase from the hyperthermophilic bacterium Thermotoga maritima.
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PMID:Indolepyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus. A new enzyme involved in peptide fermentation. 820 94

Glutamate synthase is a complex iron-sulfur flavoprotein containing one molecule each of FAD and FMN and three distinct iron-sulfur centers/alpha beta protomer. Production of the beta subunit was observed in total extracts of Escherichia coli BL21 (DE) cells harbouring a pT7-7 derivative carrying gltD, the gene encoding the Azospirillum brasilense glutamate synthase beta subunit. The protein was soluble, and the identity of the purified protein with the Azospirillum glutamate synthase beta subunit was confirmed by N-terminal sequence analysis. The kinetic and spectroscopic characterization of the glutamate synthase beta subunit confirmed that it contains the NADPH binding site, but, in contrast with earlier proposals that assigned both FAD and FMN binding sites to the alpha subunit of glutamate synthase, the beta subunit was shown to contain stoichiometric amounts of FAD. No iron-sulfur centers were detected by EPR spectroscopy measurements of the recombinant beta subunit. Under steady-state conditions, the glutamate synthase beta subunit can catalyze the NADPH-dependent reduction of several synthetic electron acceptors but no glutamate synthase or glutamate dehydrogenase reactions in either direction. The results are in agreement with previous data from our laboratory and, together with the absence of amino acid sequence similarity between glutamate synthase beta subunit and glutamate dehydrogenases, are against the hypothesis that glutamate synthase is evolutionarily derived from the association of an ancestral glutamate dehydrogenase (the beta subunit) and an amidotransferase (the alpha subunit). The protein-bound FAD is reduced by NADPH at a rate much faster than turnover with synthetic electron acceptors, leading to formation of a stable reduced flavin-NADP+ charge-transfer complex. The rate of reduction of the bound FAD by NADPH is also similar to the rate at which one of the flavins is reduced in the native glutamate synthase, as measured in a stopped-flow spectrophotometer under pre-steady-state conditions. The ability of FAD bound to the beta subunit of glutamate synthase to react with NADPH and the lack of reactivity with sulfite lead us to conclude that FAD is Flavin 1 of glutamate synthase [Vanoni, M.A., Edmondson, D.E., Zanetti, G. & Curti, B. (1992) Biochemistry 31, 4613-4623].
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PMID:Properties of the recombinant beta subunit of glutamate synthase. 866 16