Purification and properties of glutamate synthase from Bacillus licheniformis
Links to Fileshttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC214775/
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Type of Work9 pages
Citation of Original PublicationH J Schreier and R W Bernlohr, Purification and properties of glutamate synthase from Bacillus licheniformis, Journal ListJ Bacteriolv.160(2); 1984 Nov, PMC214775, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC214775/
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Glutamate synthase [L-glutamate:NADP+ oxidoreductase (transaminating); EC 126.96.36.199] (GItS) was purified to homogeneity from BaciUus licheniformis A5. The native enzyme had a molecular weight of approximately 220,000 and was composed of two nonidentical subunits (molecular weights, -158,000 and -54,000). The enzyme was found to contain 8.1 ± 1 iron atoms and 8.1 + 1 acid-labile sulfur atoms per 220,000-dalton dimer. Two flavin moieties were found per 220,000-dalton dimer, with a ratio of flavin adenine dinucleotide to flavin mononucleotide of 1.2. The UV-visible spectrum of the enzyme exhibited maxima at 263,380 and 450 nm. The GitS from B. Iicheniformis had a requirement for NADPH, oa-ketoglutarate, and glutamine. Classical hyperbolic kinetics were seen for NADPH affinity, which resulted in an apparent Km value of 13 ,uM. Nonhyperbolic kinetics were obtained for a-ketoglutarate and glutamine affinities, and the reciprocal plots obtained for these substrates were biphasic. The apparent Km values obtained for glutamine were 8 and 100 ,LM, and the apparent Km values obtained for a-ketoglutarate were 6 and 50 ,uM. GltS activity was found to be relatively insensitive to inhibition by amino acids, keto acids, or various nucleotides. L-Methionine-DLsulfoximine, L-methionine sulfone, and DL-methionine sulfoxide were found to be potent inhibitors of GltS activity, yielding 10.5 values of 150, 11, and 250 ,uM, respectively. GltSs were purified from cells grown in the presence of ammonia and nitrate as sole nitrogen sources and were compared. Both yielded identical final specific activities and identical physical (UV-visible spectra, flavin, and iron-sulfur composition) and kinetic characteristics.