Purification and properties of glutamate synthase from Bacillus licheniformis.
Glutamate synthase [L-glutamate:NADP+ oxidoreductase (transaminating); EC 1.4.1.13](GltS) was purified to homogeneity from Bacillus licheniformis A5. The native enzyme had a molecular weight of approximately 220,000 and was composed of two nonidentical subunits (molecular weights, approximately 158,000 and approximately 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 GltS from B. licheniformis had a requirement for NADPH, alpha-ketoglutarate, and glutamine. Classical hyperbolic kinetics were seen for NADPH affinity, which resulted in an apparent Km value of 13 microM. Nonhyperbolic kinetics were obtained for alpha-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 microM, and the apparent Km values obtained for alpha-ketoglutarate were 6 and 50 microM. GltS activity was found to be relatively insensitive to inhibition by amino acids, keto acids, or various nucleotides. L-Methionine-DL-sulfoximine, L-methionine sulfone, and DL-methionine sulfoxide were found to be potent inhibitors of GltS activity, yielding I0.5 values of 150, 11, and 250 microM, 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.
D000596:Amino Acids / Q000032:analysis; D001407:Bacillus / Q000201:enzymology*; D005415:Flavins / Q000032:analysis; D005970:Glutamate Synthase / Q000302:isolation & purification* / Q000378:metabolism; D007501:Iron / Q000032:analysis; D007700:Kinetics; D008982:Molybdenum / Q000032:analysis; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D000637:Transaminases / Q000302:isolation & purification*
D000596:Amino Acids; D005415:Flavins; D009249:NADP; D008982:Molybdenum; D007501:Iron; D005970:Glutamate Synthase; D000637:Transaminases