Partial Purification and Characterization of Glutamate Synthase from a Thermophilic Bacillus

Abstract

Glutamate synthase (GOGAT), glutamine synthetase (GS), NAD+-dependent glutamate dehydrogenase (GDH) and NAD+-dependent alanine dehydrogenase (AlaDH) activities were detected in cell extracts of Bacillus stearothermophilus PH24, a strain deficient in NADP+-dependent GDH. GDH and GOGAT activities were low under most growth conditions and GOGAT was not detectable in extracts of cells grown with amino acids as carbon and nitrogen source. AlaDH and GS activities were more variable, the former being high in cells grown on l-alanine as carbon and nitrogen source. GS was repressed during growth with high concentrations of NH4C1 as nitrogen source but a corresponding increase in AlaDH activities suggests that this enzyme may replace NADP+-dependent GDH as the main enzyme for ammonia assimilation under these conditions. GOGAT was purified 40-fold using affinity chromatography on NADPH-Sepharose. The molecular weight of the partially purified enzyme was estimated to be 160000 and K m values for NADPH, 2-oxoglutarate and l-glutamine were 22, 15 and 29 μm, respectively. Glutamine could be replaced by NH4C1 as nitrogen donor (K m 44 mm) but the rate was only 10% to 15% that of the l-glutamine-dependent reaction. The pH optimum for glutamine-dependent activity was 8·0 and the temperature optimum 75 C: the enzyme displayed a discontinuous Arrhenius plot over the range 30 °C to 75 °C. Azaserine, l-methionine sulphone and Cibacron Blue 3GA were all inhibitors and the enzyme was rapidly inactivated in the presence of NADPH when l-glutamine and 2-oxoglutarate were absent.