Purification and properties of glutamate synthase from Thiobacillus thioparus

Abstract

Glutamate synthase was purified .apprx. 250-fold from T. thioparus and was characterized. The MW was estimated as 280,000 g/mol. The enzyme showed absorption maxima at 280, 380 and 450 nm and was inhibited by Atebrin, suggesting that T. thioparus glutamate synthase is a flavoprotein. The enzyme activity was also inhibited by Fe chelators and thiol-binding agents. The enzyme was specific for NADPH and .alpha.-ketoglutarate, but L-glutamine was partially replaced by NH3 as the amino donor. The Km values for glutamate synthease for NADPH, .alpha.-ketoglutarate and glutamine were 3.0 .mu.M, 50 .mu.M and 1.1 mM, respectively. The enzyme had a pH optimum between 7.3-7.8. Glutamate synthase from T. thioparus was relatively insensitive to feedback inhibition by single amino acids but was sensitive to the combined effects of several amino acids. Enzymes involved in glutamate synthesis in T. thioparus were studied. Glutamine synthetase, glutamate synthase and 2 glutamate dehydrogenases (NADH and NADPH dependent) were present in this organism. The levels of glutamate synthase and glutamate dehydrogenase were similar in T. thioparus grown on 0.7 or 7.0 mM (NH4)2 SO4. The sum of the activities of both glutamate dehydrogenases was only 1/25 of that of glutamate synthase under the assay conditions. The glutamine pathway is important for NH3 assimilation in this autotrophic bacterium.