Purification and properties of glutamate synthase and glutamate dehydrogenase from Bacillus megaterium

Abstract

B. megaterium N.C.T.C. no. 10342 exhibits glutamate synthase (EC 2.6.1.53) and glutamate dehydrogenase (EC 1.4.1.4) activities. Concentrations of glutamate synthase were high when the bacteria were grown on 3 mM-NH4Cl and low when they were grown on 100 mM-NH4Cl, but glutamate dehydrogenase concentrations were higher when the bacteria were grown on 100 mM-NH4Cl than on 3 mM-NH4Cl. Glutamate synthase and glutamate dehydrogenase were purified to homogeneity from B. megaterium grown in 10 mM-glucose/10 mM-NH4Cl. The purified enzymes had MW 840,000 and 270,000 for glutamate synthase and glutamate dehydrogenase, respectively. The Km values for substrates with NADPH and coenzyme were (glutamate synthase activity shown first) 9.mu.M and 360 .mu.M for 2-oxoglutarate, 7.1 .mu.M and 8.7 .mu.M for NADPH, and 0.2 mM for glutamine and 22 mM for NH4Cl, similar values to those of enzymes from Escherichia coli. Glutamate synthase contained NH3-dependent activity (different from authentic glutamate dehydrogenase), which was enhanced 4-fold during treatment at pH 4.6. NH3-dependent activity was generally about 2% of the glutamine-dependent activity. Amidination of glutamate synthase by the bi-functional cross-linking reagent dimethyl suberimidate inactivated glutamine-dependent glutamate synthase activity, but increased NH3-dependent activity. A cross-linked structure of MW approximately 200,000 was the main product formed.