title: Purification and properties of glutamate synthase and glutamate dehydrogenase from Bacillus megaterium.
abstract: Bacillus megaterium N.C.T.C. no. 10342 exhibits glutamate synthetase (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 3mM-NH4Cl and low when they were grown on 100mM-NH4Cl, whereas glutamate dehydrogenase concentrations were higher when the bacteria were grown on 100mM-NH4Cl than on 3mM-NH4Cl. Glutamate synthase and glutamate dehydrogenase were purified to homogeneity from B. megaterium grown in 10mM-glucose/10mM-NH4Cl. The purified enzymes had mol.wts. 840000 and 270000 for glutamate synthase and glutamate dehydrogenase respectively. The Km values for substrates with NADPH and coenzyme were (glutamate synthase activity shown first) 9 micron and 360 micron for 2-oxoglutarate, 7.1 micron and 8.7 micron 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 mol.wt. approx 200000 was the main product formed.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000641:Ammonia; D001410:Bacillus megaterium / Q000201:enzymology*; D055598:Chemical Phenomena; D002621:Chemistry; D004120:Dimethyl Suberimidate; D004591:Electrophoresis, Polyacrylamide Gel; D005969:Glutamate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D005970:Glutamate Synthase / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D000637:Transaminases / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D004120:Dimethyl Suberimidate; D000641:Ammonia; D005970:Glutamate Synthase; D005969:Glutamate Dehydrogenase; D000637:Transaminases