Purification and properties of glutamate synthase and glutamate dehydrogenase from Bacillus megaterium.
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.
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*
D000596:Amino Acids; D004120:Dimethyl Suberimidate; D000641:Ammonia; D005970:Glutamate Synthase; D005969:Glutamate Dehydrogenase; D000637:Transaminases