NADPH/NADH-dependent cold-labile glutamate dehydrogenase in Azospirillum brasilense. Purification and properties.
A cold-labile glutamate dehydrogenase (GDH, EC 1.4.1.3) has been purified to homogeneity from the crude extracts of Azospirillum brasilense. The purified enzyme shows a dual coenzyme specificity, and both the NADPH and NADH-dependent activities are equally cold-sensitive. The enzyme is highly specific for the substrates 2-oxoglutarate and glutamate. Kinetic studies with GDH indicate that the enzyme is primarily designed to catalyse the reductive amination of 2-oxoglutarate. The NADP+-linked activity of GDH showed Km values 2.5 X 10(-4) M and 1.0 X 10(-2) M for 2-oxoglutarate and glutamate respectively. NAD+-linked activity of GDH could be demonstrated only for the amination of 2-oxoglutarate but not for the deamination of glutamate. The Lineweaver-Burk plot with ammonia as substrate for NADPH-dependent activity shows a biphasic curve, indicating two apparent Km values (0.38 mM and 100 mM) for ammonia; the same plot for NADH-dependent activity shows only one apparent Km value (66 mM) for ammonia. The NADPH-dependent activity shows an optimum pH from 8.5 to 8.6 in Tris/HCl buffer, whereas in potassium phosphate buffer the activity shows a plateau from pH 8.4 to 10.0. At high pH (greater than 9.5) amino acids in general strongly inhibit the reductive amination reaction by their competition with 2-oxoglutarate for the binding site on GDH. The native enzyme has a Mr = 285000 +/- 20000 and appears to be composed of six identical subunits of Mr = 48000 +/- 2000. The GDH level in A. brasilense is strongly regulated by the nitrogen source in the growth medium.
D000596:Amino Acids / Q000494:pharmacology; D001419:Bacteria / Q000201:enzymology* / Q000254:growth & development; D002021:Buffers; D003080:Cold Temperature; D004591:Electrophoresis, Polyacrylamide Gel; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D009584:Nitrogen / Q000378:metabolism; D012988:Soil Microbiology; D013379:Substrate Specificity
D000596:Amino Acids; D002021:Buffers; D009243:NAD; D009249:NADP; D005969:Glutamate Dehydrogenase; D009584:Nitrogen