NADP+-dependent glutamate dehydrogenase from the facultative methylotroph Hyphomicrobium X

Abstract NADP⁺-dependent glutamate dehydrogenase (GDH; EC 1.4.1.4) was purified using acetone precipitation, heat, DEAE-cellulose and dye-ligand Ramazol Red column chromatography. The M _r of the native enzyme was estimated to be 380 000 (± 10 000) by polyacrylamide gel electrophoresis. The same technique in the presence of sodium dodecyl sulphate (SDS) gave one subunit band with an M _r of 63 400 (±4000). Thus the enzyme has a hexameric structure. The enzyme has a pH optimum of 8.5 and has K _m apparent values of 1.6 mM, 0.015 mM and 10.2 mM for α-ketoglutarate, N NADPH and L -glutamate, respectively. Michaelis-Menten kinetics were not observed when the ammonium concentration was increased. A progressive increase in the ammonium concentration resulted in a progressively increasing K _m value. The enzyme was highly specific for all substrates and markedly insensitive to inhibitors.