Purification and Properties of NADP-dependent Glutamate Dehydrogenase from Streptomyces fradiae

Summary: Streptomyces fradiae has two chromatographically distinct forms of glutamate dehydrogenase (GDH): one GDH utilizes NAD as coenzyme, the other uses NADP. The intracellular level of both GDHs is strongly regulated by the nitrogen source in the growth medium. NADP-dependent GDH was purified to homogeneity from crude extracts of S. fradiae. The M r of the native enzyme was determined to be 200000 by size-exclusion high-performance liquid chromatography whereas after sodium dodecyl sulphate-polyacrylamide gel electrophoresis one major band of M r 49000 was found, suggesting that the enzyme is a tetramer. The enzyme was highly specific for the substrates 2-oxoglutarate and l-glutamate, and required NADP, which could not be replaced by NAD, as a cofactor. The pH optimum was 9·2 for oxidative deamination of glutamate and 8·4 for reductive amination of 2-oxoglutarate. The Michaelis constants (K m) were 28·6 mM for l-glutamate and 012 mM for NADP. K m values for reductive amination were 1·54 mM for 2-oxoglutarate, 007 mM for NADPH and 30·8 mM for NH4 +. The enzyme activity was significantly reduced by adenine nucleotides, particularly ATP.