Purification and properties of NADP-dependent glutamate dehydrogenase from Streptomyces fradiae.
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 Mr of the native enzyme was determined to be 200,000 by size-exclusion high-performance liquid chromatography whereas after sodium dodecyl sulphate-polyacrylamide gel electrophoresis one major band of Mr 49,000 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 (Km) were 28.6 mM for L-glutamate and 0.12 mM for NADP. Km values for reductive amination were 1.54 mM for 2-oxoglutarate, 0.07 mM for NADPH and 30.8 mM for NH+4. The enzyme activity was significantly reduced by adenine nucleotides, particularly ATP.
Vancurová
I
I
Institute of Microbiology, Czechoslovak Academy of Sciences, Prague.
Vancura
A
A
Volc
J
J
Kopecký
J
J
Neuzil
J
J
Basarová
G
G
Bĕhal
V
V
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D003067:Coenzymes / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D018086:Glutamate Dehydrogenase (NADP+); D008970:Molecular Weight; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity
D003067:Coenzymes; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D005969:Glutamate Dehydrogenase; D018086:Glutamate Dehydrogenase (NADP+)