Analysis of the kinetic mechanism of halophilic NADP-dependent glutamate dehydrogenase.
The amination of 2-oxoglutarate catalyzed by NADP-specific glutamate dehydrogenase (EC 1.4.1.4, L-glutamate:NADP+ oxidoreductase (deaminating)) from Halobacterium halobium has been analyzed by initial rate, graphical analysis, and product and competitive inhibition studies. Initial rate and graphical analysis reveal that a B term (representing 2-oxoglutarate) is not statistically necessary for an initial rate equation. However, the absence of a B term does not distinguish between ordered and random binding of NADPH and ammonia. The patterns of product inhibition by NADP+ and L-glutamate, and competitive inhibition by hydroxylamine and succinate permit deduction of the kinetic mechanism as ordered, with NADPH, 2-oxoglutarate and ammonia added in that order, and L-glutamate release preceding NADP+ release.
Bonete
M J
MJ
Division de Bioquímica, Facultad de Ciencias, Universidad de Alicante, Spain.
Camacho
M L
ML
Cadenas
E
E
D000641:Ammonia / Q000378:metabolism; D001667:Binding, Competitive; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D006217:Halobacterium / Q000201:enzymology*; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D009249:NADP / Q000378:metabolism*
D005971:Glutamates; D007656:Ketoglutaric Acids; D018698:Glutamic Acid; D009249:NADP; D000641:Ammonia; D005969:Glutamate Dehydrogenase