The kinetic mechanism of the reactions catalyzed by the glutamate synthase from Azospirillum brasilense.
The reactions catalyzed by glutamate synthase from Azospirillum brasilense have been investigated by a combination of absorption spectroscopy, steady-state kinetic measurements and experiments with stereospecifically labelled substrate. The data show that both L-glutamine-dependent and ammonia-dependent reactions of the glutamate synthase from A. brasilense follow an identical two-site uni-uni bi-bi kinetic mechanism, in which the enzyme is alternately reduced by NADPH and oxidized by the iminoglutarate formed on addition of ammonia to the C2 of 2-oxoglutarate. The spectroscopic experiments support the involvement of the enzyme chromophores (flavins and iron-sulfur centers) in both reactions. Finally, using stereospecifically labelled NADPH, we showed that the enzyme from Azospirillum is specific for the transfer of the 4S hydrogen of NADPH. During the catalysis of both L-glutamine-dependent and ammonia-dependent reactions, this hydrogen atom equilibrates with the solvent. The data obtained with glutamate synthase from A. brasilense, a diazotroph, differ significantly from those regarding the ammonia-dependent reaction of other glutamate synthases. The ammonia-dependent activity of glutamate synthase from Azospirillum is not physiologically significant, representing only a segment of the overall physiological L-glutamine-dependent activity and requiring the enzyme flavins and iron-sulfur centers. Finally, the data are not consistent with the hypothesis [Geary, L. E. & Meister, A. (1977) J. Biol. Chem. 252, 3501-3508] that the small subunit of glutamate synthase is endowed with a glutamate-dehydrogenase-like activity.
Vanoni
M A
MA
Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Italy.
Nuzzi
L
L
Rescigno
M
M
Zanetti
G
G
Curti
B
B
D000641:Ammonia / Q000494:pharmacology; D015806:Azospirillum brasilense / Q000201:enzymology*; D005970:Glutamate Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism / Q000494:pharmacology; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013379:Substrate Specificity; D014316:Tritium
D007656:Ketoglutaric Acids; D005973:Glutamine; D014316:Tritium; D009249:NADP; D000641:Ammonia; D005970:Glutamate Synthase