Mechanistic studies on Azospirillum brasilense glutamate synthase.
The reaction mechanism of Azospirillum brasilense glutamate synthase has been investigated by several approaches. 15N nuclear magnetic resonance studies demonstrate that the amide nitrogen of glutamine is reductively transferred to 2-oxoglutarate in an irreversible manner with no release of the transferred ammonia group into the medium. Identical results were obtained using thio-NADPH and acetylpyridine-NADPH, which are shown to be less efficient substrates of the enzyme than NADPH. Similarly, no exchange of the ammonia group being transferred with exogenous ammonium ion was observed during catalysis. The glutamate formed as the product of the iminoglutarate reduction was determined to be in the L configuration. The enzyme was also found to catalyze, under anaerobic conditions, the exchange of the 4proS H of NADPH with solvent both in the absence and in the presence of 2-oxoglutarate and glutamine. The reductive half-reaction is therefore a reversible segment of the overall irreversible amidotransferase reaction. 15N NMR studies also showed that the enzyme does not catalyze glutamate dehydrogenase/oxidase reactions or any observable glutaminase activity under neutral (pH 7.5) conditions. Glutaminase activity was also not observable with the reduced enzyme alone or in the presence of D-glutamate (a competitive inhibitor of glutamate synthase with respect to 2-oxoglutarate, with a Ki of about 11 microM) or with the oxidized enzyme in the presence of 2-oxoglutarate, D-glutamate, or NADP+. These data confirm species-dependent differences of A. brasilense glutamate synthase with respect to the enzyme from other sources.
Dipartimento di Fisiologia e Biochimica Generali, Universita degli Studi di Milano, Italy.
M A
MA
Vanoni
D E
DE
Edmondson
M
M
Rescigno
G
G
Zanetti
B
B
Curti
D000641:Ammonia / Q000378:metabolism; D015806:Azospirillum brasilense / Q000201:enzymology*; D005970:Glutamate Synthase / Q000378:metabolism*; D005971:Glutamates / Q000096:biosynthesis; D018698:Glutamic Acid; D005972:Glutaminase / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism / Q000494:pharmacology; D007656:Ketoglutaric Acids / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011725:Pyridines / Q000378:metabolism
D005971:Glutamates; D007656:Ketoglutaric Acids; D011725:Pyridines; C010835:3-acetylpyridine; D005973:Glutamine; D018698:Glutamic Acid; D009249:NADP; D000641:Ammonia; D005970:Glutamate Synthase; D005972:Glutaminase