RESUMO
The pH dependence of the kinetic parameters of the glutamine- and ammonia-dependent reactions of Azospirillum brasilense glutamate synthase revealed the presence of ionizable groups with pKa values between 6 and 10 involved in the binding of the substrates and in catalytic steps. The V profile of the glutamine-dependent reaction is complicated by a deviation from a simple bell-shaped curve between pH 8 and pH 10, which may suggest that deprotonation of a group with pKa value in this region decreases but does not abolish glutamine-dependent enzyme activity. This group does not seem to be required in the ammonia-dependent reaction of GltS, which decreases on the acidic and alkaline sides as groups with pKa values of about 8.8 and 9.9 dissociate. The V/K profile for ammonia exhibits a single pKa value of about 8.7, suggesting that ammonia is the actual substrate of the enzyme, and that ammonia binding to glutamate synthase is largely pH independent. The hypothesis that a group with pKa between 8 and 10 is involved in the glutaminase segment of the glutamine-dependent glutamate synthase activity was supported by studies of the modification of the enzyme by 6-diazo-5-oxo-L-norleucine, a glutamine analog, and iodoacetamide, a cysteine-directed reagent. Analyses of the kinetics of inactivation of the enzyme in the presence and absence of enzyme substrates and their analogs at different pH values demonstrated that iodoacetamide reacts with a group involved in glutamine binding and/or activation, most likely the cysteine residue at the N-terminus of glutamate synthase alpha subunit, which may form a Cys-His ion pair in the active site of glutamate synthase, as suggested for other amidotransferases (Mei, B., and Zalkin, H. (1989) J. Biol. Chem. 264, 16613-16619).
Assuntos
Azospirillum brasilense/enzimologia , Cisteína/química , Glutamato Sintase/metabolismo , Histidina/química , Iodoacetamida/farmacologia , Sequência de Aminoácidos , Amônia/farmacologia , Catálise , Glutamato Sintase/antagonistas & inibidores , Glutamato Sintase/química , Glutamina/metabolismo , Glutamina/farmacologia , Concentração de Íons de Hidrogênio , Iodoacetamida/metabolismo , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/farmacologia , Cinética , Dados de Sequência Molecular , NADP/metabolismo , NADP/farmacologia , Análise de SequênciaRESUMO
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.