The pH-Dependent Behavior of Catalytic Activities of Azospirillum brasilenseGlutamate Synthase and Iodoacetamide Modification of the Enzyme Provide Evidence for a Catalytic Cys-His Ion Pair

The pH dependence of the kinetic parameters of the glutamine- and ammonia-dependent reactions of Azospirillum brasilenseglutamate synthase revealed the presence of ionizable groups with pKavalues between 6 and 10 involved in the binding of the substrates and in catalytic steps. The Vprofile 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 pKavalue 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 pKavalues of about 8.8 and 9.9 dissociate. The V/Kprofile for ammonia exhibits a single pKavalue 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 pKabetween 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 α 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).