THE PH-DEPENDENT BEHAVIOR OF CATALYTIC ACTIVITIES OF AZOSPIRILLUM-BRASILENSE GLUTAMATE 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 ammo nia-dependent reactions of Azospirillum brasilense glutamate synthase revealed the presence of ionizable groups with pK(a) values between 6 and 10 involved in the binding of the substrates and in catalytic step s. 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, w hich may suggest that deprotonation of a group with pK(a) value in thi s region decreases but does not abolish glutamine-dependent enzyme act ivity. This group does not seem to be required in the ammonia-dependen t reaction of GltS, which decreases on the acidic and alkaline sides a s groups with pK(a) values of about 8.8 and 9.9 dissociate. The V/ K p rofile for ammonia exhibits a single pK, value of about 8.7, suggestin g that ammonia is the actual substrate of the enzyme, and that ammonia binding to glutamate synthase is largely pH independent. The hypothes is that a group with pK(a) between 8 and 10 is involved in the glutami nase segment of the glutamine-dependent glutamate synthase activity wa s supported by studies of the modification of the enzyme by 6-diazo-5- oxo-L-norleucine, a glutamine analog, and iodoacetamide, a cysteine-di rected 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 gro up involved in glutamine binding and/ or activation, most likely the c ysteine residue at the N-terminus of glutamate synthase alpha subunit, which may form a Cys-His ion pair in the active site of glutamate syn thase, as suggested for other amidotrans-ferases (Mei, B., and Zalkin, H. (1989) J. Biol. Chem. 264, 16613-16619). (C) 1994 Academic Press, Inc.