BETA-SUBUNIT GLU-185 OF ESCHERICHIA-COLI H-ATPASE (ATP SYNTHASE) IS AN ESSENTIAL RESIDUE FOR COOPERATIVE CATALYSIS()

Glu-beta 185 of the Escherichia coli H+-ATPase (ATP synthase) beta sub unit was replaced by 19 different amino acid residues. The rates of mu ltisite (steady state) catalysis of all the mutant membrane ATPases ex cept Asp-beta 185 were less than 0.2% of the wild type one; the Asp-be ta 185 enzyme exhibited 15% (purified) and 16% (membrane-bound) ATPase activity. The purified inactive Cys-beta 185 F-1-ATPase recovered sub stantial activity after treatment with iodoacetate in the presence of MgCl2; maximal activity was obtained upon the introduction of about 3 mol of carboxymethyl residues/mol of F-1. The divalent cation dependen ces of the S-carboxymethyl-beta 185 and Asp-beta 185 ATPase activities were altered from that of the mild type, The Asp-beta 185, Cys-beta 1 85, S-carboxymethyl-beta 185, and Gln-beta 185 enzymes showed about 13 0, 60, 20, and 50% of the mild type unisite catalysis rates, respectiv ely. The S-carboxymethyl-beta 185 and Asp-beta 185 enzymes showed alte red divalent cation sensitivities, and the S-carboxymethyl-beta 185 en zyme showed no Mg2+ inhibition. Unlike the wild type, the two mutant e nzymes showed low sensitivities to azide, which stabilizes the enzyme Mg . ADP complex. These results suggest that Glu-beta 185 may form a M g2+ binding site, and its carboxyl moiety is essential for catalytic c ooperativity. Consistent with this model, the bovine glutamate residue corresponding to Glu-beta 185 is located close to the catalytic site in the higher order structure (Abrahams, J. P., Leslie, A. G. W., Lutt er, R., and Walker, J. E. (1994) Nature 370, 621-628).