Glu-185 of the Escherichia coli H-ATPase (ATP synthase) subunit was replaced by 19 different amino acid residues. The rates of multisite (steady state) catalysis of all the mutant membrane ATPases except Asp-185 were less than 0.2% of the wild type one; the Asp-185 enzyme exhibited 15% (purified) and 16% (membrane-bound) ATPase activity. The purified inactive Cys-185 F-ATPase recovered substantial activity after treatment with iodoacetate in the presence of MgCl; maximal activity was obtained upon the introduction of about 3 mol of carboxymethyl residues/mol of F. The divalent cation dependences of the S-carboxymethyl-185 and Asp-185 ATPase activities were altered from that of the wild type. The Asp-185, Cys-185, S-carboxymethyl-185, and Gln-185 enzymes showed about 130, 60, 20, and 50% of the wild type unisite catalysis rates, respectively. The S-carboxymethyl-185 and Asp-185 enzymes showed altered divalent cation sensitivities, and the S-carboxymethyl-185 enzyme showed no Mg inhibition. Unlike the wild type, the two mutant enzymes showed low sensitivities to azide, which stabilizes the enzyme MgADP complex. These results suggest that Glu-185 may form a Mg binding site, and its carboxyl moiety is essential for catalytic cooperativity. Consistent with this model, the bovine glutamate residue corresponding to Glu-185 is located close to the catalytic site in the higher order structure (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E.(1994) Nature 370, 621-628).

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