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 subunit was replaced by 19 different amino acid residues. The rates of multisite (steady state) catalysis of all the mutant membrane ATPases except Asp-beta 185 were less than 0.2% of the wild type one; the Asp-beta 185 enzyme exhibited 15% (purified) and 16% (membrane-bound) ATPase activity. The purified inactive Cys-beta 185 F-1-ATPase recovered substantial 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 dependences 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 185, S-carboxymethyl-beta 185, and Gln-beta 185 enzymes showed about 130, 60, 20, and 50% of the mild type unisite catalysis rates, respectively. The S-carboxymethyl-beta 185 and Asp-beta 185 enzymes showed altered divalent cation sensitivities, and the S-carboxymethyl-beta 185 enzyme showed no Mg2+ inhibition. Unlike the wild type, the two mutant enzymes showed low sensitivities to azide, which stabilizes the enzyme Mg . ADP complex. These results suggest that Glu-beta 185 may form a Mg2+ binding site, and its carboxyl moiety is essential for catalytic cooperativity. 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., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628).