β Subunit Glu-185 of Escherichia coli H+-ATPase (ATP synthase) is an essential residue for cooperative catalysis

Abstract

Glu-β185 of the Escherichia coil H+-ATPase (ATP synthase) β subunit was replaced by 19 different amino acid residues. The rates of multisite (steady state) catalysis of all the mutant membrane ATPases except Asp-β185 were less than 0.2% of the wild type one; the Asp-β185 enzyme exhibited 15% (purified) and 16% (membrane-bound) ATPase activity. The purified inactive Cys-β185 F1-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 F1. The divalent cation dependences of the S-carboxymethyl-β185 and Asp-β185 ATPase activities were altered from that of the wild type. The Asp-β185, Cys- β185, S-carboxymethyl-β185, and Gln-β185 enzymes showed about 130, 60, 20, and 50% of the wild type unisite catalysis rates, respectively. The S- carboxymethyl-β185 and Asp-β85 enzymes showed altered divalent cation sensitivities, and the S-carboxymethyl-β185 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-β185 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-β185 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).