Abstract
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 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- beta 185 and Asp- beta 185 ATPase activities were altered from that of the wild 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 wild 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)
Highlights
The Hϩ-ATPase (ATP synthase) of Escherichia coli synthesizes ATP similar to those of mitochondria or chloroplasts
Properties of 19 Mutants at Position 185 (Glu, wild type) of the  Subunit—Systematic mutagenesis between Thr-156 and Lys-201 of the  subunit indicated that Glu-181 and Arg182 are essential for catalysis [6]
All the mutants except Asp-185 could not grow on succinate through oxidative phosphorylation, they exhibited substantial F0F1 assemblies in membranes (Table I)
Summary
E. coli and Growth Conditions—Strain DK8 (⌬unc B-C, ilv::Tn10, thi) [14] lacking the unc operon was used as a host for recombinant plasmids. The reaction was terminated by 200-fold dilution with a buffer (2 mM Tris-HCl, pH 8.0, 2 mM dithiothreitol, and 2 g/ml bovine serum albumin) or by removal of excess IAA using a centrifuge column. Other Procedures—Membrane vesicles were prepared as described elsewhere [18] using 10 mM Tris-HCl buffer, pH 8.0, containing 140 mM KCl, 0.5 mM dithiothreitol, 10% glycerol, 0.5 mM phenylmethanesulfonyl fluoride, 5 g/ml leupeptin, and 5 g/ml pepstatin A. ATPase activities were assayed at 37 °C in 20 mM Tris-HCl, pH 8.0, 4 mM ATP, and 2 mM MgCl2 [19] unless otherwise specified. Other reagents used were of the highest grade commercially available
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