PMID:7592742
| Citation |
Omote, H, Le, NP, Park, MY, Maeda, M and Futai, M (1995) Beta subunit Glu-185 of Escherichia coli H(+)-ATPase (ATP synthase) is an essential residue for cooperative catalysis. J. Biol. Chem. 270:25656-60 |
|---|---|
| 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) |
| Links | |
| Keywords |
Allosteric Regulation; Amino Acid Sequence; Azides/pharmacology; Binding Sites; Conserved Sequence; Dose-Response Relationship, Drug; Enzyme Activation; Escherichia coli/enzymology; Escherichia coli/genetics; Glutamic Acid/genetics; Iodoacetates/pharmacology; Iodoacetic Acid; Lithium Chloride/pharmacology; Magnesium Chloride/pharmacology; Methylation; Molecular Sequence Data; Mutagenesis, Site-Directed; Proton-Translocating ATPases/drug effects; Proton-Translocating ATPases/genetics; Proton-Translocating ATPases/metabolism; Structure-Activity Relationship |
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