title: Beta subunit Glu-185 of Escherichia coli H(+)-ATPase (ATP synthase) is an essential residue for cooperative catalysis.
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)
authors.affiliation: Department of Biological Science, Osaka University, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Omote
authors.forename: N P
authors.initials: NP
authors.lastname: Le
authors.forename: M Y
authors.initials: MY
authors.lastname: Park
authors.forename: M
authors.initials: M
authors.lastname: Maeda
authors.forename: M
authors.initials: M
authors.lastname: Futai
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001386:Azides / Q000494:pharmacology; D001665:Binding Sites; D017124:Conserved Sequence; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D018021:Lithium Chloride / Q000494:pharmacology; D015636:Magnesium Chloride / Q000494:pharmacology; D008745:Methylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D006180:Proton-Translocating ATPases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D001386:Azides; D007461:Iodoacetates; D015636:Magnesium Chloride; D018698:Glutamic Acid; D006180:Proton-Translocating ATPases; D018021:Lithium Chloride; D019807:Iodoacetic Acid