title: The role of beta-Arg-182, an essential catalytic site residue in Escherichia coli F1-ATPase.
abstract: Beta-Arg-182 in Escherichia coli F1-ATPase (beta-Arg-189 in bovine mitochondrial F1) is a residue which lies close to catalytic site bound nucleotide (Abrahams et al. (1994) Nature 370, 621-628). Here we investigated the role of this residue by characterizing two mutants, betaR182Q and betaR182K. Oxidative phosphorylation and steady-state ATPase activity of purified F1 were severely impaired by both mutations. Catalytic site nucleotide-binding parameters were measured using the fluorescence quench of beta-Trp-331 that occurred upon nucleotide binding to purified F1 from betaR182Q/betaY331W and betaR182K/betaY331W double mutants. It was found that (a) beta-Arg-182 interacts with the gamma-phosphate of MgATP, particularly at catalytic sites 1 and 2, (b) beta-Arg-182 has no functional interaction with the beta-phosphate of MgADP or with the magnesium of the magnesium-nucleotide complex in the catalytic sites, and (c) beta-Arg-182 is directly involved in the stabilization of the catalytic transition state. In these features the role of beta-Arg-182 resembles that of another positively charged residue in the catalytic site, the conserved lysine of the Walker A motif, beta-Lys-155. A further role of beta-Arg-182 is suggested, namely involvement in conformational change at the catalytic site beta-alpha subunit interface that is required for multisite catalysis.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester Medical Center, New York 14642, USA.
authors.forename: S
authors.initials: S
authors.lastname: Nadanaciva
authors.forename: J
authors.initials: J
authors.lastname: Weber
authors.forename: A E
authors.initials: AE
authors.lastname: Senior
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000535:Aluminum / Q000737:chemistry; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005461:Fluorine / Q000737:chemistry; D005973:Glutamine / Q000737:chemistry / Q000235:genetics; D008239:Lysine / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
chemical_list: D005973:Glutamine; C053629:fluoroaluminum; D005461:Fluorine; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D001120:Arginine; D000535:Aluminum; D006180:Proton-Translocating ATPases; D008239:Lysine