title: Robustness of the rotary catalysis mechanism of F1-ATPase.
abstract: F1-ATPase (F1) is the rotary motor protein fueled by ATP hydrolysis. Previous studies have suggested that three charged residues are indispensable for catalysis of F1 as follows: the P-loop lysine in the phosphate-binding loop, GXXXXGK(T/S); a glutamic acid that activates water molecules for nucleophilic attack on the γ-phosphate of ATP (general base); and an arginine directly contacting the γ-phosphate (arginine finger). These residues are well conserved among P-loop NTPases. In this study, we investigated the role of these charged residues in catalysis and torque generation by analyzing alanine-substituted mutants in the single-molecule rotation assay. Surprisingly, all mutants continuously drove rotary motion, even though the rotational velocity was at least 100,000 times slower than that of wild type. Thus, although these charged residues contribute to highly efficient catalysis, they are not indispensable to chemo-mechanical energy coupling, and the rotary catalysis mechanism of F1 is far more robust than previously thought.
authors.affiliation: From the Department of Applied Chemistry, University of Tokyo, PRESTO, Japan Science and Technology Agency, Bunkyo-ku, Tokyo 113-8656, and.
authors.forename: Rikiya
authors.initials: R
authors.lastname: Watanabe
authors.affiliation: the Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Matsukage
authors.affiliation: From the Department of Applied Chemistry, University of Tokyo.
authors.forename: Ayako
authors.initials: A
authors.lastname: Yukawa
authors.affiliation: From the Department of Applied Chemistry, University of Tokyo, PRESTO, Japan Science and Technology Agency, Bunkyo-ku, Tokyo 113-8656, and.
authors.forename: Kazuhito V
authors.initials: KV
authors.lastname: Tabata
authors.affiliation: From the Department of Applied Chemistry, University of Tokyo, hnoji@appchem.t.u-tokyo.ac.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Noji
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases
keywords: ATP Synthase; Bioenergetics; F1FO-ATPase; Molecular Motor; Single-molecule Biophysics