title: Controlled rotation of the F₁-ATPase reveals differential and continuous binding changes for ATP synthesis.
abstract: F(1)-ATPase is an ATP-driven rotary molecular motor that synthesizes ATP when rotated in reverse. To elucidate the mechanism of ATP synthesis, we imaged binding and release of fluorescently labelled ADP and ATP while rotating the motor in either direction by magnets. Here we report the binding and release rates for each of the three catalytic sites for 360° of the rotary angle. We show that the rates do not significantly depend on the rotary direction, indicating ATP synthesis by direct reversal of the hydrolysis-driven rotation. ADP and ATP are discriminated in angle-dependent binding, but not in release. Phosphate blocks ATP binding at angles where ADP binding is essential for ATP synthesis. In synthesis rotation, the affinity for ADP increases by >10(4), followed by a shift to high ATP affinity, and finally the affinity for ATP decreases by >10(4). All these angular changes are gradual, implicating tight coupling between the rotor angle and site affinities.
authors.affiliation: Department of Physics, Faculty of Science, Gakushuin University, Toshima-ku, Tokyo 171-8588, Japan. kengo@aoni.waseda.jp
authors.forename: Kengo
authors.initials: K
authors.lastname: Adachi
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Oiwa
authors.forename: Masasuke
authors.initials: M
authors.lastname: Yoshida
authors.forename: Takayuki
authors.initials: T
authors.lastname: Nishizaka
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Kinosita
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D001407:Bacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D020134:Catalytic Domain; D006868:Hydrolysis; D007700:Kinetics; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012399:Rotation
chemical_list: D001426:Bacterial Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases