title: Mechanical modulation of ATP-binding affinity of V1-ATPase.
abstract: V(1)-ATPase is a rotary motor protein that rotates the central shaft in a counterclockwise direction hydrolyzing ATP. Although the ATP-binding process is suggested to be the most critical reaction step for torque generation in F(1)-ATPase (the closest relative of V(1)-ATPase evolutionarily), the role of ATP binding for V(1)-ATPase in torque generation has remained unclear. In the present study, we performed single-molecule manipulation experiments on V(1)-ATPase from Thermus thermophilus to investigate how the ATP-binding process is modulated upon rotation of the rotary shaft. When V(1)-ATPase showed an ATP-waiting pause, it was stalled at a target angle and then released. Based on the response of the V(1)-ATPase released, the ATP-binding probability was determined at individual stall angles. It was observed that the rate constant of ATP binding (k(on)) was exponentially accelerated with forward rotation, whereas the rate constant of ATP release (k(off)) was exponentially reduced. The angle dependence of the k(off) of V(1)-ATPase was significantly smaller than that of F(1)-ATPase, suggesting that the ATP-binding process is not the major torque-generating step in V(1)-ATPase. When V(1)-ATPase was stalled at the mean binding angle to restrict rotary Brownian motion, k(on) was evidently slower than that determined from free rotation, showing the reaction rate enhancement by conformational fluctuation. It was also suggested that shaft of V(1)-ATPase should be rotated at least 277° in a clockwise direction for efficient release of ATP under ATP-synthesis conditions.
authors.affiliation: Department of Applied Chemistry, School of Engineering, the University of Tokyo, Tokyo 113-8656, Japan.
authors.forename: Naciye Esma
authors.initials: NE
authors.lastname: Tirtom
authors.forename: Daichi
authors.initials: D
authors.lastname: Okuno
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nakano
authors.forename: Ken
authors.initials: K
authors.lastname: Yokoyama
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Noji
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D001671:Biochemistry / Q000379:methods; D001703:Biophysics / Q000379:methods; D006868:Hydrolysis; D007700:Kinetics; D020409:Molecular Motor Proteins / Q000378:metabolism; D011485:Protein Binding; D013314:Stress, Mechanical; D016963:Thermus thermophilus / Q000378:metabolism; D013997:Time Factors; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry*
chemical_list: D020409:Molecular Motor Proteins; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases