title: Single-molecule study on the temperature-sensitive reaction of F1-ATPase with a hybrid F1 carrying a single beta(E190D).
abstract: F(1)-ATPase is a rotary molecular motor in which the gamma-subunit rotates against the alpha(3)beta(3) cylinder. The unitary gamma-rotation is a 120 degrees step comprising 80 and 40 degrees substeps, each of these initiated by ATP binding and ADP release and by ATP hydrolysis and inorganic phosphate release, respectively. In our previous study on gamma-rotation at low temperatures, a highly temperature-sensitive (TS) reaction step of F(1)-ATPase from thermophilic Bacillus PS3 was found below 9 degrees C as an intervening pause before the 80 degrees substep at the same angle for ATP binding and ADP release. However, it remains unclear as to which reaction step the TS reaction corresponds. In this study, we found that the mutant F(1)(beta E190D) from thermophilic Bacillus PS3 showed a clear pause of the TS reaction below 18 degrees C. In an attempt to identify the catalytic state of the TS reaction, the rotation of the hybrid F(1), carrying a single copy of beta E190D, was observed at 18 degrees C. The hybrid F(1) showed a pause of the TS reaction at the same angle as for the ATP binding of the incorporated beta E190D, although kinetic analysis revealed that the TS reaction is not the ATP binding step. These findings suggest that the TS reaction is a structural rearrangement of beta before or after ATP binding.
authors: Institute of Scientific and Industrial Research, Osaka University, 567-0047 Osaka, Japan.
authors: Sawako
authors: S
authors: Enoki
authors: Rikiya
authors: R
authors: Watanabe
authors: Ryota
authors: R
authors: Iino
authors: Hiroyuki
authors: H
authors: Noji
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases