Direct identification of the rotary angle of ATP cleavage in F1-ATPase from Bacillus PS3 |
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Institution: | 1. Tsukuba Research Center, Central Research Laboratory, Hamamatsu Photonics K.K., Ibaraki 300-2635, Japan;2. Graduate School of Arts & Sciences, The University of Tokyo, Tokyo 153-8902, Japan;3. Department of Physics, Faculty of Science, Gakushuin University, Tokyo 171-8588, Japan;4. Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Chiba 278-8510, Japan;5. Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo, 113-8656, Japan |
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Abstract: | F1-ATPase is the world’s smallest biological rotary motor driven by ATP hydrolysis at three catalytic β subunits. The 120° rotational step of the central shaft γ consists of 80° substep driven by ATP binding and a subsequent 40° substep. In order to correlate timing of ATP cleavage at a specific catalytic site with a rotary angle, we designed a new F1-ATPase (F1) from thermophilic Bacillus PS3 carrying β(E190D/F414E/F420E) mutations, which cause extremely slow rates of both ATP cleavage and ATP binding. We produced an F1 molecule that consists of one mutant β and two wild-type βs (hybrid F1). As a result, the new hybrid F1 showed two pausing angles that are separated by 200°. They are attributable to two slowed reaction steps in the mutated β, thus providing the direct evidence that ATP cleavage occurs at 200° rather than 80° subsequent to ATP binding at 0°. This scenario resolves the long-standing unclarified issue in the chemomechanical coupling scheme and gives insights into the mechanism of driving unidirectional rotation. |
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