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[摘要]：F-1-ATPase is an ATP-driven rotary motor protein in which the gamma-subunit rotates against the catalytic stator ring. Although the reaction scheme of F-1 has mostly been revealed, the timing of inorganic phosphate (P-i) release remains controversial. Here we addressed this issue by verifying the reversibility of ATP hydrolysis on arrested F-1 with magnetic tweezers. ATP hydrolysis was found to be essentially reversible, implying that P-i is released after the gamma rotation and ADP release, although extremely slow P-i release was found at the ATP hydrolysis angle as an uncoupling side reaction. On the basis of this finding, we deduced the chemomechanical coupling scheme of F-1. We found that the affinity for P-i was strongly angle dependent, implying a large contribution by P-i release to torque generation. These findings imply that under ATP synthesis conditions, P-i binds to an empty catalytic site, preventing solution ATP (though not ADP) from binding. Thus, this supports the concept of selective ADP binding for efficient ATP synthesis. |
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