Rikiya Watanabe

Phosphate release in F1-ATPase catalytic cycle follows ADP release

F(1)-ATPase is an ATP-driven rotary motor protein in which the γ-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 γ 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.

How is the Temperature Sensitive (TS) Reaction of F1-ATPase Coupled with its Rotation?

F1-ATPase (F1) is a rotary molecular motor in which γ subunit rotates against α3β3 cylinder. So far, 80° and 40° substeps in rotation of F1 have been observed, and kinetic analysis has shown that the 80° substep is initiated by concomitant ATP binding and ADP release, and the 40° substep occurs after ATP hydrolysis and inorganic phosphate release. Temperature sensitive (TS) reaction of F1 has found recently at the ATP binding angle at the temperature below 10 °C, and it has been suggested to correspond to ADP release (Watanabe et al., EMBO report 2008). In this study, TS reaction of βE190D mutant, that has much slower ATP hydrolysis rate than wild type, was found below 20 °C. Q10 factor of TS reaction of βE190D mutant was 16, that is comparable to that of wild type. Previous single-molecule rotation observations of the hybrid F1 that has only one mutant β subunit, α3β(WT)2β(E190D)γ have shown that the β subunit binds ATP at 0°, cleaves ATP at 200°, and release ADP and inorganic phosphate after cleavage of ATP (Ariga et al., NSMB 2007). In contrast to our expectation, observation of hybrid F1 α3β(WT)2β(E190D)γ rotation at 18 °C has revealed that TS reaction occurs at ATP binding angle(0°) to mutated β subunit. Furthermore, simultaneous observation at the video rate (30 frames per seconds) showed that Cy3-ATP bindings and steps in rotation occur concomitantly. We will discuss about the reaction schemes that can explain these results.