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.
Watanabe
Rikiya
R
Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan.
Iino
Ryota
R
Noji
Hiroyuki
H
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology; D001665:Binding Sites; D055162:Biocatalysis; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D010710:Phosphates / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*
D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases