Chemo-mechanical coupling in F(1)-ATPase revealed by catalytic site occupancy during catalysis.
F(1)-ATPase is a rotary molecular motor in which the central gamma subunit rotates inside a cylinder made of alpha(3)beta(3) subunits. To clarify how ATP hydrolysis in three catalytic sites cooperate to drive rotation, we measured the site occupancy, the number of catalytic sites occupied by a nucleotide, while assessing the hydrolysis activity under identical conditions. The results show hitherto unsettled timings of ADP and phosphate releases: starting with ATP binding to a catalytic site at an ATP-waiting gamma angle defined as 0 degrees , phosphate is released at approximately 200 degrees , and ADP is released during quick rotation between 240 degrees and 320 degrees that is initiated by binding of a third ATP. The site occupancy remains two except for a brief moment after the ATP binding, but the third vacant site can bind a medium nucleotide weakly.
Shimo-Kon
Rieko
R
Department of Physics, Faculty of Science and Engineering, Waseda University, Okubo, Shinjuku-ku, Tokyo, Japan. kazuhiko@waseda.jp
Muneyuki
Eiro
E
Sakai
Hiroshi
H
Adachi
Kengo
K
Yoshida
Masasuke
M
Kinosita
Kazuhiko
K
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D001665:Binding Sites; D001703:Biophysics / Q000379:methods*; D002384:Catalysis; D020134:Catalytic Domain; D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D010100:Oxygen / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013314:Stress, Mechanical
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D010100:Oxygen