Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase.
The enzyme F1-ATPase has been shown to be a rotary motor in which the central gamma-subunit rotates inside the cylinder made of alpha3beta3 subunits. At low ATP concentrations, the motor rotates in discrete 120 degrees steps, consistent with sequential ATP hydrolysis on the three beta-subunits. The mechanism of stepping is unknown. Here we show by high-speed imaging that the 120 degrees step consists of roughly 90 degrees and 30 degrees substeps, each taking only a fraction of a millisecond. ATP binding drives the 90 degrees substep, and the 30 degrees substep is probably driven by release of a hydrolysis product. The two substeps are separated by two reactions of about 1 ms, which together occupy most of the ATP hydrolysis cycle. This scheme probably applies to rotation at full speed ( approximately 130 revolutions per second at saturating ATP) down to occasional stepping at nanomolar ATP concentrations, and supports the binding-change model for ATP synthesis by reverse rotation of F1-ATPase.
Yasuda
R
R
CREST 'Genetic Programming' Team 13, Teikyo University Biotechnology Center 3F, Nogawa 907, Miyamae-Ku, Kawasaki 216-0001, Japan.
Noji
H
H
Yoshida
M
M
Kinosita
K
K
Itoh
H
H
D000255:Adenosine Triphosphate / Q000378:metabolism*; D020134:Catalytic Domain; D006868:Hydrolysis; D007700:Kinetics; D008853:Microscopy; D008863:Microspheres; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000378:metabolism*; D021122:Protein Subunits; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D012399:Rotation
D020409:Molecular Motor Proteins; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases