Stochastic high-speed rotation of Escherichia coli ATP synthase F1 sector: the epsilon subunit-sensitive rotation.
The gamma subunit of the ATP synthase F(1) sector rotates at the center of the alpha(3)beta(3) hexamer during ATP hydrolysis. A gold bead (40-200 nm diameter) was attached to the gamma subunit of Escherichia coli F(1), and then its ATP hydrolysis-dependent rotation was studied. The rotation speeds were variable, showing stochastic fluctuation. The high-speed rates of 40- and 60-nm beads were essentially similar: 721 and 671 rps (revolutions/s), respectively. The average rate of 60-nm beads was 381 rps, which is approximately 13-fold faster than that expected from the steady-state ATPase turnover number. These results indicate that the F(1) sector rotates much faster than expected from the bulk of ATPase activity, and that approximately 10% of the F(1) molecules are active on the millisecond time scale. Furthermore, the real ATP turnover number (number of ATP molecules converted to ADP and phosphate/s), as a single molecule, is variable during a short period. The epsilon subunit inhibited rotation and ATPase, whereas epsilon fused through its carboxyl terminus to cytochrome b(562) showed no effect. The epsilon subunit significantly increased the pausing time during rotation. Stochastic fluctuation of catalysis may be a general property of an enzyme, although its understanding requires combining studies of steady-state kinetics and single molecule observation.
Nakanishi-Matsui
Mayumi
M
Futai Special Laboratory, Microbial Chemistry Research Center, Microbial Chemistry Research Foundation, CREST, Japan Science and Technology Agency, Kamiosaki, Shinagawa, Tokyo.
Kashiwagi
Sachiko
S
Hosokawa
Hiroyuki
H
Cipriano
Daniel J
DJ
Dunn
Stanley D
SD
Wada
Yoh
Y
Futai
Masamitsu
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D011506:Proteins / Q000502:physiology*; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D012399:Rotation; D000097795:ATPase Inhibitory Protein
D000255:Adenosine Triphosphate; D011506:Proteins; D006180:Proton-Translocating ATPases; D000097795:ATPase Inhibitory Protein; C444660:gamma subunit, F(1) ATPase