Temperature dependence of the rotation and hydrolysis activities of F1-ATPase |
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Authors: | Furuike Shou Adachi Kengo Sakaki Naoyoshi Shimo-Kon Rieko Itoh Hiroyasu Muneyuki Eiro Yoshida Masasuke Kinosita Kazuhiko |
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Affiliation: | * Department of Physics, Faculty of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan † Institute of Industrial Science, University of Tokyo, Meguro-ku, Tokyo, Japan ‡ Tsukuba Research Laboratory, Hamamatsu Photonics KK, Tsukuba, Japan § CREST “Formation of Soft Nano-Machines” Team 13*, Tokodai, Tsukuba, Japan ¶ Department of Physics, Faculty of Science and Engineering, Chuo University, Tokyo, Japan || Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta, Yokohama, Japan ** ICORP ATP Synthesis Regulation Project, Japan Science and Technology Agency (JST), Aomi, Tokyo, Japan |
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Abstract: | F1-ATPase, a water-soluble portion of the enzyme ATP synthase, is a rotary molecular motor driven by ATP hydrolysis. To learn how the kinetics of rotation are regulated, we have investigated the rotational characteristics of a thermophilic F1-ATPase over the temperature range 4-50°C by attaching a polystyrene bead (or bead duplex) to the rotor subunit and observing its rotation under a microscope. The apparent rate of ATP binding estimated at low ATP concentrations increased from 1.2 × 106 M−1 s−1 at 4°C to 4.3 × 107 M−1 s−1 at 40°C, whereas the torque estimated at 2 mM ATP remained around 40 pN·nm over 4-50°C. The rotation was stepwise at 4°C, even at the saturating ATP concentration of 2 mM, indicating the presence of a hitherto unresolved rate-limiting reaction that occurs at ATP-waiting angles. We also measured the ATP hydrolysis activity in bulk solution at 4-65°C. F1-ATPase tends to be inactivated by binding ADP tightly. Both the inactivation and reactivation rates were found to rise sharply with temperature, and above 30°C, equilibrium between the active and inactive forms was reached within 2 s, the majority being inactive. Rapid inactivation at high temperatures is consistent with the physiological role of this enzyme, ATP synthesis, in the thermophile. |
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