Originally published as Biophys J. BioFAST on March 28, 2008.
doi:10.1529/biophysj.107.123307
OPEN ACCESS ARTICLE
Biophysical Journal 95:761-770 (2008)
© 2008 The Biophysical Societyっｓ

Temperature Dependence of the Rotation and Hydrolysis Activities of F₁-ATPase

Shou Furuike ^*, Kengo Adachi ^*, Naoyoshi Sakaki , Rieko Shimo-Kon ^*, Hiroyasu Itoh  , Eiro Muneyuki ^¶, Masasuke Yoshida ^|| ** and Kazuhiko Kinosita, Jr. ^*

^* 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, and 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; and ^** ICORP ATP Synthesis Regulation Project, Japan Science and Technology Agency (JST), Aomi, Tokyo, Japan

Correspondence: Address reprint requests to Kazuhiko Kinosita Jr., Dept. of Physics, Faculty of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan. Tel.: 81-3-5952-5871; Fax: 81-3-5952-5877; E-mail: kazuhiko_at_waseda.jp

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 x 10⁶ M^–1 s^–1 at 4°C to 4.3 x 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.