Temperature dependence of the rotation and hydrolysis activities of F1-ATPase.
F(1)-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 F(1)-ATPase over the temperature range 4-50 degrees 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 10(6) M(-1) s(-1) at 4 degrees C to 4.3 x 10(7) M(-1) s(-1) at 40 degrees C, whereas the torque estimated at 2 mM ATP remained around 40 pN.nm over 4-50 degrees C. The rotation was stepwise at 4 degrees 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 degrees C. F(1)-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 degrees 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.
Furuike
Shou
S
Department of Physics, Faculty of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan.
Adachi
Kengo
K
Sakaki
Naoyoshi
N
Shimo-Kon
Rieko
R
Itoh
Hiroyasu
H
Muneyuki
Eiro
E
Yoshida
Masasuke
M
Kinosita
Kazuhiko
K
D003198:Computer Simulation; D004789:Enzyme Activation; D006868:Hydrolysis; D008956:Models, Chemical; D008958:Models, Molecular; D020409:Molecular Motor Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000648:ultrastructure*; D012399:Rotation; D013696:Temperature
D020409:Molecular Motor Proteins; D006180:Proton-Translocating ATPases