Furuike Shou, Adachi Kengo, Sakaki Naoyoshi, Shimo-Kon Rieko, Itoh Hiroyasu, Muneyuki Eiro, Yoshida Masasuke, Kinosita Kazuhiko
Department of Physics, Faculty of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan.
Biophys J. 2008 Jul;95(2):761-70. doi: 10.1529/biophysj.107.123307. Epub 2008 Mar 28.
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.
F(1)-ATP酶是ATP合酶的水溶性部分，是一种由ATP水解驱动的旋转分子马达。为了了解旋转动力学是如何被调节的，我们通过将聚苯乙烯珠（或珠双链体）连接到转子亚基上，并在显微镜下观察其旋转，研究了嗜热F(1)-ATP酶在4至50摄氏度温度范围内的旋转特性。在低ATP浓度下估计的ATP结合表观速率从4摄氏度时的1.2×10(6) M(-1) s(-1)增加到40摄氏度时的4.3×10(7) M(-1) s(-1)，而在2 mM ATP浓度下估计的扭矩在4至50摄氏度范围内保持在40 pN·nm左右。即使在2 mM的饱和ATP浓度下，在4摄氏度时旋转也是逐步的，这表明在ATP等待角度存在一个迄今尚未解决的限速反应。我们还测量了4至65摄氏度下本体溶液中的ATP水解活性。F(1)-ATP酶倾向于通过紧密结合ADP而失活。发现失活和重新激活速率都随温度急剧上升，并且在30摄氏度以上，活性和非活性形式之间的平衡在2秒内达到，大多数为非活性。高温下的快速失活与该酶在嗜热菌中的生理作用ATP合成是一致的。
