Temperature dependence of the rotation and hydrolysis activities of F1-ATPase
Temperature dependence of the rotation and hydrolysis activities of F1-ATPase
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DOI：
10.1529/biophysj.107.123307
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发表时间：
2008-07-15
影响因子：
3.4
通讯作者：
Kinosita, Kazuhiko, Jr.
中科院分区：
文献类型：
--
作者：
Furuike, Shou;Adachi, Kengo;Kinosita, Kazuhiko, Jr.
F-1-ATPase是三磷酸腺苷合成酶的水溶性部分，是由三磷酸腺苷水解酶驱动的旋转分子马达。为了了解旋转动力学是如何调节的，我们研究了高温F-1-ATPase在4-50℃的温度范围内的旋转特性，方法是将聚苯乙烯微珠(或双链微珠)连接到转子亚单位上，并在显微镜下观察其旋转。在低浓度下估计的三磷酸腺苷结合的表观速率从4℃的1.2x10(6)M-1 S(-1)增加到40℃的4.3x10(7)M-1 S(-1)，而估计在2 mMATP的扭矩在4-50℃保持在40 pN.nm左右。在4℃时，即使在饱和的2 mM的三磷酸腺苷浓度下，旋转也是逐步的，这表明在三磷酸腺苷等待角上存在迄今未解决的限速反应。我们还测量了在4-65℃的大块溶液中的ATP水解酶活性，F1-ATPase往往会被ADP紧密结合而失活。失活和复活率均随温度升高而急剧上升，在30℃以上，在2 S内达到活性和非活性之间的平衡，大部分为非活性。高温下的快速失活与这种酶在嗜热菌中的生理作用--ATP合成是一致的。
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. 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 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.