ATP-driven stepwise rotation of FOF1,-ATP synthase
ATP-driven stepwise rotation of FOF1,-ATP synthase
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DOI：
10.1073/pnas.0407857102
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发表时间：
2005-02-01
影响因子：
11.1
通讯作者：
Yoshida, M
中科院分区：
文献类型：
--
作者：
Ueno, H;Suzuki, T;Yoshida, M
FoF1-ATP合成酶(FoF1)是一种将ATP合成/水解与跨膜质子转运结合在一起的马达酶。F-1是FoF1的一个水溶性ATPase部分，通过重复等待ATP停留、80度亚步旋转、催化停留和40度亚步旋转来旋转。与F-1相比，对FoF1的旋转还知之甚少，在这里，我们分析了ATIP驱动的FoF1旋转。在固定的FoF1中，用固定在c亚基环上的80 nm的珠子探测旋转，并用亚毫秒快相机记录。在不同的三磷酸腺苷浓度下的旋转速度服从由K-m约为30微米和V-max为350转/秒(37℃)所定义的曲线。在低三磷酸腺苷时，出现等待三磷酸腺苷的现象，k值为3.6x107M(-1)。S(-1)。在高ATP时，观察到快速的、界限不清的阶梯运动，这可能反映了催化停留。当使用可缓慢水解的底物，腺苷5‘+[伽马-硫代]三磷酸时，在大约80度的角度位置上，更清楚地看到由两个事件组成的催化空穴。FoF1的旋转行为类似于F-1。这一发现表明，在ATIP驱动的旋转过程中，F-O电机中的“摩擦”可以忽略不计。三丁基氯化锡是一种特殊的质子转移抑制剂，使旋转速度减慢了96%。然而，在这些条件下没有观察到明确定义的角度位置的住所，这表明三丁基氯化锡的抑制是复杂的。
FoF1-ATP synthase (FoF1) is a motor enzyme that couples ATP synthesis/hydrolysis with a transmembrane proton translocation. F-1, a water-soluble ATPase portion of FoF1, rotates by repeating ATP-waiting dwell, 80degrees substep rotation, catalytic dwell, and 40degrees-substep rotation. Compared with F-1, rotation of FoF1 has yet been poorly understood, and, here, we analyzed ATIP-driven rotations of FoF1. Rotation was probed with an 80-nm bead attached to the ring of c subunits in the immobilized FoF1 and recorded with a submillisecond fast camera. The rotation rates at various ATP concentrations obeyed the curve defined by a K-m of approximate to30 muM and a V-max of 350 revolutions per second (at 37degreesC). At low ATP, ATP-waiting dwell was seen and the k(on-ATP) was estimated to be 3.6 x 107 M(-1.)s(-1). At high ATP, fast, poorly defined stepwise motions were observed that probably reflect the catalytic dwells. When a slowly hydrolyzable substrate, adenosine 5'+[gamma-thio]triphosphate, was used, the catalytic dwells consisting of two events were seen more clearly at the angular position of approximate to80degrees. The rotational behavior of FoF1 resembles that of F-1. This finding indicates that "friction" in F-o motor is negligible during the ATIP-driven rotation. Tributyltin chloride, a specific inhibitor of proton translocation, slowed the rotation rate by 96%. However, dwells at clearly defined angular positions were not observed under these conditions, indicating that inhibition by tributyltin chloride is complex.