Department of Physics, Faculty of Science, Gakushuin University, Toshima-ku, Tokyo 171-8588, Japan.
Nat Commun. 2012;3:1022. doi: 10.1038/ncomms2026.
F(1)-ATPase is an ATP-driven rotary molecular motor that synthesizes ATP when rotated in reverse. To elucidate the mechanism of ATP synthesis, we imaged binding and release of fluorescently labelled ADP and ATP while rotating the motor in either direction by magnets. Here we report the binding and release rates for each of the three catalytic sites for 360° of the rotary angle. We show that the rates do not significantly depend on the rotary direction, indicating ATP synthesis by direct reversal of the hydrolysis-driven rotation. ADP and ATP are discriminated in angle-dependent binding, but not in release. Phosphate blocks ATP binding at angles where ADP binding is essential for ATP synthesis. In synthesis rotation, the affinity for ADP increases by >10(4), followed by a shift to high ATP affinity, and finally the affinity for ATP decreases by >10(4). All these angular changes are gradual, implicating tight coupling between the rotor angle and site affinities.
F(1)-ATP 合酶是一种 ATP 驱动的旋转分子马达，当它反向旋转时会合成 ATP。为了阐明 ATP 合成的机制，我们通过磁场旋转马达，在旋转过程中观察荧光标记的 ADP 和 ATP 的结合和释放。在这里，我们报告了旋转角度为 360°时三个催化位点的结合和释放速率。我们发现，这些速率与旋转方向没有显著关系，表明 ATP 合成是通过水解驱动旋转的直接反转来实现的。ADP 和 ATP 在角度相关的结合中被区分，但在释放中没有被区分。磷酸盐在角度上阻断 ATP 的结合，而 ADP 的结合对 ATP 的合成是必需的。在合成旋转中，ADP 的亲和力增加超过 10^4，然后转向高 ATP 亲和力，最后 ATP 的亲和力降低超过 10^4。所有这些角度变化都是逐渐的，这表明转子角度和位点亲和力之间存在紧密的耦合。
