Intersubunit rotation in active F-ATPase
Intersubunit rotation in active F-ATPase
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DOI：
10.1038/381623a0
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发表时间：
1996-06-13
期刊：
影响因子：
64.8
通讯作者：
Junge, W
中科院分区：
文献类型：
--
作者：
Sabbert, D;Engelbrecht, S;Junge, W
ATP合酶，或F-ATP酶，存在于细菌、叶绿体和线粒体的膜中。它的结构是二分的，有质子传导的完整膜部分F-0和外围部分F-1。溶解的F-1由五种不同的亚基（α β）（3）γ δ β组成，并具有ATP酶活性（1，2）。F-ATP酶的功能是将质子通过F-0的转运与F-1的ATP合成偶联（参考文献3）。一些证据支持F-1上ATP的自发形成（参考文献4，5）及其在协同和旋转（或至少交替）位点的吸能释放（6）（7）。以质子动力为代价释放ATP可能涉及通过（α β）（3）内较小亚基（主要是γ）的旋转将机械能从F-0转换为F-1，如电子显微镜（8），X射线晶体结构分析（9）和使用可裂解的交联剂（10）所示的F-1的催化六边形。在这里，我们通过在光漂白后对具有曙红标记的γ的固定化F-1应用偏振吸收弛豫来记录功能酶中真实的时间内的亚基间旋转，我们观察到γ相对于固定化（α β）（3）的旋转在100 ms的时间跨度内，与固定化F-1的ATP水解速率相容。它的角度范围至少为200度，有利于催化的三点机制（7，11），γ在（α β）中充当曲轴（3）。当ATP被其不可水解的类似物AMP-PNP取代时，γ的旋转被阻断。
THE enzyme ATP synthase, or F-ATPase, is present in the membranes of bacteria, chloroplasts and mitochondria, Its structure is bipartite, with a proton-conducting, integral membrane portion, F-0, and a peripheral portion, F-1. Solubilized F-1 is composed of five different subunits, (alpha beta)(3) gamma delta epsilon, and is active as an ATPase(1,2). The function of F-ATPase is to couple proton translocation through F-0 with ATP synthesis in F-1 (ref. 3), Several fines of evidence support the spontaneous formation of ATP on F-1 (refs 4, 5) and its endergonic release(6) at cooperative and rotating (or at feast alternating) sites(7). The release of ATP at the expense of protonmotive force might involve mechanical energy transduction from F-0 into F-1 by rotation of the smaller subunits (mainly gamma) within (alpha beta)(3), the catalytic hexagon of F-1 as suggested by electron microscopy(8), by X-ray crystal structure analysis(9) and by the use of cleavable crosslinkers(10). Here we record an intersubunit rotation in real time in the functional enzyme by applying polarized absorption relaxation after photobleaching to immobilized F-1 with eosin-labelled gamma, We observe the rotation of gamma relative to immobilized (alpha beta)(3) in a timespan of 100 ms, compatible with the rate of ATP hydrolysis by immobilized F-1. Its angular range, which is of at least 200 degrees, favours a triple-site mechanism of catalysis(7,11), with gamma acting as a crankshaft in (alpha beta)(3). The rotation of gamma is blocked when ATP is substituted with its non-hydrolysable analogue AMP-PNP.