Tyr39 of Ran Preserves the Ran.GTP Gradient by Inhibiting GTP Hydrolysis
Tyr39 of Ran Preserves the Ran.GTP Gradient by Inhibiting GTP Hydrolysis
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DOI：
10.1016/j.jmb.2010.05.068
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发表时间：
2010-08-06
影响因子：
5.6
通讯作者：
Koetting, Carsten
中科院分区：
文献类型：
--
作者：
Brucker, Sven;Gerwert, Klaus;Koetting, Carsten
Ran是小GTP酶超家族的成员，它在结合GTP的“开启”状态和结合GDP的“关闭”状态之间循环。Ran调节核运输。为了在核质内维持过量Ran·GTP的梯度以及在细胞质内维持过量Ran·GDP的梯度，应防止核质内Ran·GTP的水解，而在细胞质中，水解由Ran·GAP（GTP酶激活蛋白）催化。在本文中，我们通过时间分辨傅里叶变换红外光谱研究了Ran与其结合蛋白Ran结合蛋白1复合物中的GTP酶反应：我们表明，RanGTP固有水解的减缓是由酪氨酸39完成的，它可能使进攻的水分子错位。我们监测了Ran与RanGAP的相互作用，这揭示了两个反应步骤。通过对Ran和RanGAP进行同位素标记，我们能够将第一步归因于催化位点内的一个小的构象变化。随后的键断裂是水解的限速步骤。在Ras或Rap系统中发现的蛋白质结合磷酸盐中间体在动力学上未得到解析。这表明，尽管GTP酶的G结构域之间存在结构相似性，但它们利用了不同的反应机制。（C）2010爱思唯尔有限公司。保留所有权利。
Ran is a member of the superfamily of small GTPases, which cycle between a GTP-bound "on" and a GDP-bound "off" state. Ran regulates nuclear transport. In order to maintain a gradient of excess Ran.GTP within the nucleoplasm and excess Ran.GDP within the cytoplasm, the hydrolysis of Ran.GTP in the nucleoplasm should be prevented, whereas in the cytoplasm, hydrolysis is catalyzed by Ran.GAP (GTPase-activating protein). In this article, we investigate the GTPase reaction of Ran in complex with its binding protein Ran-binding protein 1 by time-resolved Fourier transform infrared spectroscopy: We show that the slowdown of the intrinsic hydrolysis of RanGTP is accomplished by tyrosine 39, which is probably misplacing the attacking water. We monitored the interaction of Ran with RanGAP, which reveals two reactions steps. By isotopic labeling of Ran and RanGAP, we were able to assign the first step to a small conformational change within the catalytic site. The following bond breakage is the rate-limiting step of hydrolysis. An intermediate of protein-bound phosphate as found for Ras or Rap systems is kinetically unresolved. This demonstrates that despite the structural similarity among the G-domain of the GTPases, different reaction mechanisms are utilized. (C) 2010 Elsevier Ltd. All rights reserved.