The GAP arginine finger movement into the catalytic site of Ras increases the activation entropy
The GAP arginine finger movement into the catalytic site of Ras increases the activation entropy
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DOI：
10.1073/pnas.0712095105
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发表时间：
2008-04-29
影响因子：
11.1
通讯作者：
Gerwert, Klaus
中科院分区：
文献类型：
--
作者：
Koetting, Carsten;Kallenbach, Angela;Gerwert, Klaus
Ras超家族成员的小G蛋白在信号转导途径中起着关键作用，它们通过GTP水解酶来控制信号转导。它们受GTP酶激活蛋白(GAP)的调节。阻止水解的突变会导致包括癌症在内的严重疾病。GAP的一个高度保守的“精氨酸手指”是一个关键残基。在这里，我们用260K下的时间分辨FTIR光谱在高时间和空间分辨率上监测了RAS-RasGAP复合体的GTPase反应。在触发反应后，我们观察到作为第一步，RAS的开关区以3 S(-1)的速率从无信号的“关”状态转移到信号的“开”状态。下一步是“精氨酸手指”以k(2)=0.8 S(-1)的速率进入RAS的活性部位。一旦精氨酸进入结合口袋，GTP的裂解就很快，蛋白质结合的PI中间体就形成了。在限速步骤k(3)=0-1 S(-1)的情况下，同时观察到开关到-1\f25“OFF”-1状态的逆转、-1\f25 PI-1的释放以及精氨酸返回到水环境中。部分反应的Arrhenius图表明，有利的正活化熵降低了裂解反应的活化能。这似乎表明，蛋白质结合的结构水分子是被“精氨酸手指”运动从结合口袋推入散装水中的。所提出的机理说明了如何通过分解成由PI中间体分隔的部分反应来降低磷酰化转移的高活化势垒。
Members of the Ras superfamily of small G proteins play key roles in signal transduction pathways, which they control by GTP hydrolysis. They are regulated by GTPase activating proteins (GAPs). Mutations that prevent hydrolysis cause severe diseases including cancer. A highly conserved "arginine finger" of GAP is a key residue. Here, we monitor the GTPase reaction of the Ras-RasGAP complex at high temporal and spatial resolution by time-resolved FTIR spectroscopy at 260 K. After triggering the reaction, we observe as the first step a movement of the switch-1 region of Ras from the nonsignaling "off" to the signaling "on" state with a rate of 3 s(-1). The next step is the movement of the "arginine finger" into the active site of Ras with a rate of k(2) = 0.8 s(-1). Once the arginine points into the binding pocket, cleavage of GTP is fast and the protein-bound Pi intermediate forms. The switch-1 reversal to the "off" state, the release of Pi, and the movement of arginine back into an aqueous environment is observed simultaneously with k(3) = 0-1 s(-1), the rate-limiting step. Arrhenius plots for the partial reactions show that the activation energy for the cleavage reaction is lowered by favorable positive activation entropy. This seems to indicate that protein-bound structured water molecules are pushed by the "arginine finger" movement out of the binding pocket into the bulk water. The proposed mechanism shows how the high activation barrier for phosphoryl transfer can be reduced by splitting into partial reactions separated by a Pi-intermediate.