Monitoring the GAP catalyzed H-Ras GTPase reaction at atomic resolution in real time
Monitoring the GAP catalyzed H-Ras GTPase reaction at atomic resolution in real time
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DOI：
10.1073/pnas.131549798
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发表时间：
2001-07-03
影响因子：
11.1
通讯作者：
Gerwert, K
中科院分区：
文献类型：
--
作者：
Allin, C;Ahmadian, MR;Gerwert, K
以笼型GTP （P-3-1-（2-硝基）苯乙基鸟苷5′- o -三磷酸）为光致触发物，采用时间分辨傅立叶变换红外（FTIR）差分光谱研究了GTP活化蛋白（GAP）催化Pas水解GTP的分子反应机理。这种方法提供了完整的GTPase反应途径，在原子水平上的时间分辨率为毫秒。到目前为止，有一种关于差距的结构模型。已知AIF(X)过渡态类似物，它代表了沿反应途径的“快照”。正如现在所揭示的，GAP与Ras - GTP的结合将负电荷从-磷酸转移到-磷酸。由于Pas结合，这种转变已经被FTIR在GTP中发现，现在被证明是由GAP结合增强的。由于GAP.Ras.GTP复合物的电荷分布类似于一个更像解离的过渡态，更像GDP中的过渡态，因此激活自由能降低。当-和-磷酸盐之间的键断裂时，在反应途径上观察到一种中间产物。在中间体中，释放的P-i与蛋白质紧密结合，并令人惊讶地显示出磷酸化酶中间体的典型带。所有这些结果提供了一个不同于Pas固有的GTPase反应的机制图。FTIR分析显示，P-i从蛋白质复合物中释放是gap催化反应的限速步骤。所提出的方法不仅可以研究单个蛋白质，还可以在非晶体状态下实时研究蛋白质与蛋白质之间的相互作用，而不需要固有的发色团。
The molecular reaction mechanism of the GTPase-activating protein (GAP)-catalyzed GTP hydrolysis by Pas was investigated by time resolved Fourier transform infrared (FTIR) difference spectroscopy using caged GTP (P-3-1-(2-nitro)phenylethyl guanosine 5 ' -O-triphosphate) as photolabile trigger. This approach provides the complete GTPase reaction pathway with time resolution of milliseconds at the atomic level. Up to now, one structural model of the GAP.Ras.GDP.AIF(X) transition state analog is known, which represents a "snap shot" along the reaction-pathway. As now revealed, binding of GAP to Ras GTP shifts negative charge from the gamma to beta phosphate. Such a shift was already identified by FTIR in GTP because of Pas binding and is now shown to be enhanced by GAP binding. Because the charge distribution of the GAP.Ras.GTP complex thus resembles a more dissociative-like transition state and is more like that in GDP, the activation free energy is reduced. An intermediate is observed on the reaction pathway that appears when the bond between beta and gamma phosphate is cleaved. In the intermediate, the released P-i is strongly bound to the protein and surprisingly shows bands typical of those seen for phosphorylated enzyme intermediates. All these results provide a mechanistic picture that is different from the intrinsic GTPase reaction of Pas. FTIR analysis reveals the release of P-i from the protein complex as the rate-limiting step for the GAP-catalyzed reaction. The approach presented allows the study not only of single proteins but of protein-protein interactions without intrinsic chromophores, in the non-crystalline state, in real time at the atomic level.