Wu Hao, Wang Zhi-Xin
National Laboratory of Biomacromolecules, Center for Molecular Biology, Institute of Biophysics, Academia Sinica, Beijing 100101, China.
J Biol Chem. 2003 Oct 24;278(43):41768-78. doi: 10.1074/jbc.M308196200. Epub 2003 Aug 7.
The p21-activated kinases (PAKs) play an important role in diverse cellular processes. PAK2 is activated by autophosphorylation upon binding of small G proteins such as Cdc42 and Rac in the GTP-bound state. However, the mechanism of PAK2 autophosphorylation in vitro is unclear. In the present study, the kinetic theory of the substrate reaction during modification of enzyme activity has been applied to a study of the autoactivation of PAK2. On the basis of the kinetic equation of the substrate reaction during the autophosphorylation of PAK2, the activation rate constants for the free enzyme and enzyme-substrate complex have been determined. The results indicate that 1) in the presence of Cdc42, PAK2 autophosphorylation is a bipartite mechanism, with the regulatory domain autophosphorylated at multiple residues, whereas activation coincides with autophosphorylation of the catalytic domain at Thr-402; 2) the autophosphorylation reactions in regulatory domain are either a nonlimiting step or not required for activation of enzyme; 3) the autophosphorylation at site Thr-402 on the catalytic domain occurs by an intermolecular mechanism and is required for phosphorylation of exogenous substrates examined; 4) binding of the exogenous protein/peptide substrates at the active site of PAK2 has little or no effect on the autoactivation of PAK2, suggesting that multiple regions of PAK2 are involved in the enzyme-substrate recognition. The present method also provides a novel approach for studying autophosphorylation reactions. Since the experimental conditions used resemble more closely the in vivo situation where the substrate is constantly being turned over while the enzyme is being modified, this new method would be particularly useful when the regulatory mechanisms of the reversible phosphorylation reaction toward certain enzymes are being assessed.
p21激活激酶（PAKs）在多种细胞过程中发挥重要作用。PAK2在结合处于GTP结合状态的小G蛋白（如Cdc42和Rac）时通过自身磷酸化被激活。然而，PAK2体外自身磷酸化的机制尚不清楚。在本研究中，酶活性修饰过程中底物反应的动力学理论已被应用于PAK2的自激活研究。基于PAK2自身磷酸化过程中底物反应的动力学方程，已确定了游离酶和酶 - 底物复合物的活化速率常数。结果表明：1）在Cdc42存在的情况下，PAK2自身磷酸化是一种双部分机制，调节结构域在多个残基处发生自身磷酸化，而活化与催化结构域在Thr - 402处的自身磷酸化同时发生；2）调节结构域中的自身磷酸化反应要么是一个非限速步骤，要么对酶的激活不是必需的；3）催化结构域上Thr - 402位点的自身磷酸化通过分子间机制发生，并且是所检测的外源底物磷酸化所必需的；4）外源蛋白质/肽底物在PAK2活性位点的结合对PAK2的自激活几乎没有影响，这表明PAK2的多个区域参与酶 - 底物识别。本方法还为研究自身磷酸化反应提供了一种新方法。由于所使用的实验条件更类似于体内情况，即在酶被修饰时底物不断周转，当评估对某些酶的可逆磷酸化反应的调节机制时，这种新方法将特别有用。
