Activation of the insulin receptor's kinase domain changes the rate-determining step of substrate phosphorylation
Activation of the insulin receptor's kinase domain changes the rate-determining step of substrate phosphorylation
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DOI：
10.1021/bi002292m
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发表时间：
2001-01-16
期刊：
影响因子：
2.9
通讯作者：
Kohanski, RA
中科院分区：
文献类型：
--
作者：
Ablooglu, AJ;Kohanski, RA
胰岛素受体和许多其他蛋白激酶通过解除由可逆磷酸化调节的内质网抑制而被激活。伴随的胰岛素受体的激酶结构域的激活的变化进行了分析，使用稳态动力学，粘度分析，和平衡结合测量。由未磷酸化的基础状态激酶催化的肽磷酸化受到化学步骤的缓慢速率的限制，并且活化的酶受到产物释放速率的限制。这些变化的基础是酶催化反应的化学步骤的速率常数增加了36倍，MgATP的亲和力增加了5倍，肽底物的亲和力增加了8倍。这导致底物的结合更有利2.2 kcal/mol，并且在活化的酶中用于过渡态形成的自由能垒降低2.1 kcal/mol。因此，自磷酸化后蛋白质中固有的构象自由能的变化[Bishop，S. M.，Ross，J. B.一、和Kohanski，R. A.（1999）Biochemistry 38，3079-3089]在底物三元复合物的形成和过渡态复合物的形成之间均匀分布。
The insulin receptor and many other protein kinases are activated by relief of intrasteric inhibition that is regulated by reversible phosphorylation. The changes accompanying activation of the insulin receptor's kinase domain were analyzed using steady-state kinetics, viscometric analysis, and equilibrium binding measurements. Peptide phosphorylation catalyzed by the unphosphorylated basal-state kinase is limited by a slow rate of the chemical step, and the activated enzyme is limited by product release rates. Underlying these changes were a 36-fold increase in the rate constant for the chemical step of the enzyme-catalyzed reaction, a 5-fold increase in the affinity for MgATP, and an 8-fold increase in the affinity for peptide substrate. This results in binding of substrates that is 2.2 kcal/mol more favorable and a free energy barrier for transition state formation that is lowered by 2.1 kcal/mol in the activated enzyme. Therefore, the change in conformational free energy inherent in the protein after autophosphorylation [Bishop, S. M., Ross, J. B. A., and Kohanski, R. A. (1999) Biochemistry 38, 3079-3089] is equally distributed between formation of the substrate ternary complex and formation of the transition state complex.