Adams Joseph A
Department of Pharmacology, University of California, San Diego, USA.
Biochemistry. 2003 Jan 28;42(3):601-7. doi: 10.1021/bi020617o.
Many protein kinases are activated strongly by the phosphorylation of a polypeptide region (activation loop) that lies outside the active-site cleft. Analysis of the X-ray crystallographic structures of the insulin receptor with the activation loop in the phosphorylated and dephosphorylated forms offers a testable model for the mechanism of activity regulation by the loop. In this model, the dephosphorylated activation loop can act as an autoinhibitor by blocking substrate access to the active site. Phosphorylation of the loop could then release the autoinhibitor from the active site, allowing substrate binding and catalysis. While this model has been widely invoked, it was not clear if solution studies would support an autoinhibitory model for kinase regulation, in general. We review the results of solution studies on six protein kinases that test the role of the activation loop in controlling active-site access. While loop phosphorylation enhances substrate binding in two cases, four protein kinases display little or no effect on substrate dissociation constants. By comparison, phosphorylation increases catalysis by 2-4 orders of magnitude in all cases. These findings can be used to place the phosphorylatable activation loops into two broad, functional subcategories. (i) Gated activation loops exhibit bifunctional properties restricting substrate access and controlling catalysis. (ii) Nongated activation loops allow free movement of the substrate in and out of the active site irrespective of phosphorylation state but potently modulate the phosphoryl transfer step. Thus, while activation loop phosphorylation greatly modulates catalytic potential, it does not necessarily affect substrate binding, as once widely believed.
许多蛋白激酶通过位于活性位点裂隙之外的多肽区域（激活环）的磷酸化而被强烈激活。对处于磷酸化和去磷酸化形式的胰岛素受体的X射线晶体结构分析，为该环调节活性的机制提供了一个可验证的模型。在这个模型中，去磷酸化的激活环可以通过阻止底物进入活性位点而作为一种自抑制剂。环的磷酸化随后可将自抑制剂从活性位点释放，从而允许底物结合和催化。虽然这个模型已被广泛引用，但总体而言，溶液研究是否会支持激酶调节的自抑制模型尚不清楚。我们回顾了对六种蛋白激酶的溶液研究结果，这些研究测试了激活环在控制活性位点可及性方面的作用。虽然在两种情况下环磷酸化增强了底物结合，但四种蛋白激酶对底物解离常数几乎没有影响。相比之下，在所有情况下磷酸化使催化作用增加2 - 4个数量级。这些发现可用于将可磷酸化的激活环分为两个宽泛的功能亚类。（i）门控激活环具有双功能特性，既能限制底物进入又能控制催化作用。（ii）非门控激活环允许底物自由进出活性位点，而与磷酸化状态无关，但能有效调节磷酰转移步骤。因此，虽然激活环磷酸化极大地调节了催化潜力，但它不一定像曾经广泛认为的那样影响底物结合。
