Stewart A E, Dowd S, Keyse S M, McDonald N Q
Structural Biology Laboratory, Imperial Cancer Research Fund, London, UK.
Nat Struct Biol. 1999 Feb;6(2):174-81. doi: 10.1038/5861.
The crystal structure of the catalytic domain from the MAPK phosphatase Pyst1 (Pyst1-CD) has been determined at 2.35 A. The structure adopts a protein tyrosine phosphatase (PTPase) fold with a shallow active site that displays a distorted geometry in the absence of its substrate with some similarity to the dual-specificity phosphatase cdc25. Functional characterization of Pyst1-CD indicates it is sufficient to dephosphorylate activated ERK2 in vitro. Kinetic analysis of Pyst1 and Pyst1-CD using the substrate p-nitrophenyl phosphate (pNPP) reveals that both molecules undergo catalytic activation in the presence of recombinant inactive ERK2, switching from a low- to high-activity form. Mutation of Asp 262, located 5.5 A distal to the active site, demonstrates it is essential for catalysis in the high-activity ERK2-dependent conformation of Pyst1 but not for the low-activity ERK2-independent form, suggesting that ERK2 induces closure of the Asp 262 loop over the active site, thereby enhancing Pyst1 catalytic efficiency.
丝裂原活化蛋白激酶磷酸酶Pyst1的催化结构域（Pyst1-CD）的晶体结构已在2.35埃分辨率下确定。该结构采用蛋白质酪氨酸磷酸酶（PTPase）折叠，具有一个浅的活性位点，在没有底物时呈现扭曲的几何形状，与双特异性磷酸酶cdc25有一定相似性。Pyst1-CD的功能表征表明它在体外足以使活化的ERK2去磷酸化。使用底物对硝基苯磷酸酯（pNPP）对Pyst1和Pyst1-CD进行动力学分析表明，在重组无活性ERK2存在的情况下，这两种分子都会发生催化活化，从低活性形式转变为高活性形式。位于活性位点远端5.5埃处的Asp 262突变表明，它对于Pyst1在高活性ERK2依赖性构象中的催化作用至关重要，但对于低活性ERK2非依赖性形式则不是必需的，这表明ERK2诱导Asp 262环在活性位点上闭合，从而提高Pyst1的催化效率。
