Mechanism of catalytic regulation and active site loop motions in related protein tyrosine phosphatases

描述(由申请者提供)：细胞必须对健康的功能、生长和分化的内外刺激做出反应。细胞做到这一点的一个主要机制是通过调节许多蛋白质的翻译后磷酸化水平。蛋白质磷酸化水平的紊乱或异常会导致多种疾病，包括癌症、II型糖尿病、肥胖症和炎症。调节蛋白质磷酸化状态的一类酶是蛋白质酪氨酸磷酸酶。由于它们的重要作用，这些酶催化除磷的速度是至关重要的，并且受到严格的调控。这种调节可以通过变构机制发生，包括小分子结合和翻译后修饰。而且，正如PI实验室的最新数据所表明的那样，调节也内在地由这些磷酸酶中酸性环的闭合动力学控制。长期目标是更详细地了解这些磷酸酶的活性是如何调节的。这一目标将通过三个目标实现：1)研究PTP1B抑制的变构机制，PTP1B是一种人类酶，其错误调节导致II型糖尿病、肥胖症和某些乳腺癌~2)表征PTP1B和YopH(来自致病耶尔森氏菌的磷酸酶)中酸环的初级序列如何控制环运动的动力学~3)表征由与伪激酶VRK-3结合而产生的人VHR磷酸酶的激活机制。对于这三个目标，酸环的运动将通过溶液核磁共振弛豫分散技术进行监测，重点是由于小分子结合、翻译后修饰、突变或辅助蛋白结合而引起的环运动的任何变化。由于这些扰动，这些磷酸酶的任何功能变化都将通过详细的动力学研究来表征，包括pH和同位素效应。这些生物物理和生化技术的结合将深入了解运动和催化之间的联系，同时提供有关医学上重要酶的调节机制的信息。

DESCRIPTION (provided by applicant): Cells must respond to internal and external stimuli for healthy function, growth, and differentiation. One primary mechanism by which cells do this is through modulation of the post-translational phosphorylation levels of many proteins. Disruption of, or aberrant protein phosphorylation levels results in numerous diseases including cancer, Type-II diabetes, obesity, and inflammation. One class of enzymes that modulate the phosphorylation state of proteins is the protein tyrosine phosphatases. Because of their important role, the rate at which these enzymes catalyze phosphate removal is crucial and is tightly regulated. This regulation can occur by allosteric mechanisms, including small molecule binding and post-translational modification. And, as recent data in the PI's lab suggests, regulation is also inherently controlled by the closing kinetics of the acid loop in these phosphatases. The long-term goal is to understand in more detail how the activity of these phosphatase enzymes is regulated. This goal will be achieved through three aims: 1) study of the allosteric mechanism of PTP1B inhibition, a human enzyme whose misregulation leads to Type-II diabetes, obesity, and certain breast cancers~ 2) characterize how the primary sequence of the acid loop in PTP1B and YopH (a phosphatase from the virulent Yersinia bacterium) controls the kinetics of loop motion~ and 3) characterize the activation mechanism of the human VHR phosphatase that results from binding to the pseudokinase, VRK-3. For these three aims the motions of the acid loop will be monitored by solution NMR relaxation dispersion techniques with focus on any alterations in loop motions due to small molecule binding, post-translational modification, mutation, or accessory protein binding. Any functional changes in these phosphatases as a result of these perturbations will be characterized by detailed kinetic studies including pH and isotope effects. The combination of these biophysical and biochemical techniques will give insight into the connections between motions and catalysis while informing on the regulatory mechanisms of medically important enzymes.