Mechanisms of Phosphoryl Transfer

修订摘要部分 该项目将利用多学科方法来研究蛋白质的作用 蛋白质酪氨酸磷酸酶（PTPs）和双- 特异性磷酸酶（DSP）。细胞磷酸化水平的控制对于细胞的生长至关重要。 调节一系列生物过程。PTP和DSP共享催化位点， 在X射线结构中可重叠。遵循相同的催化机理;化学步骤是 在所有这些酶中都是完全限速的;并且过渡态是相同的。然而，催化剂 速率跨越五个数量级。该项目将揭示蛋白质运动的细节是如何 与催化作用有关，以及这种作用在这个酶家族的成员之间是如何不同的。它将 还解决了蛋白质波动如何影响相同能力的基本问题 活性位点，使得相同反应的催化以广泛不同的效率发生。 在这个缩减的研究计划中，将有一个代表性PTP和一个代表性DSP。 考察PTP将是YopH，一种在耶尔森氏菌中负责腺病毒感染的毒力因子。 鼠疫，和最有效的（最高kcat）磷酸酶已知。用于研究的DSP是VHR， 参与调节细胞对外界压力反应的人类酶。尽管高度 相似的活性位点，这些酶在催化能力上相差近三个数量级， 我们假设这是由于，至少部分是由于，在不同的运动过程中的关键残基的差异， 与催化作用相关的蛋白质运动。这一假设将使用以下组合进行检验： 蛋白质晶体学、计算、动力学和核磁共振。晶体结构将获得的 具有结合的不可水解底物类似物的酶（或具有肽的失活突变体的酶 底物）和由肽底物+钒酸盐组成的过渡态类似物。载脂蛋白 酶的结构已经有报道。结构比较将揭示以下信息 由底物结合引起的构象变化， 在过渡状态。这些结构将被用作计算分析的起点， 确定对重要酶残基催化的积极贡献。同时 通过这些研究，使用同位素富集酶的核磁共振实验将产生解决方案 载脂蛋白形式的酶和结合底物类似物的结构，其从X射线衍射中已知， 结构来触发催化重要的构象变化。核磁共振数据也会产生 动态信息，这将揭示哪些蛋白质运动发生在相同的时间尺度上， 催化，并测量由底物结合引起的与这些的任何差异。 2

Revised Abstract Section The project will utilize a multidisciplinary approach to examine the role of protein conformational changes in binding and catalysis by protein-tyrosine phosphatases (PTPs) and dual- specific phosphatases (DSPs). The control of cellular phosphorylation levels is critical to the regulation of a host of biological processes. PTPs and DSPs share catalytic sites that are highly superimposable in X-ray structures. The same catalytic mechanism is followed; the chemical step is fully rate-limiting in all of these enzymes; and the transition states are the same. Yet, the catalytic rates span five orders of magnitude. The project will reveal how details of protein movement are associated with catalysis, and how such effects differ between members of this enzyme family. It will also address a fundamental question of how protein fluctuations can affect the ability of identical active sites such that catalysis of the same reaction occurs with widely differing efficiencies. In this reduced research plan, one representative PTP one representative DSP will be examined. The PTP will be YopH, a virulence factor in the Yersinia bacteria responsible for Bubonic Plague, and the most efficient (highest kcat) phosphatase known. The DSP for study is VHR, a human enzyme involved in the regulation of cellular response to external stresses. Despite highly similar active sites, these enzymes differ in catalytic proficiency by nearly three orders of magnitude, which we hypothesize is due, at least in part, to differences in the movement of key residues during protein movements associated with catalysis. This hypothesis will be examined using a combination of protein crystallography, computation, kinetics, and NMR. Crystal structures will be obtained of the enzymes with a bound nonhydrolyzable substrate analog (or of an inactive mutant with a peptide substrate), and of a transition state analog consisting of a peptide substrate + vanadate. The apo enzyme structures have already been reported. Structural comparisons will reveal information about conformational changes that result from substrate binding, and differences in conformation that arise in the transition state. These structures will be used as starting points for a computational analysis to ascertain the energetic contributions toward catalysis of important enzymatic residues. Simultaneous with these studies, NMR experiments using isotopically enriched enzymes will yield the solution structures of the enzymes in apo form and with a bound substrate analog that are known from X-ray structures to trigger catalytically important conformational changes. The NMR data will also yield dynamic information, which will reveal which protein motions occur on the same timescale as catalysis, and measure any differences to these that result from substrate binding. 2