Mechanisms of Phosphoryl Transfer

修订后的摘要部分 该项目将利用多学科方法来研究蛋白质的作用。 蛋白酪氨酸磷酸酶(PTPs)和双-酪氨酸磷酸酶结合和催化的构象变化 特异性磷酸酶(DSP)。细胞磷酸化水平的控制对 对一系列生物过程的调控。PTP和DSP共享高度高度的催化中心 在X射线结构中可叠加。遵循相同的催化机理；化学步骤为 在所有这些酶中都是完全限速的；过渡态是相同的。然而，催化剂 利率跨度为五个数量级。该项目将揭示蛋白质运动的细节 与催化有关，以及这种作用在该酶家族成员之间的不同之处。会的 还讨论了一个基本问题，即蛋白质波动如何影响同种异构体的能力 活性中心，使同一反应的催化效率大相径庭。 在这个精简的研究计划中，一个有代表性的PTP将一个有代表性的DSP 检查过了。PTP将是YopH，这是引起Bubonic的耶尔森氏菌中的一种毒力因子 鼠疫，以及已知的最有效(最高kcat)的磷酸酶。所研究的数字信号处理器是VHR，一种 人的酶参与调节细胞对外界压力的反应。尽管高度 类似的活性部位，这些酶的催化熟练程度相差近三个数量级， 我们推测，这至少部分归因于关键残基在不同时期的移动。 与催化有关的蛋白质运动。这一假设将通过组合进行检验。 蛋白质结晶学、计算、动力学和核磁共振。晶体结构将被获得 具有结合的不可水解底物类似物的酶(或具有肽的非活性突变体的酶 底物)，以及由多肽底物+钒酸盐组成的过渡态类似物。《阿波罗》 酶的结构已经被报道了。结构比较将揭示以下信息 底物结合引起的构象变化，以及由此产生的构象差异 处于过渡状态。这些结构将被用作计算分析的起点 确定重要的酶残基对催化的能量贡献。同时进行 有了这些研究，使用同位素富集酶的核磁共振实验将产生溶液 已知的载脂蛋白形式和结合底物类似物的酶的结构 结构触发催化重要的构象变化。核磁共振数据也将产生 动态信息，它将揭示哪些蛋白质运动发生在与 催化作用，并测量与底物结合所产生的任何差异。 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