Study of Nonenzymatic and Enzymatic Phosphoryl Transfer

描述：(申请人提供)最常见的是磷酰基转移 生物反应。原则上，蛋白激酶和磷酸酶应该是 极好的药物治疗靶点。据估计，大约有3000只 人类基因组中的蛋白激酶和磷酸酶，这些酶是 是控制基本细胞过程的中心。然而，这些酶具有 还没有成为强大的药物靶子。 这项建议的目的是增加对磷酰化的基本了解。 转移并深入探讨这些反应的酶催化作用。这 理解反过来可以帮助理解磷酰催化是如何使用的 在复杂的生物过程中，最终可能有助于设计 新药。 这项提案有两个部分，第一部分介绍了探索 非酶磷酰转移反应和第二节描述 用大肠杆菌碱性磷酸酶(AP)探索基本问题的实验 磷酰转移催化。非酶研究将提供一种 烷基磷酸盐反应的基本表征。烷基 磷酸盐是最常见的生物磷酸盐化合物，但 绝大多数对磷酸盐化合物的研究都使用了芳基以便于 测量。然而，最近的结果显示，出人意料的快速水解 令人质疑芳基磷酸盐是否提供了一种 适合烷基磷酸盐的模型。这将被测试，而烷基 用经典的物理方法表征磷酸盐反应 有机化学，如线性自由能关系和振动关系 光谱学。将测试去除水的潜在催化效果。 而在混合有机溶剂中强化水解的原因将是 调查过了。 E.ColiAp是探索酶学基本问题的理想酶 催化，因为许多先前的动力学和机理研究以及 多种高分辨率结构可用，而且由于低分辨率 特异性和开放的活性部位允许底物的系统性变化。AP 除其同系物外，还催化磷酸二酯的水解 与磷酸单酯反应。这些反应的催化作用 通过不同的过渡态在溶液中将允许的基本问题 使用线性自由能关系和同位素效应被问到：是否 酶或底物的固有性质决定了 反应的过渡态是什么？活动站点功能负责 将测试用于磷酸单酯优先水解的‘调谐’AP 通过定点突变。

DESCRIPTION: (provided by applicant) Phosphoryl transfer is the most common biological reaction. In principle, protein kinases and phosphatases should be excellent targets for drug therapy. There are estimated to be about 3000 protein kinases and phosphatases in the human genome, and these enzymes are central to control of essential cellular processes. However, these enzymes have yet to emerge as powerful drug targets. This proposal is aimed at increasing fundamental understanding of phosphoryl transfer and to probe in depth enzymatic catalysis of these reactions. This understanding can in turn aid in understanding how phosphoryl catalysis is used in complex biological processes and ultimately may contribute to the design of new drugs. This proposal has two sections, the first presenting experiments that probe nonenzymatic phosphoryl transfer reactions and the second section describing experiments with E. coli alkaline phosphatase (AP) that probe basic issues of phosphoryl transfer catalysis. The nonenzymatic studies will provide a fundamental characterization of the reactions of alkyl phosphates. Alkyl phosphates are the most common class of biological phosphate compounds, but the vast majority of studies of phosphate compounds have used aryl for ease of measurement. However, recent results showing unexpectedly fast hydrolysis of methyl phosphate have called into question whether aryl phosphates provide an appropriate model for alkyl phosphates. This will be tested, and the alkyl phosphate reactions characterized, using classical approaches of physical organic chemistry such as linear free energy relationships and also vibrational spectroscopy. Potential catalytic effects from removal of water will be tested and the origin of enhanced hydrolysis in mixed organic solvents will be investigated. E. coli AP is an ideal enzyme to probe fundamental issues in enzymatic catalysis because of the many prior kinetic and mechanistic investigations and the multiple high-resolution structures available, and because the low specificity and open active site allow systematic variation of substrates. AP catalyzes the hydrolysis of phosphate diesters in addition to its cognate reaction with phosphate monoesters. Catalysis of these reactions that proceed through different transition states in solution will allow the basic question to be asked using linear free energy relationships and isotope effects: Does the enzyme or the intrinsic properties of the substrate determine the nature of the reaction's transition state? The active site features responsible for 'tuning' AP for preferential hydrolysis of phosphate monoesters will be tested by site-directed mutagenesis.