Study of Nonenzymatic and Enzymatic Phosphoryl Transfer

描述：（由申请人提供）磷酰转移是最常见的 生物反应。原则上，蛋白激酶和磷酸酶应 药物治疗的绝佳靶点。据估计， 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 尚未成为强有力的药物靶点。 这一建议旨在增加对磷酰基的基本理解 转移和探测这些反应的酶催化。这 理解可以反过来帮助理解磷酰基催化是如何使用的 在复杂的生物过程中，最终可能有助于设计 新药 这个建议有两个部分，第一部分介绍实验， 非酶促磷酰基转移反应和第二部分描述 实验用E.大肠杆菌碱性磷酸酶（AP），探测基本问题， 磷酰基转移催化非酶研究将提供 磷酸烷基酯反应的基本特征。烷基 磷酸盐是最常见的一类生物磷酸盐化合物，但 绝大多数磷酸盐化合物的研究都使用芳基， 测量.然而，最近的结果显示， 磷酸甲酯引起了磷酸芳基酯是否提供 磷酸烷基酯的合适模型。这将是测试，和烷基 磷酸盐反应的特点，使用经典的物理方法， 有机化学，如线性自由能关系，也振动 谱将测试去除水的潜在催化作用 并且在混合有机溶剂中增强的水解的起源将是 研究了 E. coli AP是研究酶促反应中基本问题的理想酶 由于许多先前的动力学和机理研究， 多个高分辨率结构可用，并且因为低分辨率 特异性和开放活性位点允许底物系统变化。AP 催化磷酸二酯及其同源物的水解 与磷酸单酯反应。这些反应的催化作用 通过不同的过渡状态， 使用线性自由能关系和同位素效应： 酶或底物的内在性质决定了 反应的过渡态活动站点功能负责 将测试磷酸单酯优先水解的“调整”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.