MECHANISMS AND TRANSITION STATES FOR DNA GLYCOSYLASES

这项研究的长期目标是获得一个基本的 了解DNA修复糖基酶如何催化水解酶 破坏DNA中的嘧啶和嘌呤碱基。眼前的目标是 这一建议是为了了解生物多样性的机制和催化作用。 来自大肠杆菌的嘧啶专一性酶尿嘧啶DNA糖基酶(UDG)。 UDG是与健康相关的兴趣，因为它在消除 DNA中的前诱变尿嘧啶碱基，也是 抗病毒药物，因为它在病毒中起着至关重要的作用 潜伏期/重新激活周期和病毒DNA复制。具体的 这项研究的目的是：1)阐明完整的动力学和 UDG反应的热力学机理II)确定结构 对于酶结合的底物和产品，iii)建立化学物质 UDG的1012倍催化能力的基础，以及四)确定 木聚糖酶和非酶水解的过渡态结构 DNA中的尿嘧啶。沿着反应路径的微观步骤将 使用快速动力学方法进行表征，包括建议的 识别尿嘧啶的关键一步--将碱基从 DNA螺旋。一种新的荧光分析方法已经被开发出来，用于大部分 这个动量功。酶结合DNA底物的结构和 产品将通过X射线结晶学来解决，就像我们已经做的那样 为游离的酶做好了。酶促反应的化学机制 反应将通过对保守活性部位的突变进行检测。 在我们的部分晶体结构中可以看到残基的游离酶。这个 这些突变对微观动力学步骤的破坏性影响， 结合参数和pH速率曲线将被量化。因此， 这些残基的催化作用及其pKa值将是 建议。然后将直接使用异核核磁共振波谱 确定关键基团的侧链pKa值 突变，为质子建立热力学框架 参与反应的转移。过渡国家结构将 以双标记竞争同位素效应为基础进行表征 测量。对于同位素效应工作，一种新的高效方法 用于酶法合成同位素标记的脱氧尿苷- 将使用含有DNA的。酶的同位素效应和 将它们与水和酸催化的过渡态进行比较 从同位素标记的DNA中水解尿嘧啶。这是意料之中的 这项工作将提供对以下因素的基本理解 有助于稳定酶的过渡态 脱氧核糖核酸中嘧啶碱基的水解。

The long term goal of this research is to obtain a fundamental understanding how DNA repair glycosylases catalyze the hydrolysis of damaged pyrimidine and purine bases from DNA. The immediate goal of this proposal is to understand the mechanism and catalysis of the pyrimidine specific enzyme, uracil DNA glycosylase (UDG) from E. coli. UDG is of health related interest because of its role in removing premutagenic uracil bases from DNA, and is also a viable target for anti-viral drugs, because of its essential role in viral latency/reactivation cycles and viral DNA replication. The specific aims of this research are to i) elucidate the complete kinetic and thermodynamic mechanism of the UDG reaction ii) determine the structures for enzyme-bound substrate and products, iii) establish the chemical basis for the 1012-fold catalytic power of UDG, and iv) determine the transition-state structure for enzymatic and nonenzymatic hydrolysis of uracil from DNA. The microscopic steps along the reaction pathway will be characterized using rapid kinetic methods, including the suggested key step in uracil recognition-the "flipping out" of the base from the DNA helix. A novel fluorescence assay has been developed for much of this kinetic work. The structures of the enzyme-bound DNA substrate and products will be solved by X-ray crystallography, as we have already done for the free enzyme. The chemical mechanism of the enzymatic reaction will be examined by mutagenesis of conserved active-site residues seen in our partial crystal structure of the free enzyme. The damaging effects of these mutations on the microscopic kinetic steps, binding parameters, and the pH-rate profiles will be quantified. Thus, the catalytic roles of these residues and their pKa values will be suggested. Heteronuclear NMR spectroscopy will then be used directly to determine the side-chain pKa values of key groups identified by mutagenesis, establishing a thermodynamic framework for the proton transfers involved in the reaction. The transition-state structure will be characterized on the basis of dual-label competitive isotope effect measurements. For the isotope effect work, a new and efficient method for the enzymatic synthesis of isotopically labeled deoxyuridine- containing DNA will be used. The enzymatic isotope effects and transition-state will them be compared with the water and acid-catalyzed hydrolysis of uracil from isotopically labeled DNA. It is anticipated that this work will provide a basic understanding of the factors that contribute to stabilization of the enzymatic transition-state for hydrolysis of pyrimidine bases from DNA.