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修复糖基化酶如何催化 破坏DNA中的嘧啶和嘌呤碱基。 这一近期目标 这个建议是为了了解的机制和催化的 嘧啶特异性酶，尿嘧啶DNA糖基化酶（UDG）。杆菌 UDG具有与健康相关的意义，因为它在消除 预诱变尿嘧啶碱基从DNA，也是一个可行的目标， 抗病毒药物，由于其在病毒中的重要作用， 潜伏期/再激活周期和病毒DNA复制。 具体 这项研究的目的是i）阐明完整的动力学和 UDG反应的热力学机制ii）确定结构 对于酶结合的底物和产物，iii）建立化学品 UDG的1012倍催化能力的基础，以及iv）确定 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沿着将 使用快速动力学方法进行表征，包括建议的 尿嘧啶合成的关键步骤--碱基从 DNA螺旋。 一种新的荧光分析方法已经开发出来， 这个动能的工作。 酶结合的DNA底物的结构和 产品将通过X射线晶体学来解决，正如我们已经解决的那样， 对游离酶的作用。酶促反应的化学机理 将通过保守活性位点的诱变来检查反应 在游离酶的部分晶体结构中看到的残基。 的 这些突变对微观动力学步骤的破坏作用， 结合参数和pH速率曲线将被定量。因此，在本发明中， 这些残基的催化作用及其pKa值将是 建议道。 然后将直接使用异频NMR光谱法 测定通过以下方法鉴定的关键基团的侧链pKa值 诱变，建立一个热力学框架的质子 参与反应的转移。 过渡态结构将 基于双标记竞争同位素效应进行表征 测量. 对于同位素效应工作， 用于同位素标记的脱氧尿苷的酶促合成， 将使用含有DNA的材料。 酶的同位素效应和 过渡态将它们与水和酸催化相比 从同位素标记的DNA水解尿嘧啶。 预计 这项工作将提供一个基本的了解的因素， 有助于酶促过渡态的稳定， 从DNA水解嘧啶碱基。

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.