OROTATE PHOSPHORIBOSYLTRANSFERASE--STRUCTURE & MECHANISM

这一合作项目将首次提供详细的 磷酸核糖转移酶的结构和功能研究进展 (PRTase)，参与核苷酸形成的酶。这些酶是 抗癌化疗的靶点及其功能障碍是其根源 包括Lesch-Nyhan病在内的几种代谢紊乱。尽管 它们的关键代谢作用和药理意义，没有三个- 这些酶的空间结构是已知的，并且是机械的 分析仅限于对单个酶的零星观察。 一群人。我们选择了Orotate磷酸核糖转移酶(OPRTase)作为 我们综合研究的对象，因为它的体积小，动力学 易受操控。这种酶催化从头开始的核苷酸形成步骤 合成嘧啶核苷酸(UTP、TTP和CTP)，是研究的目标 基于抗癌药物5-致死合成的化疗 氟尿嘧啶。缺乏OPRTase，无论是遗传性的还是药物诱导的，都会导致 奥罗里奥利酸雨。我们已经克隆、测序并过表达了OPRTase 从鼠伤寒沙门氏菌中分离到(Pre)基因，并对其进行了纯化 为同质性干杯。这种酶很容易结晶成一种卓越的形式。 适用于x射线结晶学研究，衍射谱好于 2.0解决方案。人们发现了一种重原子的衍生物。该系统是 准备进行下文所述类型的详细调查。 我们的研究采用了动力学同位素效应和位置同位素交换 (Pix)研究反应的过渡态的性质， 为今后的缓蚀剂设计提供了重要信息。这一机制 进一步探索使用pH研究来定位酶质子 与催化有关的受体/供体基团。三度空间 OPRTase的结构将被确定为高分辨率，并用于 对紧密相似的OPRTase结构域进行建模 药理上重要的人类UMP合成酶。使用该结构和 提出的机制，进行了一系列的修饰/诱变实验 计划探索底物专一性的起源 PRTase，尤其是OPRTase。我们预计，结构性的 所产生的信息将允许预测其他PRTase结构， 我们发现的催化原理将适用于其他 PRTase也是如此。

This collaborative project will provide, for the first time, a detailed view of the structure and function of the phosphoribosyltransferases (PRTases), the enzymes involved in nucleotide formation. These enzymes are targets for anticancer chemotherapies, and their dysfunction is the origin of several metabolic disorders including Lesch-Nyhan disease. Despite their key metabolic role and pharmacological significance, no three- dimensional structures are known for these enzymes, and mechanistic analysis is limited to scattered observations on individual enzymes of the group. We have chosen orotate phosphoribosyltransferase (OPRTase) as the subject for our comprehensive study because of its small size and kinetic tractability. The enzyme catalyzes the nucleotide-forming step in de novo synthesis of pyrimidine nucleotides (UTP, TTP and CTP), and is a target for chemotherapy based on lethal synthesis from the anticancer drug 5- fluorouracil. Lack of OPRTase, either hereditary or drug-induced, leads to orotic acidurea. We have cloned, sequenced, and overexpressed the OPRTase (pyrE) gene from Salmonella typhimurium, and purified the resultant enzyme to homogeneity. The enzyme crystallizes readily in a form eminently suitable for x-ray crystallographic studies, and diffracts to better than 2.0 A resolution. A heavy atom derivative has been found. The system is poised for detailed investigations of the type described below. Our studies employ kinetic isotope effects and positional isotope exchange (PIX) to study the nature of the transition state for the reaction, providing important information for future inhibitor design. The mechanism is further explored using pH studies to localize enzymic proton acceptor/donor groups involved in catalysis. The three-dimensional structure of OPRTase will be determined to high resolution, and used to model the structure of the closely similar OPRTase domain of the pharmacologically important human UMP synthase. Using the structure and proposed mechanism, a series of modification/mutagenesis experiments is planned to explore the origin of the substrate specificity exhibited by the PRTases, and OPRTase in particular. We expect that the structural information generated will allow predictions of other PRTase structures, and that the principles of catalysis that we uncover will hold for other PRTases as well.