OROTATE PHOSPHORIBOSYLTRANSFERASE--STRUCTURE & MECHANISM

这一合作项目将首次提供详细的 磷酸核糖基转移酶的结构和功能 （PRTases），参与核苷酸形成的酶。 这些酶 抗癌化学疗法的靶点，它们的功能障碍是 包括莱-尼二氏病在内的多种代谢紊乱 尽管 他们的关键代谢作用和药理学意义，没有三个- 已知这些酶的三维结构， 分析仅限于对酶的单个酶的分散观察。 组 我们选择乳清酸磷酸核糖基转移酶（OPRTase）作为 由于其体积小，动力学特性好， 易处理性 该酶催化核苷酸从头形成步骤， 嘧啶核苷酸（UTP、TTP和CTP）的合成，是 化疗的基础上致命的合成抗癌药物5- 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 乳清酸尿 我们已经克隆、测序并过表达了OPRTase （pyrE）基因，并纯化所得酶 同质性。 这种酶很容易以一种 适用于X射线晶体学研究，衍射性能优于 2.0一项决议。 发现了一种重原子衍生物， 准备进行以下类型的详细调查。 我们的研究采用动力学同位素效应和位置同位素交换 (PIX)为了研究反应过渡态的性质， 为今后的缓蚀剂设计提供了重要信息。 机制 进一步探索使用pH研究定位酶质子 参与催化的受体/供体基团。 三维 OPRTase的结构将被确定为高分辨率，并用于 模型的结构密切相似OPRTase结构域的 重要的人UMP合酶。 使用结构和 提出的机制，一系列的修饰/诱变实验， 计划探索的起源底物特异性所表现出的 PRTase，特别是OPRTase。 我们预计， 所产生的信息将允许预测其它PRTase结构， 我们发现的催化原理也适用于其他 PRTases也是。

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.