MECHANISMS OF DTMP SYNTHASE AND DCMP HYDROXYMETHLASE

DTMP 合成酶和 DCMP 羟甲基酶的机制

• 批准号: 3301947
• 负责人: LARRY W HARDY
• 金额: $ 16.88万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3301947
• 关键词: DNA replication; Escherichia coli; Lactobacillaceae; X ray crystallography; adduct; deuterium; enzyme mechanism; enzyme structure; enzyme substrate; hybrid enzyme; nonradiation isotope effect; nuclear magnetic resonance spectroscopy; protein engineering; protein sequence; site directed mutagenesis; tetrahydrofolates; thymidylate synthase; transferase; ultraviolet spectrometry

Thymidylate synthase (TS) is an essential enzyme for DNA synthesis and is a target for anticancer and antimicrobial drugs. The major goal of this proposal is to understand the structural correlates of TS function, which will ultimately improve efforts to design more potent and specific inhibitors of the enzyme or use as drugs. The roles proposed for certain amino acid residues in TS, based upon its known three- dimensional structure, will be tested by making site-directed mutant variants of the enzyme and quantitating their kinetic and structural differences with the wild type enzyme. Random mutagenesis will be used to identify additional residues involved in catalysis and in nucleotide and folate binding, and residues that are essential for the structural integrity TS. This information will be essential to understanding the extensive conformational changes which TS undergoes during catalysis. X-ray diffraction studies of certain variants of TS will be performed in collaboration with colleagues at the University of California, San Francisco. Parallel mutagenesis studies will be conducted on another enzyme, dCMP hydroxymethylase (CH), which catalyzes a reaction which is similar but distinct from that catalyzed by TS. The strategy is to compare the structural correlates of the functions of these two enzymes, in order to understand the stereochemical factors that determine the catalytic specificity of each. The amino sequence of CH, an essential enzyme for DNA synthesis by bacteriophage T4, indicates that CH and TS are structural homologs. The initial site-directed mutagenesis experiments on CH, the three-dimensional structure of which is unsolved, will be based on the presumed structural homology between CH and TS. Attempts will be made to crystallize CH, for x-ray structural study in collaboration with colleagues at the University of Oregon, Eugene. Kinetic approaches previously used to unravel the TS mechanism will be employed with CH to test the hypothesis that the two enzymes share certain mechanistic features, and to reveal aspects of their mechanisms that differ. The kinetic studies will in addition provide quantitative monitors, which already exist for TS, of the changes which are effected by mutagenesis of CH. The comparison of TS and CH will reveal basic principles of enzyme design.

胸苷酸合酶 (TS) 是 DNA 合成和合成过程中必需的酶 是抗癌和抗菌药物的靶点。 主要目标是 这个建议是为了理解 TS 功能的结构相关性， 这最终将改进设计更有效和更具体的努力 该酶的抑制剂或用作药物。 提议的角色 TS 中的某些氨基酸残基，基于其已知的三- 维度结构，将通过制作定点突变体来测试 酶的变体并定量其动力学和结构 与野生型酶的差异。 将使用随机诱变 鉴定参与催化和核苷酸的其他残基 和叶酸结合，以及结构所必需的残基 完整性 TS。 这些信息对于理解 TS 在催化过程中经历广泛的构象变化。 TS 某些变体的 X 射线衍射研究将在 与加州大学圣何塞分校的同事合作 弗朗西斯科. 将对另一种酶 dCMP 进行平行诱变研究 羟甲基酶（CH），它催化类似的反应，但 与 TS 催化的不同。 策略是比较 这两种酶的功能的结构相关性，以便 了解决定催化作用的立体化学因素 每个的特殊性。 CH 的氨基酸序列，一种必需的酶 噬菌体 T4 的 DNA 合成表明 CH 和 TS 是 结构同系物。 最初的定点诱变实验 在CH上，其三维结构未解，将是 基于 CH 和 TS 之间假定的结构同源性。 尝试 将使CH结晶，用于X射线结构研究 与俄勒冈大学尤金分校的同事合作。 先前用于解开 TS 机制的动力学方法将是 与 CH 一起使用来检验这两种酶共有的假设 某些机械特征，并揭示其机制的各个方面 有所不同。 动力学研究还将提供定量 监视已存在的 TS 所发生的变化 通过CH的诱变。 TS 和 CH 的比较将揭示基本 酶设计原理。