MECHANISMS OF DTMP SYNTHASE AND DCMP HYDROXYMETHLYLASE

DTMP 合成酶和 DCMP 羟甲基化酶的机制

• 批准号: 3301949
• 负责人: LARRY W HARDY
• 金额: $ 15.59万
• 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/3301949
• 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 是抗癌和抗菌药物的靶标。 主要目标 该建议是了解TS功能的结构相关性， 最终将改善设计更有效和具体的努力 酶的抑制剂或用作药物。 提出的角色 TS中的某些氨基酸残基，基于其已知的三个 尺寸结构将通过使位于位置的突变体进行测试 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 确定参与催化和核苷酸中涉及的其他残基 和叶酸结合，以及对结构必不可少的残基 完整性TS。 这些信息对于理解 TS在催化过程中发生的广泛构象变化。 TS的某些变体的X射线衍射研究将在 与加利福尼亚大学SAN分校的同事合作 弗朗西斯科。 平行诱变研究将在另一种酶DCMP上进行 羟基基酶（CH），催化反应相似但 与TS催化的那个不同。 策略是比较 这两种酶的功能的结构相关性，以便 了解决定催化的立体化学因子 每个的特异性。 CH的氨基序列，一种必需酶 噬菌体T4合成DNA，表明CH和TS是 结构同源物。 初始位置定向的诱变实验 在CH上，其三维结构未解决，将是 基于CH和TS之间的假定结构同源性。 尝试 将制作以结晶的ch，用于X射线结构研究 与俄勒冈大学尤金大学的同事合作。 以前用于阐明TS机制的动力学方法将是 用CH使用，以检验两种酶共享的假设 某些机械特征，并揭示其机制的各个方面 那不同。 动力学研究还将提供定量 已针对TS的监视器已实现的更改 通过CH的诱变。 TS和CH的比较将揭示基本 酶设计原理。