MECHANISMS OF DTMP SYNTHASE AND DCMP HYDROXYMETHLASE

胸苷酸合成酶（TS）是DNA合成的必需酶， 是抗癌和抗微生物药物的靶点。 的主要目标 这个建议是为了理解TS功能的结构相关性， 这将最终改善设计更有效和更具体的 酶的抑制剂或用作药物。 拟议的作用 根据已知的三个氨基酸残基， 三维结构，将进行测试，使定点突变 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 为了鉴定参与催化和核苷酸合成的其他残基， 和叶酸结合，以及结构必需的残基， 完整性TS。 这些信息对于理解 TS在催化过程中经历的广泛的构象变化。 TS某些变体的X射线衍射研究将在 与加州大学旧金山分校的同事合作， 弗朗西斯科。 将对另一种酶dCMP进行平行诱变研究 羟甲基化酶（CH），其催化类似但 与TS催化的不同。 策略是比较 这两种酶功能的结构相关性，以便 了解决定催化剂的立体化学因素 每个人的特异性。 CH的氨基酸序列，一种必需的酶， 通过噬菌体T4的DNA合成，表明CH和TS是 结构同源物。 最初的定点突变实验 在CH上，其三维结构尚未解决，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， 与同事在俄勒冈州，尤金大学的合作。 以前用于解开TS机制的动力学方法将被 与CH一起使用，以检验这两种酶共享 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的 已经存在的用于TS的监视器，用于监视所影响的改变 通过比较TS和CH的突变， 酶设计原理

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.