MECHANISMS OF DTMP SYNTHASE AND DCMP HYDROXYMETHLYLASE

胸苷酸合成酶(TS)是DNA合成和代谢的重要酶。 是抗癌和抗微生物药物的靶标。的主要目标是 这项建议是为了了解TS功能的结构相关性， 这最终将改进设计更有力和更具体的努力 酶的抑制剂或用作药物。建议担任的角色 根据已知的三种氨基酸，TS中的某些氨基酸残基- 空间结构，将通过制造定点突变来测试 酶的变异体及其动力学和结构定量 与野生型酶不同。将使用随机诱变 确定与催化和核苷酸有关的其他残基 和叶酸结合，以及结构上必不可少的残基 诚信TS。这些信息对于理解 TS在催化过程中会经历广泛的构象变化。 某些TS变种的X射线衍射研究将在#年进行 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进行平行的突变研究。 羟甲基酶(CH)，催化类似但 与TS催化的不同。我们的策略是比较 这两种酶功能的结构相关性，以便 了解决定催化作用的立体化学因素 每种方法的特殊性。CH的氨基酸序列，这是一种关键的酶 噬菌体T4的DNA合成表明，CH和TS是 结构上的同源。初步的定点诱变实验 在其三维结构尚未解决的CH上，将是 基于假设的CH和TS之间的结构同源性。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。 以前用来解开TS机制的动力学方法将是 与CH一起使用来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 这是不同的。动力学研究还将提供定量的 监控已存在的TS的更改 通过对CH.的诱变。TS和CH的对比将揭示基本的 酶的设计原理。

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.