THYMIDYLATE SYNTHASE

胸苷酸合成酶(TS)催化DUMP和5，10-DUMP的转化 亚甲基四氢叶酸(CH2/H/2叶酸)为DTMP和7，8-二氢叶酸。 它对于从头合成DTMP是必不可少的，因此是 是合成DNA所必需的。对这种酶的持续深入研究是 重要(I)对于增加TS的知识库，一个重要的 化疗靶点，尤其在癌症化疗中；(2)作为一种 相关酶的范例，(Iii)作为酶的模型 结构/功能研究；以及(四)作为技术平台 发展。 在本建议中，我们计划：(1)研究机制和 抑制TS，(II)定点突变研究以了解 重要氨基酸残基的功能，(三)组合 二聚体界面的突变，以及iv)试图进化酶 通过基因重组或分子进化变成更有效的催化剂。 将进行几项抑制剂研究，包括搜索 可能抑制亚单位结合的多肽和小分子。 诱变将主要通过多个盒式磁带完成 人工合成干酪乳杆菌ts基因的诱变；此外， 提出了组合诱变和基因改组的方法。突变酶 将被提纯并接受详细的表征，以便 确定关键部位氨基酸替代的效果。 我们将对有趣的突变体进行X射线晶体分析 酶和酶抑制物复合体，与Robert博士合作 加州大学旧金山分校斯特劳德。寻找新的小分子抑制剂将是 使用与欧文·昆茨博士合作的程序对接进行的， 加州大学旧金山分校。利用核磁共振光谱学将探讨一些机理问题 与加州大学旧金山分校的托马斯·詹姆斯博士合作。

Thymidylate synthase (TS) catalyzes the conversion of dUMP and 5, 10- methylenetetrahydrofolate (CH2/H/2 folate) to dTMP and 7,8-dihydrofolate. It is essential for de novo synthesis of dTMP and consequently is required for DNA synthesis. Continued in-depth studies of this enzyme are important (i) for increasing the knowledge base of TS, an important chemotherapeutic target, especially in cancer chemotherapy, (ii) as a paradigm for related enzymes, (iii) as a model for enzyme structure/function studies, and (iv) as a platform for technology development. In the present proposal we plan: (i) studies on the mechanism and inhibition of TS, (ii) site directed mutagenesis studies to understand functions of important amino acid residues, (iii) combinatorial mutagenesis of the dimer interface, and iv) attempts to evolve the enzyme into a more effect catalyst by gene-shuffling, or molecular evolution. Several inhibitor studies will be performed, including a search for peptides and small molecules that may inhibit subunit association. Mutagenesis will be accomplished primarily by cassette multiple mutagenesis of a synthetic L. casei Ts gene; in addition, experiments in combinatorial mutagenesis and gene shuffling are proposed. Mutant enzymes will be purified and subjected to detailed characterization in order to determine the effects of amino acid replacement at key positions. We will carry out X-ray crystallographic analysis of interesting mutant enzymes, and enzyme-inhibitor complexes in collaboration with Dr. Robert Stroud, UCSF. The search for novel small molecule inhibitors will be carried out using the program DOCK in collaboration with Dr. Irwin Kunts, UCSF. Some mechanistic question will be approached using NMR spectroscopy in collaboration with Dr. Thomas James, UCSF.