THYMIDYLATE SYNTHETASE & RELATED ENZYMES

胸苷酸合成酶

• 批准号: 6308801
• 负责人: DANIEL V. SANTI
• 金额: $ 0.99万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2002-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6308801
• 关键词: biological products; biomedical resource; biotechnology; hematology; immunology; infection; inflammation; lymphatic system; spectrometry; structural biology

This project involves a multi-disciplinary research effort directed at structure/function/inhibition studies of the enzyme thymidylate synthase (TS). This enzyme has gained increased interest over the past five years because of several major accomplishments/findings: (i) The X-ray crystal structures of TS from several different sources and in different bound forms have been solved. Now, structures of mutants can be readily solved by molecular replacement. (ii) The human TS has been expressed. (iii) The Lactobacillus casei TS gene has been chemically synthesized, serving as an ideal mutagenesis/expression vector. (iv) Conditions have been found to unfold/refold TS. Our studies of TS can be subdivided into several categories: (1) We are carrying out structure-function studies of TS using a mutational approach. Here, we mutate a chosen amino acidto all 19 other residues using "mixture-cassette mutagenesis" of the synthetic gene; a segment of the synthetic gene is replaced by mixtures of oligonucleotides containing all codons at the target site. The mutants are identified, individually purified and characterized. X-ray and other biophysical studies are undertaken for the interesting mutants. A similar approach is taken to prepare combinatorial (multiple) mutations, including combinatorial libraries. (2) In other studies, we are attempting the rational design of peptide and other inhibitors of TS with unique modes of action. One approach will utilize computational methods for the design and development of novel inhibitors. (3) We are performing folding studies of TS mutants to try to understand molecular features of subunit dimerization. (4) We are attempting to determine the pKa of the catalytic thiol by 13C NMR. MassSpectrometry is used for analysis and identification of compounds which are potential inhibitors of TS or products of the reaction of TS with unusual inhibitors. It is also used in the analysis of mutant enzymes and confirmation of predicted molecular weights. In the case of the human enzyme, a cleaved enzyme is formed under some conditions. We are using Mass spec to determine the exact molecular weight in order to identify the cleavage site.

该项目涉及多学科研究工作 针对酶的结构/功能/抑制研究 胸苷酸合酶（TS）。 这种酶已经增加了兴趣 在过去的五年中，由于几个专业 成就/发现：（i）TS的X射线晶体结构 几种不同的来源和不同的形式已经 已解决。 现在，突变体的结构很容易通过分子求解 替代品。 （ii）人类TS已被表达。 （iii） 乳杆菌基因已经化学合成，服务 作为理想的诱变/表达载体。 （iv）条件已经 发现展开/重新折叠ts。 我们对TS的研究可以细分为 几个类别：（1）我们正在执行结构功能 使用突变方法对TS进行研究。 在这里，我们突变一个选择 氨基酸到所有其他19个残留物，都使用“混合物” 合成基因的诱变；合成基因的一部分是 取而代之的是包含所有密码子的寡核苷酸的混合物 目标站点。 突变体被识别，单独纯化和 特征。 进行X射线和其他生物物理研究 有趣的突变体。 采取类似的方法来准备 组合（多个）突变，包括组合文库。 （2）在其他研究中，我们正在尝试合理设计肽 以及具有独特作用模式的TS的其他抑制剂。 一种方法 将利用计算方法的设计和开发 新型抑制剂。 （3）我们正在进行TS突变体的折叠研究 试图了解亚基二聚化的分子特征。 （4） 我们试图通过13C确定催化硫醇的PKA NMR。 大量光谱法用于分析和识别 化合物是TS或产品的潜在抑制剂 TS与异常抑制剂的反应。 它也用于 分析突变酶并确认预测的分子 权重。 对于人酶，形成裂解酶 在某些情况下。 我们正在使用质量规格来确定确切的 分子量以识别裂解位点。