THYMIDYLATE SYNTHETASE & RELATED ENZYMES

该项目涉及多学科的研究工作 针对酶的结构/功能/抑制研究 胸苷酸合成酶（TS）。 这种酶引起了越来越多的兴趣 在过去的五年里，由于几个主要的 成果/发现：（一）TS的X射线晶体结构， 几个不同的来源和不同的绑定形式已经被 解决了 现在，突变体的结构可以很容易地通过分子 更换. (ii)人TS已表达。 (iii)的 干酪乳杆菌TS基因已被化学合成， 作为理想的诱变/表达载体。 (iv)条件已经 发现TS展开/重新折叠。 我们对TS的研究可以细分为 几个方面：（1）我们正在进行结构-功能 使用突变方法的TS研究。 在这里，我们变异一个被选中的 使用“混合盒”将氨基酸与所有19个其他残基连接 合成基因的“诱变”;合成基因的区段是 替换为含有所有密码子的寡核苷酸的混合物， 目标部位。 鉴定突变体，单独纯化， 表征了 进行X射线和其他生物物理研究， 有趣的变种人 采用类似的方法来准备 组合（多个）突变，包括组合文库。 (2)在其他的研究中，我们正在尝试合理设计肽， 以及具有独特作用模式的TS的其它抑制剂。 一种方法 将利用计算方法设计和开发 新型抑制剂。 (3)我们正在进行TS突变体的折叠研究 试图理解亚基二聚化的分子特征。 （四） 我们试图通过13 C测定催化硫醇的pKa 匪r 质谱法用于分析和鉴定 作为TS的潜在抑制剂或TS的产物的化合物， TS与不寻常的抑制剂的反应。 它也用于 突变酶的分析和预测分子的确认 权重 在人类酶的情况下，形成切割的酶， 在某些条件下。 我们正在用质谱仪确定 分子量，以确定切割位点。

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.