THYMIDYLATE SYNTHETASE & RELATED ENZYMES

这个项目涉及多学科的研究工作。 针对酶的结构/功能/抑制研究 胸苷合成酶(TS)。这种酶获得了越来越多的兴趣 在过去的五年里，因为几个主要的 成就/发现：(I)来自 几种不同的来源和不同的束缚形式 解决了。现在，突变体的结构可以很容易地通过分子解决 替补。(Ii)人的TS已表达。(Iii) 化学合成干酪乳杆菌TS基因，服务于 作为理想的诱变/表达载体。(四)情况 发现要展开/重新折叠TS。我们对TS的研究可以细分为 几个类别：(1)我们正在进行结构功能 用突变方法研究TS。在这里，我们变异了一个被选中的人 用“混合盒”对所有19个其他残基进行氨基酸分析 合成基因的突变；合成基因的一段是 替换为包含所有密码子的寡核苷酸混合物 目标站点。对突变体进行鉴定、单独纯化和 特色化的。进行X射线和其他生物物理研究是为了 有趣的变种人。采取了类似的方法来准备 组合(多重)突变，包括组合文库。 (2)在其他研究中，我们正在尝试多肽的合理设计 以及其他具有独特作用模式的TS抑制剂。一种方法 将利用计算方法来设计和开发 新型抑制剂。(3)我们正在进行TS突变体的折叠研究 试图了解亚基二聚的分子特征。(4) 我们正在尝试用~(13)C测定催化硫醇的pKa 核磁共振。质谱学用于分析和鉴定 是TS的潜在抑制剂的化合物或其产物 TS与不寻常的抑制剂的反应。它也被用在 突变酶的分析及预测分子的验证 重量。在人类酶的情况下，形成了一种被切割的酶 在某些情况下。我们正在使用质谱学来确定 分子量，以确定裂解位点。

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.