THYMIDYLATE SYNTHASE REGULATION

胸苷酸合酶(TS)的生物合成最近被发现 通过将蛋白质与其自身的mRNA结合而下调，而这 调节参与了癌细胞的耐药性的发展 几种以这种酶为靶点的抗代谢药物。我们目前正在 从事对这一现象的物理表征和量化 进程。NCI-NMOB观察到TS/mRNA结合和TS 活性受到巯基乙醇(ME)存在的强烈影响 和其他还原剂。我们假设有约束力的和 活性可能依赖于可逆的巯基开关 涉及酶活性口袋附近的一个或多个半胱氨酸的。 为了检验这一假设，我们构建了这样一个动力学模型 过程，并检查它是否能够解释实验性的 观察。该模型允许将TS绑定到两个不同的 在分析过程中被T1-RNase化学切割的mRNA位置： 可逆氧化还原开关，引入未知数量的活性TS 添加ME前的分析和合成的有序机制 尿苷和亚甲基四氢叶酸中的胸苷。我们估计 结合常数为0.76 nm，氧化还原平衡常数为4×10-9， 初始TS活性组份为1.1%。最重要的是，我们发现 参数之间没有显著差异，从 结合和酶的组合数据集或单独的配件的拟合， 这表明同一个还原位点参与了结合和 活跃的袖珍区域设置。此外，计算的酶活性具有 发现与从以下来源获得的TS活性非常一致 干酪乳杆菌。

The biosynthesis of thymidylate synthase (TS) has recently been found to be down-regulated by binding of the protein to its own mRNA, and this regulation is involved in the development by cancer cells of resistance to several antimetabolites targeted at this enzyme. We are presently engaged in the physical characterization and quantification of this process. NCI-NMOB has observed that both the TS/mRNA binding and TS activity are strongly affected by the presence of mercaptoethanol (ME) and other reducing agents. We have hypothesized that both binding and activity are dependent on a reversible sulfhydryl switch, probably involving one or more cysteines near the active pocket of the enzyme. To test this hypothesis, we have constructed a kinetic model of such a process and examined it for its ability to account for experimental observation. The model allows for the binding of TS to two different mRNA locations chemically cleaved by T1-RNAse during assay: the reversible redox switch, an unknown amount of active TS introduced into the assay before addition of ME; and an ordered mechanism for synthesis of thymidine from uridylate and methylenetetrahydrofolate. We estimated a binding constant of 0.76 nM, a redox equilibrium constant of 4 x 10-9, and an initial TS active fraction of 1.1%. Most importantly, we found no significant difference between parameters estimated either from a fitting of combined binding and enzyme data sets or individual fittings, suggesting that the same reducing site is involved in both binding and active pocket locales. Furthermore,the calculated enzyme activity has been found to be in very close agreement with TS activity obtained from lactobacillus caseii.