THYMIDYLATE SYNTHASE REGULATION

胸苷酸合成酶（TS）的生物合成最近被发现， 通过蛋白质与其自身mRNA的结合而下调， 调节与癌细胞的耐药性发展有关 几种针对这种酶的抗代谢物 我们目前正在 从事物理表征和量化， 过程 NCI-NMOB已经观察到TS/mRNA结合和TS/mRNA结合都可以被抑制。 巯基乙醇（ME）的存在强烈影响活性 和其它还原剂。 我们假设约束和 活性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。 为了验证这一假设，我们构建了一个动力学模型， 过程，并检查它的能力，以占实验 观察. 该模型允许TS绑定到两个不同的 在测定期间被T1-RNAse化学切割的mRNA位置： 可逆氧化还原开关，未知量的活性TS引入到 添加ME之前的测定;以及合成的有序机制 由尿苷酸和亚甲基四氢叶酸合成的胸苷。 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活性分数为1.1%。 最重要的是，我们发现 估计的参数之间无显著差异， 组合的结合和酶数据集的拟合或单独的拟合， 这表明相同的还原位点参与结合和 活跃的口袋区域。 此外，计算的酶活性具有 被发现是在非常密切的协议，从TS活动获得 你好

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.