THYMIDYLATE SYNTHASE REGULATION

胸苷酸合酶调节

• 批准号: 3789467
• 负责人: P F MORRISON
• 金额: --
• 依托单位: NATIONAL CENTER FOR RESEARCH RESOURCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3789467
• 关键词: DNA binding protein; chemical binding; chemical kinetics; chemical models; enzyme activity; enzyme biosynthesis; enzyme induction /repression; enzyme mechanism; reducing agents; thymidylate synthase

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.

最近发现胸苷酸合酶（TS）的生物合成 通过蛋白质与其自身 mRNA 的结合而下调，并且这 调节参与癌细胞耐药性的发展 几种针对该酶的抗代谢物。 我们目前 从事该物质的物理表征和量化 过程。 NCI-NMOB 观察到 TS/mRNA 结合和 TS 活性受到巯基乙醇 (ME) 的存在的强烈影响 和其他还原剂。 我们假设绑定和 活性取决于可逆的巯基开关，可能 涉及酶活性口袋附近的一个或多个半胱氨酸。 为了验证这个假设，我们构建了一个这样的动力学模型 过程并检查其解释实验的能力 观察。 该模型允许将 TS 绑定到两个不同的 检测过程中被 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 添加 ME 之前的测定；以及有序的合成机制 从尿苷酸和亚甲基四氢叶酸中提取胸苷。 我们估计 结合常数为 0.76 nM，氧化还原平衡常数为 4 x 10-9， 初始 TS 活性分数为 1.1%。 最重要的是，我们发现 估计的参数之间没有显着差异 组合结合和酶数据集的拟合或单独拟合， 表明相同的还原位点参与结合和 活跃的口袋区域。 此外，计算出的酶活性为 被发现与获得的 TS 活性非常接近 干酪乳杆菌。