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首页> 外文期刊>Biochemistry >Effects of subunit occupancy on partitioning of an intermediate in thymidylate synthase mutants.
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Effects of subunit occupancy on partitioning of an intermediate in thymidylate synthase mutants.

机译：亚基占用对胸苷酸合酶突变体中中间体分配的影响。

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摘要

Experimental evidence for a 5-exocyclic methylene-dUMP intermediate in the thymidylate synthase reaction was recently obtained by demonstrating that tryptophan 82 mutants of the Lactobacillus casei enzyme produced 5-(2-hydroxyethyl)thiomethyl-dUMP (HETM-dUMP) (Barret, J. E., Maltby, D. A., Santi, D. V., and Schultz, P. G. (1998) J. Am. Chem. Soc. 120, 449-450). The unusual product was proposed to emanate from trapping of the intermediate with beta-mercaptoethanol in competition with hydride transfer from H(4)folate to form dTMP. Using mutants of the C-terminal residue of thymidylate synthase, we found that the ratio of HETM-dUMP to dTMP varies as a function of CH(2)H(4)folate concentration. This observation seemed inconsistent with the conclusion that both products arose from a common intermediate in which CH(2)H(4)folate was already bound to the enzyme. The enigma was resolved by a kinetic model that allowed for differential partitioning of the intermediate formed on each of the two subunits of the homodimeric enzyme in forming the two different products. With three C-terminal mutants of L. casei TS, HETM-dUMP formation was consistent with a model in which product formation occurs upon occupancy of the first completely bound subunit, the rate of which is unaffected by occupancy of the second subunit. With one analogous E. coli TS mutant, HETM-dUMP formation occurred upon occupancy of the first subunit, but was inhibited when both subunits were occupied. With all mutants, dTMP formation occurs from occupied forms of both subunits at different rates; here, binding of cofactor to the first subunit decreased affinity for the second, but the reaction occurred faster in the enzyme form with both subunits bound to dUMP and CH(2)H(4)folate. The model resolves the apparent enigma of the cofactor-dependent product distribution and supports the conclusion that the exocyclic methylene intermediate is common to both HETM-dUMP and dTMP formation.
机译：最近通过证明酪乳杆菌酶的色氨酸82突变体产生了5-（2-羟乙基）硫代甲基-dUMP（HETM-dUMP）（胸腺肽，JE）获得了胸苷酸合酶反应中5-外环亚甲基-dUMP中间体的实验证据。 ，Maltby，DA，Santi，DV，和Schultz，PG（1998）J.Am.Chem.Soc.120，449-450）。提出了这种不寻常的产物是由于中间体与β-巯基乙醇的捕获而产生的，它与H（4）叶酸的氢化物转移形成dTMP竞争。使用胸苷酸合酶的C端残基的突变体，我们发现HETM-dUMP与dTMP的比率随CH（2）H（4）叶酸浓度的变化而变化。该观察结果似乎与以下结论不一致：两种产物均来自一个共同的中间体，其中CH（2）H（4）叶酸已与该酶结合。通过动力学模型解析谜团，该动力学模型允许在形成两种不同产物的同二聚酶的两个亚基中的每一个上形成的中间体进行差异分配。对于干酪乳杆菌TS的三个C末端突变体，HETM-dUMP形成与其中第一完全结合的亚基占据时产物形成发生的模型是一致的，其速率不受第二亚基的占有率的影响。对于一个类似的大肠杆菌TS突变体，HETM-dUMP的形成在第一个亚基占据时发生，但在两个亚基均被占据时被抑制。对于所有突变体，dTMP的形成均以不同的速率从两个亚基的占据形式中产生。在这里，辅因子与第一个亚基的结合降低了对第二个亚基的亲和力，但该反应以酶形式更快地发生，两个亚基均与dUMP和CH（2）H（4）叶酸结合。该模型解决了辅因子依赖性产物分布的表观谜团，并支持以下结论：环外亚甲基中间体对于HETM-dUMP和dTMP形成均是共有的。

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