Variath P, Liu Y, Lee T T, Stroud R M, Santi D V
Departments of Biochemistry and Biophysics and Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0448, USA.
Biochemistry. 2000 Mar 14;39(10):2429-35. doi: 10.1021/bi991802d.
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.
最近，通过证明干酪乳杆菌胸苷酸合酶的色氨酸82突变体产生5-(2-羟乙基)硫代甲基-dUMP（HETM-dUMP），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（巴雷特，J.E.，马尔蒂比，D.A.，桑蒂，D.V.，和舒尔茨，P.G.（1998年）《美国化学会志》120，449 - 450）。有人提出，这种不寻常的产物是由于中间体与β-巯基乙醇捕获，与氢化物从H(4)叶酸转移形成dTMP相互竞争而产生的。使用胸苷酸合酶C末端残基的突变体，我们发现HETM-dUMP与dTMP的比例随CH(2)H(4)叶酸浓度而变化。这一观察结果似乎与两种产物都来自CH(2)H(4)叶酸已与酶结合的共同中间体这一结论不一致。通过一个动力学模型解决了这个谜团，该模型允许在同二聚体酶的两个亚基上形成的中间体在形成两种不同产物时进行差异分配。对于干酪乳杆菌TS的三个C末端突变体，HETM-dUMP的形成与一个模型一致，即在第一个完全结合的亚基被占据时发生产物形成，其速率不受第二个亚基占据的影响。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被占据时发生，但当两个亚基都被占据时受到抑制。对于所有突变体，dTMP的形成以不同速率从两个亚基的占据形式发生；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，但在两个亚基都与dUMP和CH(2)H(4)叶酸结合的酶形式中反应发生得更快。该模型解决了辅因子依赖性产物分布的明显谜团，并支持外环亚甲基中间体是HETM-dUMP和dTMP形成所共有的这一结论。
