Garrett C, Wataya Y, Santi D V
Biochemistry. 1979 Jun 26;18(13):2798-804. doi: 10.1021/bi00580a017.
Tymidylate synthetase catalyzes the facile dehalogenation of 5-bromo-2'-deoxyuridylate (BrdUMP) and 5-iodo-2'-deoxyuridylate )IdUMP) to give 2'-deoxyuridylate (dUMP), the natural substrate of the enzyme. The reaction does not require folate cofactors and stoichiometrically consumes 2 equiv of thiol. In addition to dUMP, a minor product is formed during the debromination of BrdUMP which has been identified as a 5-alkylthio derivative formed by displacement of bromide ion by thiolate. The reaction has been found to proceed with a substantial alpha-secondary inverse tritium isotope effect (kT/kH = 1.212--1.258) with [2-14C,6-3H]-BrdUMP as the substrate. Similarly, an inverse tritiumisotope effect of 1.18 was observed in the nonenzymatic chemical counterpart of this reaction, the cysteine-promoted dehalogenation of [2-14C,6-3H]-5-bromo-2'-deoxyuridine. Previous evidence for the mechanism of action of this enzyme has rested largely on chemical model studies and on information obtained from its stoichiometric interaction with the inhibitor 5-fluoro-2'-deoxyuridylate. The magnitude of the secondary isotope effect during the enzymatic dehalogenation described here provides direct proof for nucleophilic catalysis and formation of 5,6-dihydroprimidine intermediates in a reaction catalyzed by thymidylate synthetase.
胸苷酸合成酶催化5-溴-2'-脱氧尿苷酸（BrdUMP）和5-碘-2'-脱氧尿苷酸（IdUMP）轻松脱卤生成2'-脱氧尿苷酸（dUMP），后者是该酶的天然底物。该反应不需要叶酸辅因子，化学计量地消耗2当量的硫醇。除了dUMP，在BrdUMP脱溴过程中还形成了一种次要产物，已鉴定为硫醇盐取代溴离子形成的5-烷基硫代衍生物。已发现以[2-14C,6-3H]-BrdUMP为底物时，该反应具有显著的α-二级反向氚同位素效应（kT/kH = 1.212--1.258）。同样，在该反应的非酶化学对应物，即[2-14C,6-3H]-5-溴-2'-脱氧尿苷的半胱氨酸促进脱卤反应中，观察到了1.18的反向氚同位素效应。此前关于该酶作用机制的证据主要基于化学模型研究以及从其与抑制剂5-氟-2'-脱氧尿苷酸的化学计量相互作用中获得的信息。此处描述的酶促脱卤过程中二级同位素效应的大小为胸苷酸合成酶催化的反应中亲核催化和5,6-二氢嘧啶中间体的形成提供了直接证据。
