Department of Chemistry, The University of Iowa, Iowa City, IA 52242-1727;
Department of Chemistry, The University of Iowa, Iowa City, IA 52242-1727.
Proc Natl Acad Sci U S A. 2018 Oct 9;115(41):10311-10314. doi: 10.1073/pnas.1811059115. Epub 2018 Sep 24.
Thymidylate synthase was one of the most studied enzymes due to its critical role in molecular pathogenesis of cancer. Nevertheless, many atomistic details of its chemical mechanism remain unknown or debated, thereby imposing limits on design of novel mechanism-based anticancer therapeutics. Here, we report unprecedented isolation and characterization of a previously proposed intact noncovalent bisubstrate intermediate formed in the reaction catalyzed by thymidylate synthase. Free-energy surfaces of the bisubstrate intermediates interconversions computed with quantum mechanics/molecular mechanics (QM/MM) methods and experimental assessment of the corresponding kinetics indicate that the species is the most abundant productive intermediate along the reaction coordinate, whereas accumulation of the covalent bisubstrate species largely occurs in a parallel nonproductive pathway. Our findings not only substantiate relevance of the previously proposed noncovalent intermediate but also support potential implications of the overstabilized covalent intermediate in drug design targeting DNA biosynthesis.
胸苷酸合成酶是研究最为广泛的酶类之一，因为它在癌症的分子发病机制中起着关键作用。然而，其化学机制的许多原子细节仍然未知或存在争议，从而限制了基于新机制的抗癌治疗药物的设计。在这里，我们报告了前所未有的胸腺嘧啶合成酶催化反应中形成的先前提出的完整非共价双底物中间产物的分离和表征。用量子力学/分子力学（QM/MM）方法计算双底物中间产物转化的自由能表面，并对相应的动力学进行实验评估，表明该物质是反应坐标上最丰富的产物性中间产物，而共价双底物物质的积累主要发生在平行的非产物性途径中。我们的发现不仅证实了先前提出的非共价中间产物的相关性，而且支持了在针对 DNA 生物合成的药物设计中，过稳定的共价中间产物的潜在影响。
