Departament de Química Física i Analítica, Universitat Jaume I, 12071 Castellón, Spain.
Phys Chem Chem Phys. 2010 Oct 7;12(37):11657-64. doi: 10.1039/c003799k. Epub 2010 Aug 16.
A theoretical study of the temperature dependence of dynamic effects in the rate limiting step of the reaction catalyzed by thymidylate synthase is presented in this paper. From hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) optimizations of transition state structures within a fully flexible molecular model, free downhill molecular dynamics trajectories have been performed at four different temperatures. The analysis of the reactive and non-reactive trajectories in the enzyme environment has allowed us to study the geometric and electronic coupling between the substrate, the cofactor and the protein. The results show how the contribution of dynamic effects to the rate enhancement measured by the transmission coefficients is, at the four studied temperatures, negligible. Nevertheless, the rare event trajectories performed have shown how the hydride transfer and the scission of the conserved active site cysteine residue (Cys146 in E. coli) take place in a concerted but asynchronous way; the latter takes place once the transfer has occurred. The analysis of the dynamics of the protein reveals also how the relative movements of some amino acids, especially Arg166, and a water molecule, promotes the departure of the Cys146 from the dUMP. Finally, it seems that the protein environment creates an almost invariant electric field in the active site of the protein that stabilizes the transition state of the reaction, thus reducing the free energy barrier.
本文对胸苷酸合酶催化反应限速步骤中动态效应的温度依赖性进行了理论研究。通过在完全柔性分子模型中对过渡态结构进行混合量子力学/分子力学（QM/MM）优化，在四个不同温度下进行了自由下坡分子动力学轨迹计算。在酶环境中对反应和非反应轨迹的分析，使我们能够研究底物、辅因子和蛋白质之间的几何和电子耦合。结果表明，在四个研究温度下，通过传输系数测量的动态效应对速率增强的贡献可以忽略不计。然而，进行的稀有事件轨迹表明，氢化物转移和保守活性位点半胱氨酸残基（大肠杆菌中的 Cys146）的断裂是如何协同但异步发生的；一旦转移发生，后者就会发生。对蛋白质动力学的分析还揭示了蛋白质中一些氨基酸（特别是 Arg166）和一个水分子的相对运动如何促进 Cys146 离开 dUMP。最后，似乎蛋白质环境在蛋白质的活性位点中创建了一个几乎不变的电场，稳定了反应的过渡态，从而降低了自由能垒。
