Graduate School of Pure and Applied Sciences, University of Tsukuba , Tennodai 1-1-1, Tsukuba, Ibaraki 305-8571, Japan.
Center for Computational Sciences, University of Tsukuba , Tennodai 1-1-1, Tsukuba, Ibaraki 305-8577, Japan.
J Phys Chem B. 2017 Jun 8;121(22):5536-5543. doi: 10.1021/acs.jpcb.7b02932. Epub 2017 May 24.
Threonine synthase (ThrS) catalyzes the final chemical reaction of l-threonine biosynthesis from its precursor, O-phospho-l-homoserine. As the phosphate ion generated in its former half reaction assists its latter reaction, ThrS is recognized as one of the best examples of product-assisted catalysis. In our previous QM/MM study, the chemical reactions for the latter half reactions, which are critical for the product-assisted catalysis, were revealed. However, accurate free energy changes caused by the conformational ensembles and entrance of water molecules into the active site are unknown. In the present study, by performing long-time scale MD simulations, the free energy changes by the divalent anions (phosphate or sulfate ions) and conformational states of the intermediate states were theoretically investigated. We found that the calculated free energy double differences are in good agreement with the experimental results. We also revealed that the phosphate ion contributes to forming hydrogen bonds that are suitable for the main reaction progress. This means that the conformation of the active site amino acid residues and the substrate, and hence, the tunable catalysis, are controlled by the product phosphate ion, and this clearly demonstrates a molecular mechanism of the product-assisted catalysis in ThrS.
苏氨酸合酶（ThrS）催化 L-苏氨酸生物合成的最后一步化学反应，其前体为 O-磷酸-L-高丝氨酸。由于在前半反应中生成的磷酸离子有助于后半反应，因此 ThrS 被认为是产物辅助催化的最佳范例之一。在我们之前的QM/MM 研究中，揭示了对于产物辅助催化至关重要的后半反应的化学反应。然而，对于构象整体和水分子进入活性位点所引起的准确自由能变化仍然未知。在本研究中，通过进行长时间尺度的 MD 模拟，理论上研究了二价阴离子（磷酸或硫酸根离子）和中间状态构象状态引起的自由能变化。我们发现计算得到的自由能双差异与实验结果非常吻合。我们还揭示了磷酸离子有助于形成适合主反应进程的氢键。这意味着活性位点氨基酸残基和底物的构象，以及因此而可调的催化作用受到产物磷酸离子的控制，这清楚地证明了 ThrS 中产物辅助催化的分子机制。
