Liang Guangchao, Webster Charles Edwin
Department of Chemistry and Center for Computational Sciences, Mississippi State University, Mississippi State, Mississippi 39762, USA.
Org Biomol Chem. 2017 Oct 18;15(40):8661-8668. doi: 10.1039/c7ob02098h.
As a member of the histidine triad (HIT) protein superfamily, human histidine triad nucleotide binding protein 1 (hHint1) serves as an efficient enzyme in the hydrolysis of phosphoramidate. In particular, hHint1 has been utilized to activate nucleotide prodrugs (proTides). Understanding the mechanism of hHint1 will aid in the future design of proTides. Density functional theory (DFT) computations on a 228-atom cluster active-site model were performed to investigate the hydrolysis mechanism of a phosphoramidate substrate. The overall proposed mechanism included the key involvement of the histidine triad as a proton shuttle. Protonated methylphosphoramidate was first formed by proton transfer of protonated His114 species. A penta-coordinated phosphoryl intermediate, protonated methylphosphorodiamidate, was generated by a nucleophilic attack of His112. After the release of amine and the generation of a phosphorylated histidine intermediate, the nucleophilic attack of an active-site water produced a hydrolyzed intermediate that subsequently transferred a proton back to His114. A rate-determining fully associative pathway with a free energy of activation of 21.7 kcal mol formed the penta-coordinated phosphoryl intermediate. A non-rate determining associative-interchange transition state was involved in the formation of transient tetra-coordinated phosphoryl intermediate. The overall hydrolysis was favorable by -16.1 kcal mol.
作为组氨酸三联体（HIT）蛋白超家族的一员，人组氨酸三联体核苷酸结合蛋白1（hHint1）是一种高效的氨基磷酸水解酶。特别地，hHint1已被用于激活核苷酸前药（前体药物）。了解hHint1的作用机制将有助于未来前体药物的设计。我们对一个包含228个原子的簇活性位点模型进行了密度泛函理论（DFT）计算，以研究氨基磷酸底物的水解机制。所提出的总体机制包括组氨酸三联体作为质子穿梭体的关键作用。质子化的甲基氨基磷酸首先通过质子化的His114物种的质子转移形成。His112的亲核攻击产生了一个五配位的磷酰中间体，即质子化的甲基磷二酰胺。在胺释放和磷酸化组氨酸中间体生成后，活性位点水的亲核攻击产生了一个水解中间体，随后该中间体将一个质子转移回His114。形成五配位磷酰中间体的速率决定步骤是一个自由能为21.7 kcal/mol的完全缔合途径。瞬态四配位磷酰中间体的形成涉及一个非速率决定的缔合交换过渡态。总体水解反应的自由能为-16.1 kcal/mol，是有利的。
