Departament de Química Física, Universitat de València, 46100 Burjassot, Spain.
J Am Chem Soc. 2010 May 26;132(20):6955-63. doi: 10.1021/ja908391v.
We here present a theoretical study of the alkaline hydrolysis of methyl p-nitrophenyl phosphate (MpNPP(-)) in aqueous solution and in the active site of nucleotide pyrophosphatase/phosphodiesterase (NPP). The analysis of our simulations, carried out by means of hybrid quantum mechanics/molecular mechanics (QM/MM) methods, shows that the reaction takes place through different reaction mechanisms depending on the environment. Thus, while in aqueous solution the reaction occurs by means of an A(N)D(N) mechanism, the enzymatic process takes place through a D(N)A(N) mechanism. In the first case, we found associative transition-state (TS) structures, while in the enzyme TS structures have dissociative character. The reason for this change is rationalized in terms of the very different nature of the electrostatic interactions established in each of the environments: while the aqueous solution reduces the repulsion between the negatively charged reacting fragments, assisting their approach, the NPP active site stabilizes the charge distribution of dissociative TS structures, allowing the reaction to proceed with a significantly reduced free energy cost. Interestingly, the NPP active site is able to accommodate different substrates, and it seems that the nature of the TSs depends on their electronic characteristics. So, in the case of the MpNPP(-) substrate, the nitro group establishes hydrogen-bond interactions with water molecules and residues found in the outer part of the catalytic site, while the leaving group oxygen atom does not coordinate directly with any of the zinc atoms of the active site. If methyl phenyl phosphate is used as substrate, then the charge on the leaving group is supported to larger extent by the oxygen atom and the phenolate anion can be then coordinated to one of the two zinc atoms present in the active site.
我们在此提出了一个关于在水溶液中和核苷酸焦磷酸酶/磷酸二酯酶（NPP）活性部位中甲基对硝基苯磷酸酯（MpNPP(-)）碱性水解的理论研究。通过混合量子力学/分子力学（QM/MM）方法进行的分析表明，反应根据环境的不同而通过不同的反应机制发生。因此，虽然在水溶液中反应通过 A(N)D(N) 机制发生，但酶促过程通过 D(N)A(N) 机制发生。在第一种情况下，我们发现了缔合过渡态（TS）结构，而在酶中 TS 结构具有离解特征。这种变化的原因可以根据每种环境中建立的静电相互作用的非常不同的性质来合理化：虽然水溶液减少了带负电荷的反应片段之间的排斥，促进了它们的接近，但 NPP 活性部位稳定了离解 TS 结构的电荷分布，允许反应以大大降低的自由能成本进行。有趣的是，NPP 活性部位能够容纳不同的底物，并且似乎 TS 的性质取决于它们的电子特性。因此，对于 MpNPP(-)底物，硝基与水分子和催化部位外部的残基建立氢键相互作用，而离去基团氧原子不与活性部位的任何一个锌原子直接配位。如果使用甲基苯磷酸酯作为底物，则离去基团上的电荷在更大程度上得到氧原子的支持，并且酚盐阴离子可以与活性部位中存在的两个锌原子之一配位。
