Departament de Química Física, Universitat de València, 46100 Burjassot, Spain.
J Am Chem Soc. 2011 Aug 10;133(31):12050-62. doi: 10.1021/ja2017575. Epub 2011 Jun 14.
We here present a theoretical study of the alkaline hydrolysis of a phosphodiester (methyl p-nitrophenyl phosphate or MpNPP) in the active site of Escherichia coli alkaline phosphatase (AP), a monoesterase that also presents promiscuous activity as a diesterase. The analysis of our simulations, carried out by means of molecular dynamics (MD) simulations with hybrid quantum mechanics/molecular mechanics (QM/MM) potentials, shows that the reaction takes place through a D(N)A(N) or dissociative mechanism, the same mechanism employed by AP in the hydrolysis of monoesters. The promiscuous activity observed in this superfamily can be then explained on the basis of a conserved reaction mechanism. According to our simulations the specialization in the hydrolysis of phosphomonoesters or phosphodiesters, developed in different members of the superfamily, is a consequence of the interactions established between the protein and the oxygen atoms of the phosphate group and, in particular, with the oxygen atom that bears the additional alkyl group when the substrate is a diester. A water molecule, belonging to the coordination shell of the Mg(2+) ion, and residue Lys328 seem to play decisive roles stabilizing a phosphomonoester substrate, but the latter contributes to increase the energy barrier for the hydrolysis of phosphodiesters. Then, mutations affecting the nature or positioning of Lys328 lead to an increased diesterase activity in AP. Finally, the capacity of this enzymatic family to catalyze the reaction of phosphoesters having different leaving groups, or substrate promiscuity, is explained by the ability of the enzyme to stabilize different charge distributions in the leaving group using different interactions involving either one of the zinc centers or residues placed on the outer side of the catalytic site.
我们在这里提出了一个关于在大肠杆菌碱性磷酸酶（AP）活性部位中磷酸二酯（甲基对硝基苯磷酸酯或 MpNPP）的碱性水解的理论研究，AP 是一种单酯酶，也具有作为二酯酶的混杂活性。通过使用混合量子力学/分子力学（QM/MM）势的分子动力学（MD）模拟进行的分析表明，反应通过 D（N）A（N）或离解机制发生，该机制也被 AP 用于单酯的水解。在这个超家族中观察到的混杂活性可以基于保守的反应机制来解释。根据我们的模拟，在不同超家族成员中发展的磷酸单酯或磷酸二酯的水解专业化是由于蛋白质与磷酸基团的氧原子之间建立的相互作用的结果，特别是与氧原子的相互作用，当底物是二酯时，该氧原子带有额外的烷基基团。属于 Mg（2+）离子配位壳的水分子和残基 Lys328似乎在稳定磷酸单酯底物方面起着决定性的作用，但后者有助于增加磷酸二酯水解的能量障碍。然后，影响 Lys328的性质或定位的突变导致 AP 中二酯酶活性增加。最后，该酶家族能够催化具有不同离去基团的磷酸酯的反应，或者底物混杂性，这是由于酶能够使用涉及锌中心之一或位于催化位点外侧的不同相互作用来稳定不同的离去基团中的不同电荷分布。
