Formation of Catalytically Active Binuclear Center of Glycerophosphodiesterase: A Molecular Dynamics Study
Formation of Catalytically Active Binuclear Center of Glycerophosphodiesterase: A Molecular Dynamics Study
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甘油磷酸二酯酶催化活性双核中心的形成：分子动力学研究
DOI：
10.1021/acs.jpcb.8b02046
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Prabhakar, Rajeev
中科院分区：
文献类型：
--
作者：
Paul, Thomas J.;Schenk, Gerhard;Prabhakar, Rajeev
甘油磷酸二酯酶（GpdQ）是一种双核金属磷酸酶，其催化多种关键分子的单、二和三磷酸酯键的水解裂解。在底物结合时，这种酶经历了从无活性单核形式（Em，其中金属驻留在α位点）到活性双核中心（Eb-S，金属结合到α和β位点）的复杂转化，通过单核、底物结合的中间状态（Em-S）。在这项研究中，全原子分子动力学模拟已被用来研究结构和动力学转换在这一过程中使用八个不同的变体，即，酶的五种野生型和三种突变形式。此外，一个实际的基板，双-（对硝基苯基）磷酸盐（bpNPP），金属桥接亲核羟基，和特定的第一和第二配位壳残基的影响进行了研究。底物与E的初始结合增强了α位点的金属结合亲和力，并为第二金属离子的配位准备了β位点。这些结果与停流荧光和量热数据一致。在Eb-S中，计算的衬底和金属（α和β）结合能的增加也与实验数据一致。然而，发现从该络合物中去除底物会导致α和β金属的结合能显著降低。在这项研究中预测的活性位点的创建和稳定的基板的作用是支持的动力学测量使用停流和核磁共振技术。重要的是，残基Asn 80，β位点中金属的配体，通过在Eb-S的形成中充当门而表现出配位灵活性，与诱变和光谱数据良好一致。
Glycerophosphodiesterase (GpdQ) is a binuclear metallophosphatase that catalyzes the hydrolytic cleavage of mono-, di-, and triphosphoester bonds of a wide range of critical molecules. Upon substrate binding, this enzyme undergoes a complex transformation from an inactive mononuclear form (Em, where the metal resides in the α site) to an active binuclear center (Eb-S, with metals bound to both the α and β sites) through a mononuclear, substrate-bound intermediate state (Em-S). In this study, all-atom molecular dynamics simulations have been employed to investigate structures and dynamical transformations in this process using eight different variants, i.e., five wild-type and three mutant forms of the enzyme. Additionally, the effects of an actual substrate, bis-(para-nitrophenyl) phosphate (bpNPP), a metal-bridging nucleophilic hydroxyl, and specific first and second coordination shell residues have been investigated. The initial binding of the substrate toEmenhances the metal binding affinity of the α site and prepares the β site for coordination of the second metal ion. These results are in agreement with stopped-flow fluorescence and calorimetry data. InEb-S, the computed increase in the substrate and metal (both α and β) binding energies is also in line with the experimental data. However, removal of the substrate from this complex is found to cause substantial reduction in binding energies of both α and β metals. The role of the substrate in the creation and stabilization of the active site predicted in this study is supported by the kinetic measurements using both stopped-flow and nuclear magnetic resonance techniques. Importantly, residue Asn80, a ligand of the metal in the β site, exhibits coordination flexibility by acting as a gate in the formation ofEb-S, in good agreement with mutagenesis and spectroscopic data.