基于分子动力学模拟提高嗜热磷酸三酯酶样内酯酶非特异性底物活力的理论研究

采用分子动力学模拟和拉伸分子动力学模拟方法, 结合分子力学-广义玻恩表面积(MM-GB/SA)方法进行自由能计算和结构交互指纹分析, 研究了模拟过程中非特异性底物(对氧磷/内酯)分别与嗜热磷酸三酯酶样内酯酶(SsoPox)野生型和突变体(W263F/W263T)结合的构象变化, 分析了Loop8中重要残基Trp263的突变提高SsoPox非特异性底物活力的原因, 发现其能够影响门控残基Phe229的构象变化, 导致活性口袋入口变宽(Phe229与Tyr99之间的距离变大), 使对氧磷和内酯更容易结合到蛋白质的活性位点上; Asp256和Arg223形成盐桥的几率高于野生型(WT)SsoPox, 在Arg223(位于Loop7)的协助下质子更加高效地从活性中心的Asp256(位于Loop8)传递到溶剂中去, 因而能够提高SsoPox水解底物的效率. 通过比较2个野生型复合物的结构稳定性和结合自由能差异, 发现在模拟过程中SsoPox与内酯的复合物体系更加稳定并且具有更低的结合自由能, 有利于SsoPox识别底物并使其埋在活性部位的疏水环境中, 促进氢氧化物亲核进攻底物的亲电中心. 因此, 底物与酶稳定的相互作用可能是SsoPox具有天然内酯酶活性的原因之一.

Exploration of Increasing the Non-specificity Substrates Activity for the Phosphotriesterase-like Lactonase Using Molecular Dynamics Simulations†

The structural changes of phosphotriesterase-like lactonase induced by paraoxon and undecanoic-γ-lactone binding to WT SsoPox and W263F/W263T mutant were compared using molecular dynamics simulation and steered molecular dynamics simulation combined with molecular mechanics-generalized Born/surface area(MM-GB/SA) and structural interaction fingerprints(SIFt) methods. The reasons that the important residue Trp263(located in Loop 8) in SsoPox could determine the enzymatic promiscuity were listed as follow, (1) the displacements in two mutant complex could lead to a widening of the active site entrance, which allowed for tighter fitting of the substrate into the enzyme’s active site; (2) the salt bridge between Asp256 and Arg223 in the two mutant complexes occurred with a higher probability compared to two WT SsoPox complexes, which could offer a potential pathway for proton relay after catalysis. The results of comparing conformational stabilities and binding free energies between WT SsoPox and W263F/W263T mutant indicated that the complex of SsoPox and lactone was more stable and had lower binding free energy during the simulation, which was beneficial to the recognition of the substrate and nucleophilic attack of the hydroxide on the electrophilic center of the substrate. Therefore, the stable interaction of substrate with enzyme may be a reason for the natural lactonase activity of SsoPox.