Hamza Adel, Cho Hoon, Tai Hsin-Hsiung, Zhan Chang-Guo
Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, USA.
J Phys Chem B. 2005 Mar 17;109(10):4776-82. doi: 10.1021/jp0447136.
Molecular dynamics (MD) simulations were carried out to study cocaine binding with wild-type human butyrylcholinesterase (BChE) and its mutants based on a recently reported X-ray crystal structure of human BChE. For each BChE-cocaine system, we simulated both the nonprereactive and prereactive complexes in water. Despite the significant difference found at the acyl binding pocket, the simulated structures confirm the fundamental structural and mechanistic insights obtained from earlier computational studies of wild-type BChE with cocaine based on a homology model, e.g. the rate-determining step for BChE-catalyzed hydrolysis of biologically active (-)-cocaine is the (-)-cocaine rotation in the active site from the nonprereactive BChE-(-)-cocaine complex to the prereactive complex. It has been demonstrated that the MD simulations on both the nonprereactive and prereactive BChE-cocaine complexes can clearly reveal whether specific mutations produce the desired BChE-(-)-cocaine binding structures in which the (-)-cocaine rotation is less hindered while the required prereactive BChE-(-)-cocaine binding is maintained. Based on the MD simulations, both A328W/Y332A and A328W/Y332G BChE's are expected to have catalytic activity for (-)-cocaine hydrolysis higher than that of wild-type BChE and the activity of A328W/Y332G BChE should be slightly higher than that of A328W/Y332A BChE due to the less-hindered (-)-cocaine rotation in the mutant BChE's. However, the less-hindered (-)-cocaine rotation is only a necessary condition for a higher activity mutant BChE. The (-)-cocaine rotation is also less hindered in A328W/Y332A/Y419S BChE, but (-)-cocaine binds with A328W/Y332A/Y419S BChE in a way that is not suitable for the catalysis. Thus, A328W/Y332A/Y419S BChE is expected to lose the catalytic activity. The computational predictions were confirmed by our experimental kinetic data, demonstrating that the MD simulation-based computational protocol used in this study is reliable in prediction of the catalytic activity of BChE mutants for (-)-cocaine hydrolysis.
基于最近报道的人丁酰胆碱酯酶（BChE）的X射线晶体结构，进行了分子动力学（MD）模拟，以研究可卡因与野生型人丁酰胆碱酯酶及其突变体的结合情况。对于每个BChE-可卡因系统，我们在水中模拟了非预反应复合物和预反应复合物。尽管在酰基结合口袋处发现了显著差异，但模拟结构证实了基于同源模型对野生型BChE与可卡因进行早期计算研究获得的基本结构和机制见解，例如，BChE催化生物活性（-）-可卡因水解的速率决定步骤是活性位点中（-）-可卡因从非预反应性BChE-（-）-可卡因复合物旋转到预反应复合物。已经证明，对非预反应性和预反应性BChE-可卡因复合物进行的MD模拟可以清楚地揭示特定突变是否产生所需的BChE-（-）-可卡因结合结构，其中（-）-可卡因的旋转受到的阻碍较小，同时维持所需的预反应性BChE-（-）-可卡因结合。基于MD模拟，预计A328W/Y332A和A328W/Y332G BChE对（-）-可卡因水解的催化活性高于野生型BChE，并且由于突变体BChE中（-）-可卡因的旋转受到的阻碍较小，A328W/Y332G BChE的活性应略高于A328W/Y332A BChE。然而，（-）-可卡因旋转受到的阻碍较小只是更高活性突变体BChE的必要条件。在A328W/Y332A/Y419S BChE中（-）-可卡因的旋转也受到较小阻碍，但（-）-可卡因与A3,28W/Y332A/Y419S BChE的结合方式不适合催化。因此，预计A328W/Y332A/Y419S BChE会失去催化活性。我们的实验动力学数据证实了计算预测，表明本研究中使用的基于MD模拟的计算方案在预测BChE突变体对（-）-可卡因水解的催化活性方面是可靠的。
