Department of Chemistry & Biochemistry, University of Mississippi, University, MS 38677, USA.
J Mol Model. 2012 Jun;18(6):2869-83. doi: 10.1007/s00894-011-1308-9. Epub 2011 Nov 30.
Carboxylesterases (CEs) are ubiquitous enzymes responsible for the detoxification of xenobiotics. In humans, substrates for these enzymes are far-ranging, and include the street drug heroin and the anticancer agent irinotecan. Hence, their ability to bind and metabolize substrates is of broad interest to biomedical science. In this study, we focused our attention on dynamic motions of a CE from B. subtilis (pnbCE), with emphasis on the question of what individual domains of the enzyme might contribute to its catalytic activity. We used a 10 ns all-atom molecular dynamics simulation, normal mode calculations, and enzyme kinetics to understand catalytic consequences of structural changes within this enzyme. Our results shed light on how molecular motions are coupled with catalysis. During molecular dynamics, we observed a distinct C-C bond rotation between two conformations of Glu310. Such a bond rotation would alternately facilitate and impede protonation of the active site His399 and act as a mechanism by which the enzyme alternates between its active and inactive conformation. Our normal mode results demonstrate that the distinct low-frequency motions of two loops in pnbCE, coil_5 and coil_21, are important in substrate conversion and seal the active site. Mutant CEs lacking these external loops show significantly reduced rates of substrate conversion, suggesting this sealing motion prevents escape of substrate. Overall, the results of our studies give new insight into the structure-function relationship of CEs and have implications for the entire family of α/β fold family of hydrolases, of which this CE is a member.
羧酸酯酶(Carboxylesterases,CEs)是广泛存在的酶,负责解毒外来物质。在人类中,这些酶的底物范围很广,包括街头毒品海洛因和抗癌药物伊立替康。因此,它们结合和代谢底物的能力引起了生物医学科学的广泛关注。在这项研究中,我们专注于枯草芽孢杆菌(B. subtilis)的 CE(pnbCE)的动态运动,重点关注酶的个别结构域可能对其催化活性有何贡献的问题。我们使用了 10 ns 的全原子分子动力学模拟、正常模式计算和酶动力学来了解该酶内结构变化的催化后果。我们的结果阐明了分子运动如何与催化相耦合。在分子动力学过程中,我们观察到 Glu310 两种构象之间明显的 C-C 键旋转。这种键旋转会交替促进和阻碍活性位点 His399 的质子化,并作为酶在其活性和非活性构象之间交替的机制。我们的正常模式结果表明,pnbCE 中的两个环(coil_5 和 coil_21)的独特低频运动对于底物转化很重要,并封闭了活性位点。缺乏这些外环路的突变 CE 显示出显著降低的底物转化速率,表明这种密封运动阻止了底物的逃逸。总的来说,我们研究的结果为 CE 的结构-功能关系提供了新的见解,并对整个α/β折叠家族水解酶家族产生了影响,该 CE 是该家族的成员。