Computational Molecular Biophysics, IWR, University of Heidelberg, Im Neuenheimer Feld 368, 69120 Heidelberg, Germany.
J Comput Chem. 2012 Jul 15;33(19):1603-14. doi: 10.1002/jcc.22993. Epub 2012 May 2.
The Diels-Alderase ribozyme is an in vitro-evolved ribonucleic acid enzyme that catalyzes a [4 + 2] cycloaddition reaction between an anthracene diene and a maleimide dienophile. The ribozyme can in principle be used to selectively synthesize only one product enantiomer, depending on which of the two entrances to the catalytic pocket, "front" or "back", the substrate is permitted to use. Here, we investigate stereoselection and substrate recognition in the ribozyme by means of multiple molecular dynamics simulations, performed on each of the two substrates individually in the pocket, on the reactant state, and on the product state. The results are consistent with a binding mechanism in which the maleimide likely binds first followed by the anthracene, which enters preferentially through the front door. The free energy profiles for anthracene binding indicate that the pre-(R,R)-enantiomer conformation is slightly preferred, in agreement with the experimentally observed small enantiomeric excess of the (R,R)-enantiomer of the product. The reactant state is stabilized by the simultaneous presence of both substrates bound to their binding sites in the hydrophobic pocket as well as by stacking interactions between them.
Diels-Alderase 核酶是一种体外进化的核糖核酸酶，可催化蒽二烯和马来酰亚胺二烯之间的 [4 + 2] 环加成反应。该核酶原则上可以用于选择性合成仅有一种产物对映异构体，具体取决于底物被允许使用催化口袋的两个入口（“前”或“后”）中的哪一个。在这里，我们通过分子动力学模拟研究了核酶中的立体选择性和底物识别，对口袋中的两种底物分别在反应物状态和产物状态下进行了模拟。结果与一种结合机制一致，其中马来酰亚胺可能首先结合，然后是蒽，蒽优先通过前门进入。蒽结合的自由能曲线表明，(R,R)-前手性对映体构象略有优势，这与实验观察到的产物 (R,R)-对映体的小对映过量一致。反应物状态通过同时存在结合在疏水性口袋的结合位点上的两种底物以及它们之间的堆积相互作用来稳定。
