Pal Nibedita, Wu Meiling, Lu H Peter
Department of Chemistry, Center for Photochemical Sciences, Bowling Green State University, Bowling Green, OH 43403.
Department of Chemistry, Center for Photochemical Sciences, Bowling Green State University, Bowling Green, OH 43403
Proc Natl Acad Sci U S A. 2016 Dec 27;113(52):15006-15011. doi: 10.1073/pnas.1613404114. Epub 2016 Dec 8.
Unraveling the conformational details of an enzyme during the essential steps of a catalytic reaction (i.e., enzyme-substrate interaction, enzyme-substrate active complex formation, nascent product formation, and product release) is challenging due to the transient nature of intermediate conformational states, conformational fluctuations, and the associated complex dynamics. Here we report our study on the conformational dynamics of horseradish peroxidase using single-molecule multiparameter photon time-stamping spectroscopy with mechanical force manipulation, a newly developed single-molecule fluorescence imaging magnetic tweezers nanoscopic approach. A nascent-formed fluorogenic product molecule serves as a probe, perfectly fitting in the enzymatic reaction active site for probing the enzymatic conformational dynamics. Interestingly, the product releasing dynamics shows the complex conformational behavior with multiple product releasing pathways. However, under magnetic force manipulation, the complex nature of the multiple product releasing pathways disappears and more simplistic conformations of the active site are populated.
在催化反应的关键步骤（即酶 - 底物相互作用、酶 - 底物活性复合物形成、新生产物形成和产物释放）中，由于中间构象状态的瞬态性质、构象波动以及相关的复杂动力学，解析酶的构象细节具有挑战性。在此，我们报告了使用单分子多参数光子时间标记光谱结合机械力操纵（一种新开发的单分子荧光成像磁镊纳米方法）对辣根过氧化物酶构象动力学的研究。一个新生形成的荧光产物分子用作探针，完美适配于酶促反应活性位点以探测酶的构象动力学。有趣的是，产物释放动力学显示出具有多种产物释放途径的复杂构象行为。然而，在磁力操纵下，多种产物释放途径的复杂性质消失，活性位点出现更简单的构象。
