Xu Yan, Gao Yanjing, Su Yingying, Sun Lele, Xing Feifei, Fan Chunhai, Li Di
School of Chemistry and Molecular Engineering , East China Normal University , Shanghai 200241 , China.
Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation Facility, Key Laboratory of Interfacial Physics and Technology , Shanghai Institute of Applied Physics, Chinese Academy of Sciences , Shanghai 201800 , China.
J Phys Chem Lett. 2018 Dec 6;9(23):6786-6794. doi: 10.1021/acs.jpclett.8b02992. Epub 2018 Nov 16.
Unraveling the conformational changes of enzymes together with inhibition kinetics during an enzymatic reaction has great potential in screening therapeutic candidates; however, it remains challenging due to the transient nature of each intermediate step. We report our study on the noncompetitive inhibition of horseradish peroxidase with single-turnover resolution using single-molecule fluorescence microscopy. By introducing DNA origami as an addressable nanoreactor, we observe the coexistence of nascent-formed fluorescent product on both catalytic and docking sites. We further propose a single-molecule kinetic model to reveal the interplay between product generation and noncompetitive inhibition and find three distinct inhibitor releasing pathways. Moreover, the kinetic isotope effect experiment indicates a strong correlation between catalytic and docking sites, suggesting an allosteric conformational change in noncompetitive inhibition. A memory effect is also observed. This work provides an in-depth understanding of the correlation between enzyme behavior and enzymatic conformational fluctuation, substrate conversion, and product releasing pathway and kinetics.
在酶促反应过程中解析酶的构象变化以及抑制动力学，在筛选治疗候选药物方面具有巨大潜力；然而，由于每个中间步骤的瞬态性质，这仍然具有挑战性。我们报告了使用单分子荧光显微镜对辣根过氧化物酶进行单周转分辨率的非竞争性抑制的研究。通过引入DNA折纸作为可寻址的纳米反应器，我们观察到新生形成的荧光产物在催化位点和对接位点上共存。我们进一步提出了一个单分子动力学模型，以揭示产物生成与非竞争性抑制之间的相互作用，并发现了三种不同的抑制剂释放途径。此外，动力学同位素效应实验表明催化位点和对接位点之间存在强相关性，这表明在非竞争性抑制中存在别构构象变化。还观察到了记忆效应。这项工作深入了解了酶行为与酶促构象波动、底物转化、产物释放途径和动力学之间的相关性。
