Single-Molecule Studies of Allosteric Inhibition of Individual Enzyme on a DNA Origami Reactor,The Journal of Physical Chemistry Letters

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Single-Molecule Studies of Allosteric Inhibition of Individual Enzyme on a DNA Origami Reactor
The Journal of Physical Chemistry Letters ( IF 4.8 ) Pub Date : 2018-11-09 00:00:00 , DOI: 10.1021/acs.jpclett.8b02992
Yan Xu _{1,

2,

3} , Yanjing Gao _{2,

4} , Yingying Su _{1,

5} , Lele Sun _{2,

4} , Feifei Xing ₅ , Chunhai Fan ₂ , Di Li _{1,

2}

Affiliation

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.

中文翻译：

单个酶对DNA折纸反应器的变构抑制作用的单分子研究

在酶促反应过程中揭示酶的构象变化以及抑制动力学具有筛选治疗候选物的巨大潜力。然而，由于每个中间步骤的瞬时性，它仍然具有挑战性。我们用单分子荧光显微镜报告了我们的研究对辣根过氧化物酶的非竞争性抑制作用，其具有单周转率的分辨率。通过引入DNA折纸作为可寻址的纳米反应器，我们观察到了新生的荧光产物在催化位点和对接位点上的共存。我们进一步提出了一个单分子动力学模型来揭示产物生成和非竞争性抑制之间的相互作用，并发现三种不同的抑制剂释放途径。而且，动力学同位素效应实验表明，催化位点和对接位点之间具有很强的相关性，这表明非竞争性抑制作用中的构构构象变化。还观察到记忆效应。这项工作提供了对酶行为与酶构象波动，底物转化以及产物释放途径和动力学之间的相关性的深入理解。

更新日期：2018-11-09

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