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Single-Molecule Observation of Selenoenzyme Intermediates in a Semisynthetic Seleno-α-Hemolysin Nanoreactor
journal contributionposted on 2022-05-27, 18:33 authored by Xiaojia Jiang, Tiezheng Pan, Chao Lang, Chao Zeng, Jinxing Hou, Jiayun Xu, Quan Luo, Chunxi Hou, Junqiu Liu
The development of monitoring methods to capture short-lived intermediates is crucial for kinetic mechanism validation of enzymatic reaction steps. In this work, a semisynthetic selenoenzyme nanoreactor was constructed by introducing the unnatural amino acid (Sec) into the lumen of the α-hemolysin (αHL) nanopore. This nanoreactor not only created a highly confined space to trap the enzyme–substrate complex for a highly efficient antioxidant activity but also provided a single channel to characterize a series of selenoenzyme intermediates in the whole catalytic cycle through electrochemical analysis. In particular, the unstable intermediate of SeOH can be clearly detected by the characteristic blocking current. The duration time corresponding to the lifetime of each intermediate that stayed within the nanopore was also determined. This label-free approach showed a high detection sensitivity and temporal–spatial resolution to scrutinize a continuous enzymatic process, which would facilitate uncovering the mysteries of selenoenzyme catalysis at the single-molecule level.
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