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Download fileSingle-Molecule Observation of Selenoenzyme Intermediates in a Semisynthetic Seleno-α-Hemolysin Nanoreactor
journal contribution
posted 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 LiuThe
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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would facilitate uncoveringwhole catalytic cycleunnatural amino acidkinetic mechanism validationhighly confined spacehigh detection sensitivityfree approach showedenzymatic reaction stepsduration time correspondingcontinuous enzymatic processcharacteristic blocking currentsemisynthetic selenoenzyme nanoreactorsemisynthetic selenoselenoenzyme intermediatesselenoenzyme catalysisstayed withinmonitoring methodsmolecule observationmolecule levellived intermediateselectrochemical analysisclearly detectedcapture shortalso providedalso determined