A Nanopore Sensing Assay Resolves Cascade Reactions in a Multienzyme System

Enzymatic cascade reactions are widely used to synthesize complex molecules from simple precursors. The major underlying mechanism of cascade reactions is substrate channelling, where intermediates of different enzymatic steps are not in equilibrium with the bulk solution. Here, we report a nanopore...

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Bibliographic Details
Published in:Angewandte Chemie International Edition 2022-05, Vol.61 (20), p.e202200866-n/a
Main Authors: Sheng, Yingying, Zhou, Ke, Liu, Lei, Wu, Hai‐Chen
Format: Article
Language:English
Subjects:
Cascade chemical reactions
Cascade Reactions
Channeling
Chemical synthesis
Confined spaces
Deoxyribonucleic acid
DNA
Intermediates
Multienzyme Systems
Nanopore Sensing
Nanopores
Reaction intermediates
Substrate Channelling
Substrates
Systems analysis
Translocation
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Summary:Enzymatic cascade reactions are widely used to synthesize complex molecules from simple precursors. The major underlying mechanism of cascade reactions is substrate channelling, where intermediates of different enzymatic steps are not in equilibrium with the bulk solution. Here, we report a nanopore sensing assay that allows accurate quantification of all the reaction intermediates and the product of an artificial three‐enzyme system. A DNA–peptide complex is used as the initial substrate which undergoes sequential enzymatic cleavages in solution. All the temporal changes of the intermediates and product can be obtained through nanopore translocation recordings. Furthermore, we find that in a confined environment such as liposome, substrate channelling occurs between two sets of the three enzymes. Our results demonstrate a novel and powerful approach to determine and quantify substrate channelling effects, which is potentially useful for designing and evaluating multienzyme systems. A nanopore sensing assay is developed to resolve cascade reactions of an artificial three‐enzyme system. In a confined environment such as liposomes, substrate‐channelling effects are observed between two sets of the three enzymes.
ISSN:1433-7851
1521-3773
DOI:10.1002/anie.202200866