Sheng Yingying, Zhou Ke, Liu Lei, Wu Hai-Chen
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
Angew Chem Int Ed Engl. 2022 May 9;61(20):e202200866. doi: 10.1002/anie.202200866. Epub 2022 Mar 16.
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.
酶促级联反应被广泛用于从简单前体合成复杂分子。级联反应的主要潜在机制是底物通道化，即不同酶促步骤的中间体与本体溶液不处于平衡状态。在此，我们报告了一种纳米孔传感测定法，该方法能够准确量化人工三酶系统的所有反应中间体和产物。一种DNA-肽复合物用作初始底物，它在溶液中经历顺序酶切。中间体和产物的所有时间变化都可以通过纳米孔转位记录获得。此外，我们发现，在脂质体等受限环境中，两组三酶之间会发生底物通道化。我们的结果展示了一种新颖且强大的方法来确定和量化底物通道化效应，这对于设计和评估多酶系统可能是有用的。
