Megarity Clare F, Weald Thomas R I, Heath Rachel S, Turner Nicholas J, Armstrong Fraser A
Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QR, U.K.
School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K.
ACS Catal. 2022 Aug 5;12(15):8811-8821. doi: 10.1021/acscatal.2c00999. Epub 2022 Jul 8.
The importance of energized nanoconfinement for facilitating the study and execution of enzyme cascades that feature multiple exchangeable cofactors is demonstrated by experiments with carboxylic acid reductase (CAR), an enzyme that requires both NADPH and ATP during a single catalytic cycle. Conversion of cinnamic acid to cinnamaldehyde by a package of four enzymes loaded into and trapped in the random nanopores of an indium tin oxide (ITO) electrode is driven and monitored through the simultaneous delivery of electrical and chemical energy. The electrical energy is transduced by ferredoxin NADP reductase, which undergoes rapid, direct electron exchange with ITO and regenerates NADP(H). The chemical energy provided by phosphoenolpyruvate, a fuel contained in the bulk solution, is cotransduced by adenylate kinase and pyruvate kinase, which efficiently convert the AMP product back into ATP that is required for the next cycle. The use of the two-kinase system allows the recycling process to be dissected to evaluate the separate roles of AMP removal and ATP supply during presteady-state and steady-state catalysis.
通过对羧酸还原酶（CAR）进行实验，证明了能量化纳米限域对于促进具有多个可交换辅因子的酶级联反应的研究和实施的重要性。CAR是一种在单个催化循环中需要NADPH和ATP的酶。通过将四种酶封装并捕获在氧化铟锡（ITO）电极的随机纳米孔中，肉桂酸转化为肉桂醛的过程通过同时传递电能和化学能来驱动和监测。电能由铁氧化还原蛋白NADP还原酶转换，该酶与ITO进行快速直接电子交换并再生NADP（H）。磷酸烯醇丙酮酸（一种本体溶液中含有的燃料）提供的化学能由腺苷酸激酶和丙酮酸激酶共同转换，它们有效地将AMP产物转化回下一个循环所需的ATP。双激酶系统的使用使得可以剖析循环过程，以评估在预稳态和稳态催化过程中AMP去除和ATP供应的各自作用。
