Single-Molecule Studies of Allosteric Inhibition of Individual Enzyme on a DNA Origami Reactor

Unraveling the conformational changes of enzymes together with inhibition kinetics during an enzymatic reaction has great potential in screening therapeutic candidates; however, it remains challenging due to the transient nature of each intermediate step. We report our study on the noncompetitive inhibition of horseradish peroxidase with single-turnover resolution using single-molecule fluorescence microscopy. By introducing DNA origami as an addressable nanoreactor, we observe the coexistence of nascent-formed fluorescent product on both catalytic and docking sites. We further propose a single-molecule kinetic model to reveal the interplay between product generation and noncompetitive inhibition and find three distinct inhibitor releasing pathways. Moreover, the kinetic isotope effect experiment indicates a strong correlation between catalytic and docking sites, suggesting an allosteric conformational change in noncompetitive inhibition. A memory effect is also observed. This work provides an in-depth understanding of the correlation between enzyme behavior and enzymatic conformational fluctuation, substrate conversion, and product releasing pathway and kinetics.