Imaging monomeric enzyme trajectories with and without substrate

Enhanced diffusion in catalytic enzymes is key to understanding how molecular machines derive work from non-equilibrium chemical activity. Although it has been experimentally observed in various oligomeric enzymes, dissociation under non-biologically relevant conditions introduces uncertainty. In contrast, monomeric enzymes, which are free from subunit interactions, provide a simpler model for studying enhanced diffusion. Here, we captured the freely diffusing trajectories of monomeric apyrases, which catalyze the cascade hydrolysis of ATP to ADP and then to AMP, in a usual buffer, and found its enhanced diffusion correlated with the reaction rate. Compared to oligomeric enzymes, apyrase exhibited a more significant degree of enhancement, likely due to its asymmetric clamp-like structure and butterfly-like motion, indicating a more efficient extraction of work from catalytic activity.