Enzyme Activity Regulates Substrate Diffusion by Modulating Viscosity in Crowded Milieu

Abstract Enzymatic activity and its tight regulation are fundamental for cellular metabolism and life. While classical models of enzyme kinetics explain the behaviour of enzymes in buffer solutions, there are elusive properties that emerge from enzymes in their native, crowded environment. In this study, we harness liquid-liquid phase separation (LLPS) to create in vitro droplets that mimic cytosolic protein crowding, offering a unique window to understand enzyme kinetics within well controlled microenvironments. We discover that the enzyme self-regulates its activity by modifying the shear viscosity of both the protein-rich droplets and the surrounding bulk solution. Through microscopy and rheology techniques, we discovered a significant correlation between enzyme activity and the shear viscosity of the droplets and bulk solution, influencing substrate diffusion. Our findings suggest that enzymes control their activity, influencing the organization and dynamics of macromolecular crowding within droplets. These results provide new insights into how enzymes regulate both their physical environment and metabolic processes in cell.