Abstract
Enzymatic activity and its tight regulation are fundamental to cellular metabolism and life. While classical models of enzyme kinetics explain the behavior of enzymes in dilute buffer solutions, there are elusive properties that emerge from enzymes in their native, crowded environments. In this study, we harness liquid-liquid phase separation (LLPS) to create controlled in vitro droplets that mimic cytosolic protein crowding, offering a unique system to understand enzyme kinetics in complex microenvironments. We uncover a mechanism in which enzyme-induced changes in shear viscosity arise from dynamic interactions among the substrate, product, and the protein crowder. Using fluorescence microscopy, bulk shear rheometry and microrheology, we show that enzymatic activity modifies the apparent viscosity of both protein-rich droplets and the surrounding PEG-rich phase, enhancing substrate mobility and improving substrate access to catalytic sites. Our findings suggest that this enzymatic-viscosity coupling affects substrate availability and influences the organization and dynamics of macromolecular crowding within droplets. These results provide new insights into how enzymes impact both their physical environment and metabolic processes in the cell.