Enzyme Activity Regulates Substrate Diffusion by Modulating Viscosity in Crowded Milieu

Abstract

Enzymatic activity and its tight regulation are fundamental to life, yet how enzymes maintain efficient substrate availability within the highly crowded, heterogeneous native cytosol remains poorly understood. Here, we harness liquid-liquid phase separation (LLPS) to generate controlled in vitro protein droplets that mimic the crowding of cytosolic proteins and demonstrate that enzymatic activity enhances substrate diffusion within the crowded environment by up to three-fold. Using fluorescence microscopy, fluorescence recovery after photobleaching (FRAP), molecular docking, and particle-tracking microrheology, we show that this enhanced diffusion arises from a reduction in apparent shear viscosity driven by enzymatic disruption of transient non-covalent interactions between substrate and protein crowder. Our findings reveal a physical feedback mechanism by which enzymatic activity actively reshapes its local environment to sustain substrate availability, providing new insight into how enzymatic activity creates a positive physical feedback on its own substrate supply under the diffusion constraints of the crowded cell.