Biomolecular Condensates Regulate Enzymatic Activity under a Crowded Milieu: Synchronization of Liquid–Liquid Phase Separation and Enzymatic Transformation

Cellular crowding plays a key role in regulating the enzymatic reactivity in physiological conditions, which is challenging to realize in the dilute phase. Enzymes drive a wide range of complex metabolic reactions with high efficiency and selectivity under extremely heterogeneous and crowded cellular environments. However, the molecular interpretation behind the enhanced enzymatic reactivity under a crowded milieu is poorly understood. Herein, using the horseradish peroxidase (HRP) and glucose oxidase (GOx) cascade pair, we demonstrate for the first time that macromolecular crowding induces liquid-liquid phase separation (LLPS) via the formation of liquid-like condensates/droplets and thereby increases the intrinsic catalytic efficiencies of HRP and GOx. Both these enzymes undergo crowding induced homotypic LLPS via enthalpically driven multivalent electrostatic as well as hydrophobic interactions. Using a set of kinetic and microscopic experiments, we show that precise synchronization of spontaneous LLPS and enzymatic transformations is key to realize the enhanced enzymatic activity under the crowded environments. Our findings reveal an unprecedented enhancement (91- to 205-fold) in the catalytic efficiency (kcat/Km) of HRP at pH 4.0 within the droplet phase relative to that in the bulk aqueous phase in the presence of different crowders. In addition, we have shown that other enzymes also undergo spontaneous LLPS under macromolecular crowding, signifying the generality of this phenomenon under the crowded environments. More importantly, coalescence driven highly regulated GOx/HRP cascade reactions within the fused droplets have been demonstrated with enhanced activity and specificity under the crowded environments. The present discovery highlights the active role of membraneless condensates in regulating the enzymatic efficacy for complex metabolic reactions under the crowded cellular environments and may find significant importance in the field of biocatalysis.