Nonspecific interaction and overlap concentration influence macromolecular crowding effect on glucose oxidase activity.

Macromolecular crowding can change kinetics of enzyme catalysis. How interaction between enzymes and neighboring macromolecules contributes to the crowding effect on enzyme catalysis has not been quantitatively revealed. In this study, crowding effects of dextran and poly(ethylene glycol) (PEG) on glucose oxidase (GOx) are studied. Fluorescence resonance energy transfer experiments show the high transfer efficiency and stable interaction between the dextran and GOx. Further fluorescence quenching analysis also proves that the association of the dextran-GOx pair can become stronger than that of the PEG-GOx pair. Dextrans with concentrations above or below their chain overlap concentrations (c*) reduce Michaelis constants (Km) of GOx catalysis by 90 % or 45 %, respectively, through volume exclusion mechanism, and in the meantime elevate the enzymatic efficiency (kcat/Km) by 8-fold or by 3-fold, respectively, which is more dramatic than that found in other enzymes before. Strong association between the enzyme and the dextran results in slow turnover rates (kcat). Intermediate crowding with weak to moderate affinity to the enzyme below the c* can tune the kcat higher than in the free state. Catalysis under crowded conditions is a joint effect of the enzyme-crowder nonspecific interaction, volume exclusion and overlap condition of the crowders.