Harnessing Water Competition to Drive Enzyme Crosstalk

In nature, enzymatic pathways often involve compartmentalization effects that can modify the intrinsic activity and specificity of the different enzymes involved. Consequently, extensive research has focused on replicating and studying the compartmentalization effects on individual enzymes and on multistep enzyme “cascade” reactions. This study explores the influence of compartmentalization achieved using molecular crowding on the glucose oxidase/horseradish peroxidase (GOx/HRP) cascade reaction. The crowder tested is methoxy poly(ethylene glycol) (mPEG) that can, depending on conditions, promote GOx and HRP coassociation at the nanoscale and extend their contact time. Low-molecular-weight mPEG (0.35 kDa), but not mPEG of higher molecular weights (5 or 20 kDa), significantly enhanced the cascade reaction where up to a 20-fold increase in the rate of the cascade reaction was observed under some conditions. The combined analyses emphasize the particularity of low-molecular-weight mPEG and point toward mPEG-induced coassociation of HRP and GOx, producing nearest crowded neighbor effects of HRP on GOx, and vice versa. These altered the nanoscale environments of these enzymes, which influenced substrate affinity. Using mPEG to promote protein coassociation is simple and does not chemically modify the proteins studied. This approach could be of interest for more broadly characterizing nearest crowded neighbor effects (i.e., protein–protein interactions) for multiprotein systems (i.e., more than just two), thus making it an interesting tool for studying very complex systems, such as those found in nature.