Macromolecular crowding effect on the β-galactosidase cascade reaction under multivalency and chain overlapping conditions of glycopolymers

Enzymes catalyze cascade reactions in a crowded cellular environment where as much as half of cell volume is occupied by various macromolecules. Nonspecific interaction between enzymes and crowders and excluded volume of the crowders can superimpose on each other and complicate catalytic activity of the enzymes. This study investigates the interaction between a β-galactosidase (β-Gal) and glycosylated acrylamide polymer (P(Glc-β-EAAm)) and unravels the macromolecular crowding effect of the glycopolymer on enzymatic cascade catalysis consisting of β-Gal, glucose oxidase and hydrogen peroxidase. Binding of the glycopolymer induced conformational alterations in β-Gal and gave rise to static fluorescence quenching and stable complex formation with β-Gal via nonspecific interaction. Fluorescence resonance energy transfer assays further demonstrated a compact association between β-Gal and glycopolymer crowders, with binding affinity correlating with the molecular weight of glycopolymer. The reduction of the apparent kinetic constants of the catalysis with the glycopolymer size showed the role of the multivalent effect in enzyme-substrate interactions. The kinetics also depended on the chain overlapping conditions of the crowders. Therefore, the macromolecular crowding effect on the cascade reaction is jointly associated with the crowder's multivalent effect, chain overlapping conditions of the crowders, in addition to the volume exclusion effect of the crowders.