Encapsulation of an Enzyme Cascade within the Bacteriophage P22 Virus-Like Particle

Developing methods for investigating coupled enzyme systems under conditions that mimic the cellular environment remains a significant challenge. Here we describe a biomimetic approach for constructing densely packed and confined multienzyme systems through the co-encapsulation of 2 and 3 enzymes within a virus-like particle (VLP) that perform a coupled cascade of reactions, creating a synthetic metabolon. Enzymes are efficiently encapsulated <i>in vivo</i> with known stoichiometries, and the kinetic parameters of the individual and coupled activities are characterized. From the results we develop and validate a mathematical model for predicting the expected kinetics for coupled reactions under co-localized conditions