Exploring mechanism of enzyme catalysis by on-chip transient kinetics coupled with global data analysis and molecular modeling

Summary The ability to engineer enzymes for industrial and biomedical applications is primarily limited by a paucity of mechanistic understanding. To gain insight into the mechanisms enzyme catalysis, one must screen enormous numbers discrete reaction conditions, which laborious task using conventional technologies. address such limitations, we develop droplet-based microfluidic platform high-throughput acquisition transient kinetic data over range substrate concentrations temperatures. When compared with methods, our reduces assay volumes six orders magnitude increases throughput 9,000 reactions/min. demonstrate their utility, measure kinetics three model enzymes, namely, ?-galactosidase, horseradish peroxidase, microperoxidase. Additionally, conduct complex thermodynamic study engineered variants haloalkane dehalogenases. Datasets are globally analyzed complemented molecular dynamics simulations, providing new insights basis specificity role hydration-related entropy.