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

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

Automated summary

The development of a droplet-based microfluidic platform has enabled high-throughput acquisition of transient kinetic data for enzymes under various substrate concentrations and temperatures. This platform significantly reduces assay volumes and increases throughput when compared to conventional methods. Through studying model enzymes and engineered variants, new insights into enzyme design and applications have been gained, complemented by molecular dynamics simulations.