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

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HESS David DOČKALOVÁ Veronika KOKKONEN Piia Pauliina BEDNÁŘ David DAMBORSKÝ Jiří DEMELLO Andrew PROKOP Zbyněk STAVRAKIS Stavros

Rok publikování 2021
Druh Článek v odborném periodiku
Časopis / Zdroj CHEM
Fakulta / Pracoviště MU

Přírodovědecká fakulta

Citace
www https://www.sciencedirect.com/science/article/abs/pii/S2451929421000917?via%3Dihub
Doi http://dx.doi.org/10.1016/j.chempr.2021.02.011
Klíčová slova HALOALKANE DEHALOGENASES; MICROFLUIDIC DEVICES; WATER; BIOCATALYSIS; DROPLETS; ENTROPY; BINDING
Popis 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.
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