Exploring mechanism of enzyme catalysis by on-chip transient kinetics coupled with global data analysis and molecular modeling
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Date
2021-04-08
Publication Type
Journal Article
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yes
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Abstract
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, β-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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published
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Journal / series
Volume
7 (4)
Pages / Article No.
1066 - 1079
Publisher
Elsevier
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Edition / version
Methods
Software
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Date collected
Date created
Subject
Enzyme kinetics; haloalkane dehalogenases; fluorescence imaging; droplet microfluidics; high throughput; global data analysis; thermodynamics; molecular dynamics
Organisational unit
03914 - deMello, Andrew / deMello, Andrew
Notes
Funding
857560 - CETOCOEN Excellence / Teaming Phase 2 (EC)