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@article{1790417,
author = {Hess, David and Dočkalová, Veronika and Kokkonen, Piia Pauliina and Bednář, David and Damborský, Jiří and DeMello, Andrew and Prokop, Zbyněk and Stavrakis, Stavros},
article_location = {Cambridge},
article_number = {4},
doi = {http://dx.doi.org/10.1016/j.chempr.2021.02.011},
keywords = {HALOALKANE DEHALOGENASES; MICROFLUIDIC DEVICES; WATER; BIOCATALYSIS; DROPLETS; ENTROPY; BINDING},
language = {eng},
issn = {2451-9294},
journal = {Chem},
title = {Exploring mechanism of enzyme catalysis by on-chip transient kinetics coupled with global data analysis and molecular modeling},
url = {https://www.sciencedirect.com/science/article/abs/pii/S2451929421000917?via%3Dihub},
volume = {7},
year = {2021}
}
TY - JOUR
ID - 1790417
AU - Hess, David - Dočkalová, Veronika - Kokkonen, Piia Pauliina - Bednář, David - Damborský, Jiří - DeMello, Andrew - Prokop, Zbyněk - Stavrakis, Stavros
PY - 2021
TI - Exploring mechanism of enzyme catalysis by on-chip transient kinetics coupled with global data analysis and molecular modeling
JF - Chem
VL - 7
IS - 4
SP - 1066-1079
EP - 1066-1079
PB - Cell Press
SN - 24519294
KW - HALOALKANE DEHALOGENASES
KW - MICROFLUIDIC DEVICES
KW - WATER
KW - BIOCATALYSIS
KW - DROPLETS
KW - ENTROPY
KW - BINDING
UR - https://www.sciencedirect.com/science/article/abs/pii/S2451929421000917?via%3Dihub
N2 - 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.
ER -
HESS, David, Veronika DOČKALOVÁ, Piia Pauliina KOKKONEN, David BEDNÁŘ, Jiří DAMBORSKÝ, Andrew DEMELLO, Zbyněk PROKOP and Stavros STAVRAKIS. Exploring mechanism of enzyme catalysis by on-chip transient kinetics coupled with global data analysis and molecular modeling. \textit{Chem}. Cambridge: Cell Press, 2021, vol.~7, No~4, p.~1066-1079. ISSN~2451-9294. Available from: https://dx.doi.org/10.1016/j.chempr.2021.02.011.
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