Abis, G;
Pacheco-Gomez, R;
Bui, TT;
Conte, MR;
(2019)
Isothermal Titration Calorimetry Enables Rapid Characterization of Enzyme Kinetics and Inhibition for the Human Soluble Epoxide Hydrolase.
Analytical Chemistry
, 91
(23)
pp. 14865-14872.
10.1021/acs.analchem.9b01847.
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Abstract
Isothermal titration calorimetry (ITC) is conventionally used to acquire thermodynamic data for biological interactions. In recent years, ITC has emerged as a powerful tool to characterise enzyme kinetics. In this study, we have adapted a single-injection method (SIM) to study the kinetics of human soluble epoxide hydrolase (hsEH), an enzyme involved in cardiovascular homeostasis, hypertension, nociception and insulin sensitivity through the metabolism of epoxy-fatty acids (EpFAs). In the SIM method, the rate of reaction is determined by monitoring the thermal power while the substrate is being depleted, overcoming the need for synthetic substrates, and reducing postreaction processing. Our results show that ITC enables the detailed, rapid and reproducible characterisation of the hsEH-mediated hydrolysis of several natural EpFA substrates. Furthermore, we have applied a variant of the single-injection ITC method for the detailed description of enzyme inhibition, proving the power of this approach in the rapid screening and discovery of new hsEH inhibitors using the enzyme’s physiological substrates. The methods described herein will enable further studies on EpFAs metabolism and biology, as well as drug discovery investigations to identify and characterise hsEH inhibitors. This also promises to provide a general approach for the characterisation of lipid catalysis, given the challenges that lipid metabolism studies pose to traditional spectroscopic techniques.
| Type: | Article |
|---|---|
| Title: | Isothermal Titration Calorimetry Enables Rapid Characterization of Enzyme Kinetics and Inhibition for the Human Soluble Epoxide Hydrolase |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1021/acs.analchem.9b01847 |
| Publisher version: | https://doi.org/10.1021/acs.analchem.9b01847 |
| Language: | English |
| Additional information: | This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions. |
| Keywords: | Enzyme kinetics, Peptides and proteins, Hydrolysis, Inhibitors, Inhibition |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10085386 |
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