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 characterize 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 characterization 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 characterize hsEH inhibitors. This also promises to provide a general approach for the characterization of lipid catalysis, given the challenges that lipid metabolism studies pose to traditional spectroscopic techniques.
Highlights
The C-terminal domain (CTD) is responsible for the hydrolysis of numerous epoxyfatty acids (EpFAs), bioactive epoxidation products of monoand polyunsaturated fatty acids with essential roles in cellular and organism homeostasis.[2−4] human soluble epoxide hydrolase (hsEH) CTD hydrolyzes EpFAs via an SN2 nucleophilic attack by D335 on the more accessible carbon of the epoxide ring, forming an alkyl-enzyme intermediate, which is released by the assisted action of D496 and H524.1,2,5 The catalytic triad is located in the vertex of a large “L-shaped” active site and is surrounded by two hydrophobic surfaces dubbed the W334 niche and the F265 pocket, wherein the aliphatic chains of the EpFAs are accommodated.[1,2,4−6]
To probe the kinetics of EpFA hydrolysis catalyzed by hsEH CTD, we employed an isothermal titration calorimetry (ITC) single-injection method (SIM).[22,23]
We have presented a novel, versatile, expedient, and reliable ITC SIM application that has the potential to be adopted as the method of choice to perform such characterizations
Summary
(CTD) is responsible for the hydrolysis of numerous epoxyfatty acids (EpFAs), bioactive epoxidation products of monoand polyunsaturated fatty acids with essential roles in cellular and organism homeostasis.[2−4] hsEH CTD hydrolyzes EpFAs via an SN2 nucleophilic attack by D335 on the more accessible carbon of the epoxide ring, forming an alkyl-enzyme intermediate, which is released by the assisted action of D496 and H524.1,2,5 The catalytic triad is located in the vertex of a large “L-shaped” active site and is surrounded by two hydrophobic surfaces dubbed the W334 niche and the F265 pocket, wherein the aliphatic chains of the EpFAs are accommodated.[1,2,4−6]. The best characterized EpFAs substrates of hsEH CTD are the epoxyeicosatrienoic acids (EETs), epoxy derivatives of arachidonic acid (ARA;[7] Figure S1A). Four EET regioisomers, namely, 5(6)EET, 8(9)EET, 11(12)EET, and 14(15)EET, have been isolated in several organs,[8] the latter two have been shown to be the predominant ARA epoxidation metabolites.[9] EETs function primarily as endothelial-derived hyperpolarizing factors in the cardiovascular system and kidneys.[7] They play a role in vasorelaxation and vascular homeostasis, exerting anti-inflammatory and pro-angiogenic actions.[7] The bioavailability of EETs is reduced by hsEHmediated hydrolysis of their epoxy ring to generate the corresponding vicinal diols, namely, dihydroxyeicosatrienoic acids (DHETs; Figure S1A), which possess a considerably reduced biological activity.[7]. By measuring the intrinsic heat of hsEH-mediated hydrolysis of the epoxy-fatty acids in a continuous manner,[19−23] our method circumvents the limiting issue of the lack of physicochemical properties of EpFAs substrates/ products that can be monitored in real time in a continuous manner.[19−23] This new ITC application shows promise in the complete and highly reproducible characterization of hsEHmediated catalysis of epoxy-fatty acids, with relatively low sample amounts, low costs, and rapid acquisition times
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
