Isothermal Titration Calorimetry Enables Rapid Characterization of Enzyme Kinetics and Inhibition for the Human Soluble Epoxide Hydrolase
Isothermal Titration Calorimetry Enables Rapid Characterization of Enzyme Kinetics and Inhibition for the Human Soluble Epoxide Hydrolase
复制标题
DOI：
10.1021/acs.analchem.9b01847
复制
发表时间：
2019-12-03
影响因子：
7.4
通讯作者：
Conte, Maria R.
中科院分区：
文献类型：
--
作者：
Abis, Giancarlo;Pacheco-Gomez, Raul;Conte, Maria R.
等温滴定量热法（ITC）通常用于获取生物相互作用的热力学数据。近年来，ITC已成为表征酶动力学的有力工具。在这项研究中，我们已经适应了单次注射法（SIM）的动力学研究的人可溶性环氧化物水解酶（hsEH），参与心血管稳态，高血压，伤害感受和胰岛素敏感性通过代谢的环氧脂肪酸（EpFA）的酶。在SIM方法中，通过监测热功率来确定反应速率，同时底物被耗尽，克服了对合成底物的需要并减少了反应后处理。我们的研究结果表明，ITC使详细的，快速的，和可重复的几个天然EpFA底物的hsEH介导的水解表征。此外，我们已经应用了一个变种的单次注射ITC方法的酶抑制的详细描述，证明了这种方法的力量，在快速筛选和发现新的hsEH抑制剂使用酶的生理底物。本文所述的方法将使得能够进一步研究EpFA的代谢和生物学，以及药物发现研究以鉴定和表征hsEH抑制剂。这也有望提供一个通用的方法来表征脂质催化，脂质代谢研究带来的挑战，传统的光谱技术。
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.