Isothermal Titration Calorimetry Enables Rapid Characterization of Enzyme Kinetics and Inhibition for the Human Soluble Epoxide Hydrolase

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.


Recombinant hsEH C-Terminal Domain (CTD) preparation
Recombinant hsEH CTD was expressed in E. coli Ros2(DE3) cells and purified as previously described. 3 After the last purification step, the enzyme was dialysed overnight at 4°C in the reaction buffer, consisting of 50 mM HEPES pH 7.4, 300 mM NaCl, 5% glycerol, 10 μM tris(2-carboxyethyl)phosphine (TCEP). The protein was then concentrated to 1 mg mL -1 and stored at -80°C in small aliquots upon flash freezing in liquid nitrogen. Protein concentration was assessed using a theoretical extinction coefficient, obtained from ProtPARAM ExPASY. 4

Substrate and inhibitor sample preparation
EpFAs were purchased from Bertin Pharma, dissolved in ethanol. The ethanol was evaporated using a Savant SpeedVac (ThermoScientific) at 30°C for 3 hours.
Substrate stock solutions were prepared dissolving the lipids to a final concentration of 1 mg mL -1 in the identical reaction buffer used for protein dialysis, to avoid heat effects due to differences in buffer composition and/or pH (buffer mismatch effects).
Solutions were stored in small aliquots at -20°C for a maximum time of four weeks.

Substrate single-injection ITC kinetics theoretical background
The theoretical basis of kinetic rates determination by ITC has been previously described. [5][6][7][8] In brief, in the ITC experiment, the reaction rate (aka heat rate dQ/dt) is described by the heat variation (dQ) over time (dt). The total heat variation measured by the calorimeter is the sum of all the events occurring during the reaction under analysis, which include the catalysis, the interaction between substrate and enzyme (and putative cofactors), and the proton release or uptake from the buffer. The total heat measured in an ITC experiment is proportional to the enthalpy of all these molecular events, the apparent enthalpy (ΔH app ), and to the number of moles of product generated (n), which in turn is given by the total volume multiplied by the concentration of product (eq 1):

S4
The reaction rate v (d[P]/dt), can be related to the amount of heat generated over the same time (dQ/dt) through eq 2: In single-injection ITC measurements, saturating concentrations of substrate (greater than the Michaelis-Menten constant K M ) are injected in an enzyme solution. The injection generates negative or positive heat signals, and the heat rate is monitored in a continuous manner, until the signal returns to baseline (reference power), indicating that the substrate is completely depleted, and the enzymatic reaction is complete. The ΔH app is measured by the integration of the single peak: where [S] 0 is the total substrate concentration in the single injection experiments.
[S] i can be therefore extrapolated at any given time i from the integral of heat evolved (eq 3).
By determining the v i from eq 2 and the [S] i from eq 3 at any given time, plots of reaction rate versus substrate concentration, corresponding to Michaelis-Menten plots, can be obtained: where [E] tot is the total concentration of enzyme. Eq 4 provides affinity for the substrate (K M ), turnover rate (k cat ), and catalytic efficiency (k cat /K M = K sp ) values.

Isothermal titration calorimetry (ITC) set-up
ITC experiments were performed on MicroCal PEAQ-ITC and MicroCal iTC200 calorimeters (Malvern), both set up to high-feedback mode, reference power 5 μcal sec -1 , stirring speed of 750 rpm, and experimental temperature 25°C. A 600 seconds pre-injection delay was applied for baseline stabilisation after equilibration.

Substrate single-injection ITC kinetics experiments
Preliminary test experiments were carried out to determine the final measurement parameters, including temperature, substrate-to-enzyme ratio concentration, injection volume, reference power and injection speed. Particular attention was paid to S5 substrate-to-enzyme ratio concentration, by measuring the enzymatic rate reaction at increasing concentrations of each substrate, until no changes in k cat (and K sp ) were observed, indicating that steady state was reached (Fig. S7). The optimised experiments were performed as follows: hsEH CTD and substrate were diluted in identical reaction buffer to final concentrations of 250 nM and 0.5-1.5 mM respectively (Table S1)

Progressive inhibition ITC enzyme kinetics measurements
To evaluate the inhibitory constant (K i ) of hsEH antagonists and their mode of inhibition we employed a previously developed progressive inhibition method 9 and optimised it for our system, as follows. As a test inhibitor, we used the well-known

Inhibitory constant measurements with spectrofluorometric method
AUDA inhibitory potency was also tested with a spectrofluorometric method which monitors the hsEH-mediated hydrolysis of the synthetic substrate PHOME