Conformational study of purified epoxide hydrolase from rat liver

Hepatic epoxide hydrolase (EC 3.3.2.3) was purified from phenobarbital-treated rats by ion-exchange chromatography followed by hydrophobic chromatography. The enzyme had a specific activity of 300--400 nmol min-1 mg-1 protein with benzo[a]pyrene-4,5-oxide as the substrate. Circular dichroism (CD) spectra of the purified enzyme gave two negative bands, centered at 210 nm and 222 nm, respectively. The mean residue ellipticity at 222 nm was 12,9000 deg X cm(2) X dmol(-1), which indicated the presence of about 35% alpha-helical structures. Sodium dodecyl sulfate (SDS) greatly affected the shape of the CD spectra, which were gradually shifted to the blue. This suggested a decrease in the aggregation state of the protein. Electrostatic interactions were important in the organization of the enzyme structure since the conformation was stable between pH 7.4 and pH 10. At pH-values 5.0, 6.0 and 12.0, the CD bands underwent considerable changes in both amplitude and shape. Moreover there was a good correlation between the optimal pH range of the epoxide hydrolase activity and the organization state of the protein. After membrane reconstitution with liposomes, the conformation of the enzyme was not significantly modified by the presence of dimyristoyl L-alpha-phosphatidylcholine or other phospholipids. This constancy was obtained over a wide range of molar ratios of phospholipids to protein (0--500). However, phospholipids did increase the thermal stability of the enzyme. Fluorescence measurements of diphenylhexatriene (DPH) bound to dimyristoyl L-alpha-phosphatidylcholine indicated that addition of epoxide hydrolase modified the thermal transition of the lipid phase. On the other hand, electron paramagnetic resonance (EPR) signals of the nitroxide-labelled fatty acid, 2-(14-carboxy-tetradecyl)-2-ethyl-4,4-dimethyl-3,3,-oxazolidiny-oxyl, bound to the phospholipid, indicated that the presence of the protein decreased by about 53% the correlation time of the label, suggesting that its motion had increased. In conclusion, phospholipid-epoxide hydrolase interactions enhanced the fluidity of dimyristoyl L-alpha-phosphatidylcholine liposomes without changing the secondary structure of the enzyme. Electrostatic interactions also played an important role in the conformational stability of the protein.