Analysis of Enoyl-Coenzyme A Hydratase Activity and Its Stereospecificity Using High-Performance Liquid Chromatography Equipped with Chiral Separation Column

Enoyl-coenzyme A (CoA) hydratase catalyzes the hydration of trans-2-enoyl-CoA to yield 3-hydroxyacyl-CoA during fatty acid degradation (β-oxidation). Although much research has focused on the stereospecificities of 2-enoyl-CoA hydratases, a direct quantification of the production of 3(R)- and 3(S)-hydroxyacyl-CoA has not yet been established. Therefore, we developed a method of concurrently quantifying 3(R)- and 3(S)-hydroxyacyl-CoA using high-performance liquid chromatography (HPLC) equipped with a chiral separation column. The optimized conditions for the separation of 3(R)-, 3(S)-hydroxyhexadecanoyl-CoA and trans-2-hexadecenoyl-CoA, were determined to be as follows: mobile phase of 35/65 (v/v) of 50 mM phosphate buffer (pH 5.0)/methanol; flow rate of 0.5 mL/min; detection at 260 nm; and column temperature of 25°C. This method was applied to subcellular fractions of rat liver; the results directly confirmed that 3(S)-hydroxyhexadecanoyl-CoA is the dominant product obtained from the heat-stable enoyl-CoA hydratase-catalyzed reaction of trans-2-hexadecenoyl-CoA. Finally, the stereospecificities of L-bifunctional protein (L-BP) and D-bifunctional protein (D-BP) were reinvestigated using this method, and it was confirmed that L- and D-BP yielded 3(S)- and 3(R)-hydroxyhexadecanoyl-CoA were yielded from trans-2-hexadecenoyl-CoA, respectively. 3(R)-Hydroxyacyl-CoA is a peroxisomal β-oxidation-specific intermediate. Therefore, this method is potentially useful not only studies regarding the stereochemistry of enoyl-CoA hydratase but also for the diagnosis of diseases caused by defects of peroxisomal enoyl-CoA hydratase.