Enzymes converting D-3-hydroxyacyl-CoA to trans-2-enoyl-CoA: microsomal and peroxisomal isoenzymes in rat liver

Epimerization of 3-hydroxyacyl-CoA, which has been shown to occur as a two-step dehydration-hydration reaction (Hiltunen, J.K., Palosaari, P.M., and Kunau, W.-H. (1989) J. Biol. Chem. 264, 13536-13540; Smeland, E., Jianxun, L., Chu, C., Cuebas, D., and Schulz, H. (1989) Biochem. Biophys. Res. Commun. 160, 988-992) was studied in rat liver. Subcellular fractionations of rat liver on different density gradients revealed a dual distribution of activity, catalyzing dehydration of D-3-hydroxydecanoyl-CoA to trans-2-decenoyl-CoA (hydratase 2) in both peroxisomal and microsomal compartments. Both hydratase 2 activity peaks were separated by dye ligand chromatography from the extract of the combined heavy and light mitochondrial fractions. The activity eluted at low salt was identified as the microsomal isoform and was purified to apparent homogeneity. The Mr of the native protein (subunit) was found to be 60,000 (31,500), indicating that it is homodimeric. The enzyme activity was inhibited by IgGs isolated from antisera raised against the denatured subunit. The activity eluted at high salt was tentatively identified to be peroxisomal of origin, and the Mr of the native protein (subunit) was determined to be 62,000 (33,500). The peroxisomal enzyme was not recognized by the antibody to its microsomal counterpart. Analysis of the reaction products of microsomal enzyme activity by gas chromatography-mass spectrometry showed that the enzyme catalyzed reversibly hydration/dehydration between trans-2-enoyl-CoA and D-3-hydroxyacyl-CoA, but L-3-hydroxydecanoyl-CoA was not dehydrated to delta 2-enoyl-CoA compounds. Similar reaction characteristics were also determined for the peroxisomal hydratase by using stereospecific auxiliary enzymes. The present data demonstrate that rat liver contains microsomal and peroxisomal proteins possessing hydratase 2 activities. Although their kinetic properties are similar, immunological data, subunit sizes, and chromatographic evidence clearly indicate that they are different enzymes. Comparisons with other hydratases revealed that the microsomal and peroxisomal hydratase 2 described in the present work are proteins that have not been previously purified.