Rat liver peroxisomal D-3-hydroxyacyl-CoA
dehydratase, which in combination with
enoyl-CoA hydratase catalyzes the epimerization of 3-hydroxyacyl-CoA, was purified by a five-step procedure to yield a highly purified preparation as judged by gel electrophoresis of the native and denatured
enzyme. Since the molecular mass of the native
dehydratase was estimated to be twice that of its 44-kDa subunit, the
enzyme seems to be composed of two, possibly identical subunits. This
dehydratase catalyzes the reversible
dehydration of D-3-hydroxyacyl-CoA to 2-trans-enoyl-CoA, but, in contrast to
enoyl-CoA hydratase, does not act on 2-cis-enoyl-CoA. The
dehydratase is virtually inactive toward
crotonyl-CoA, but exhibits high activity with 2-trans-hexenoyl-CoA as a substrate and acts with decreasing efficiency on all 2-enoyl-CoAs tested from 2-hexenoyl-CoA to
2-hexadecenoyl-CoA. The pH optimum of the
enzyme is close to 8. Equilibrium ratios of 3-hydroxyoctanoyl-
CoA/2-trans-
octenoyl-CoA and 3-hydroxyoctanoyl-
CoA/2-cis-
octenoyl-CoA were found to be close to 3 and 137, respectively. It is suggested that 2-cis-enoyl-CoA intermediates formed during the beta-oxidation of
polyunsaturated fatty acids in peroxisomes are hydrated by
enoyl-CoA hydratase to D-3-hydroxyacyl-CoAs which are epimerized to their L-isomers by the sequential actions of D-3-hydroxyacyl-CoA
dehydratase and
enoyl-CoA hydratase.