Substrate specificity of THCA-CoA oxidases from rat liver light mitochondrial fractions on dehydrogenation of 3α,7α,12α-trihydroxy-5β-cholestanoic acid CoA thioester

The substrate specificity of rat liver peroxisomal 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoyl-CoA (THCA-CoA) oxidases, which catalyze the dehydrogenation of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) CoA thioester, having an asymmetric center at C-25, to form (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-enoic acid (Delta(24)-THCA) CoA thioester, Ir as studied. The stable isotope labeled substrates, [3,7,12-O-18(3)]-(25R)- and (25S)-THCA CoA thioesters were synthesized by an exchange reaction of carbonyl oxygens on a steroid nucleus of 3,7,12-trioxo-5 beta-cholestanoic acid, followed by metal hydride reduction and condensation reaction with CoA. After incubation of a mixture of unlabeled (25R)- and O-18-labeled (25S)-THCA CoA thioester, or vice versa, with hepatic peroxisomal THCA-CoA oxidases, biotransformed Delta(24)-THCA was determined by liquid chromatography/ atmospheric pressure chemical ionization mass spectrometry. The Delta(24)-THCA was derived only from (25S)-THCA CoA thioester, indicating that the 25S epimer of THCA is a preferential substrate on dehydrogenation by THCA-CoA oxidases. (Steroids 63:603607, 1998) (C) 1998 by Elsevier Science Inc.