D-3-hydroxyacyl coenzyme A dehydratase from rat liver peroxisomes. Purification and characterization of a novel enzyme necessary for the epimerization of 3-hydroxyacyl-CoA thioesters.
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.
Li
J X
JX
Department of Chemistry City College, City University of New York, New York 10031.
Smeland
T E
TE
Schulz
H
H
D000818:Animals; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D017886:Durapatite; D006836:Hydro-Lyases / Q000302:isolation & purification* / Q000378:metabolism; D006882:Hydroxyapatites; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008830:Microbodies / Q000201:enzymology* / Q000648:ultrastructure; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D006882:Hydroxyapatites; D017886:Durapatite; C065185:D-3-hydroxyacyl CoA dehydratase; D006836:Hydro-Lyases