Long Chain Enoyl Coenzyme A Hydratase from Pig Heart

Abstract

Abstract A long chain enoyl-CoA hydratase, which in addition to crotonase is present in pig heart muscle, has been isolated and partially purified. This enzyme appears to be located in the mitochondria and is tighter membrane-bound than crotonase. It catalyzes the hydration of medium and long chain trans-Δ2,3-enoyl-CoA substrates to their corresponding l-3-hydroxy derivatives but it is nearly inactive toward crotonyl-CoA. The highest Vmax was observed with Δ2,3-octenoyl-CoA, while longer chain substrates gave progressively decreasing values. However, the same Km value of 24 µm was obtained for all even numbered Δ2,3-enoic acid derivatives containing 8 to 14 carbons. The pH optimum of this enzyme was found to be 8.5. Inhibition studies showed that this hydratase, in contrast to bovine liver crotonase, is not significantly inhibited by acetoacetyl-CoA but is strongly inhibited by long chain enoylCoA substrates. This inhibition can be at least partially prevented by the presence of bovine serum albumin. The enzyme is also inhibited by sulfhydryl inhibitors, as, for example, p-chloromercuribenzoate or N-methylmaleimide, which at concentrations of 1 mm inhibited the enzyme to the extent of 100% and 69%, respectively.