Enoyl Coenzyme A Hydratase (Crotonase)

Abstract

Abstract The substrate specificity of bovine liver crotonase has been examined with seven Δ2, 3-trans-enoyl-CoA substrates, containing an even number of carbon atoms. The Vmax for this series decreases progressively from a value of about 340,000 moles per min per mole of enzyme for crotonyl-CoA, the C4 derivative, to 2,300 for the C16 derivative. The action of several CoA derivatives on crotonase has been tested. None were found to stimulate the enzyme and only one derivative, acetoacetyl-CoA, was found to be markedly inhibitory. Evidence was obtained that the enolate form of acetoacetyl CoA was the inhibitory species and acted as a competitive inhibitor with a Ki of 1.6 x 10-6 m, a value about ten times lower than the Km for the best substrate, crotonyl-CoA. The interaction of acetoacetyl-CoA with crotonase was studied by ultraviolet difference spectroscopy and it was found that 6 molecules of inhibitor were bound per molecule of enzyme, or an average of one per subunit. This suggests that there are six active sites per molecule of native enzyme. The binding constant for the inhibitor was about equal to the kinetically determined value for Ki. The catalytic properties of crotonase have been compared with the turnover numbers and substrate specificities of the other enzymes acting in β oxidation of fatty acyl-CoA derivatives. This comparison suggests that crotonase, by virtue of its substrate specificity and its sensitivity to feedback inhibition by acetoacetyl-CoA, may play a regulatory role in fatty acid oxidation. The effects of acetoacetyl-CoA on the rate of oxidation of butyric, octanoic, and palmitic acids by heart muscle or liver mitochondria were those expected if crotonase is acting, at least in part, to regulate fatty acid oxidation.