Rates of the
NAD+-dependent oxidation of 2-trans,4-trans-decadienoyl-CoA, a metabolite of trans-omega-6-unsaturated
fatty acids, catalyzed by the mitochondrial
enoyl-CoA hydratase plus
3-hydroxyacyl-CoA dehydrogenase and by the corresponding
enzymes from peroxisomes, as well as Escherichia coli, were compared. The study of the mitochondrial system revealed that the conventional kinetic theory of coupled
enzyme reactions cannot be applied to systems in which the primary reaction has a small equilibrium constant, and/or the concentration of coupling
enzyme is higher than 0.01 Km for the intermediate and higher than the steady-state concentration of the intermediate. In contrast to the results obtained with the mitochondrial beta-oxidation system of unlinked
enzymes, the steady-state velocities of 2-trans,4-trans-decadienoyl-CoA degradation catalyzed by either the
peroxisomal bifunctional enzyme or by the E. coli
fatty acid oxidation complex were found to be equal to the activities of
enoyl-CoA hydratase even though the concentration of coupling
enzyme was equal to that of the primary
enzyme, and the quotient of Vmax/Km for the
dehydration of
3-hydroxy-4-trans-decenoyl-CoA is much larger than the Vmax/Km for its dehydrogenation. The extraordinarily high efficiencies of these two multifunctional
proteins in catalyzing the degradation of 2-trans,4-trans-decadienoyl-CoA is best explained by the direct transfer of the
3-hydroxy-4-trans-decenoyl-CoA intermediate from the active site of
enoyl-CoA hydratase to that of
3-hydroxyacyl-CoA dehydrogenase. The discovery of an intermediate channeling mechanism on the
peroxisomal bifunctional enzyme explains on the molecular level why the peroxisomal beta-oxidation system is well suited for the degradation of
trans-fatty acids.