Channeling of a beta-oxidation intermediate on the large subunit of the fatty acid oxidation complex from Escherichia coli.

The kinetic properties of the fatty acid oxidation complex from Escherichia coli were studied with the aim of elucidating the functional consequence of having enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase associated with a multifunctional polypeptide. The kinetic parameters of individual enzymes were determined and used in model calculations based on a published theory (Storer, A. C., and Cornish-Bowden, A. (1974) Biochem. J. 141, 205-209) to predict the kinetic behavior of a system of functionally unlinked enzymes. The validity of the theory for making these calculations was proven by demonstrating a good agreement between the calculated and observed rates of intermediate and product formation for the conversion of 2-decenoyl-CoA to 3-ketodecanoyl-CoA catalyzed by a mixture of bovine liver enoyl-CoA hydratase and pig heart L-3-hydroxyacyl-CoA dehydrogenase. The conversion of 2-decenoyl-CoA to 3-ketodecanoyl-CoA catalyzed by the sequential action of the hydratase and dehydrogenase of the complex from E. coli was determined by measuring the rate of NADH formation. Stopped-flow measurements showed the rate of NADH formation to be linear without any lag period. When the initial velocity of the hydratase was 10.2 microM min-1, that of the overall reaction was 8.41 microM min-1. In contrast, the results calculated by use of the Storer and Cornish-Bowden equation for a system of unlinked enzymes predicted the overall reaction to exhibit a lag time of 30 s and to result in the accumulation of 2.1 microM 3-hydroxydecanoyl-CoA before reaching a velocity corresponding to 82.5% of that of the hydratase reaction. The high initial rate and the unusual kinetic properties of the overall reaction observed in the present study are best explained by a channeling mechanism on the large subunit of the E. coli fatty acid oxidation complex. When the apparent degree of channeling is corrected for the percentage of the dehydrogenase active sites saturated with NAD+, more than 90% of the intermediate appears to be transferred directly from the active site of enoyl-CoA hydratase to that of 3-hydroxyacyl-CoA dehydrogenase.