Abstract
The present study was designed to determine the action of the 2-acetylenic acid thioester on mitochondrial fatty acid chain elongation and β-oxidation. Addition of 2-decynoyl CoA to a rat liver mitochondrial suspension resulted in a significant stimulation of the rate of oxidation of NADPH and NADH. This enhanced oxidation rate was not due to the mitochondrial
trans-2-enoyl CoA reductase-catalyzed conversion of the 2-acetylenic acid thioester to the saturated product, decanoate, as measured by gas-liquid chromatography. On the contrary, the mitochondrial
trans-2-enoyl CoA reductase activity was markedly inhibited by the 2-acetylenic acid derivative, as evidenced by the decrease in the reduction of
trans-2-decenoyl CoA to decanoic acid. Incubation of the mitochondrial fraction with either NADPH or NADH and 2-decynol CoA resulted in the gas Chromatographic identification of three products: β-ketodecanoate, β-hydroxydecanoate, and
trans-2-decenoate. In the absence of reduced pyridine nucleotide, a single product was formed and identified as β-ketodecanoate. Confirmation of the identity of this product was obtained by the observation of the formation of the Mg
2+-enolate complex (303-nm absorbance peak). These results suggest that, although the 2-decynoyl CoA is an inhibitor of mitochondrial
trans-2-enoyl CoA reductase activity, it is a substrate for the mitochondrial
trans-2-enoyl CoA hydratase (crotonase). This was confirmed by incubation of 2-decynoyl CoA with commercially purified liver mitochondrial crotonase. The β-ketodecanoate is formed in a two-step process: hydration of the 2-decynoyl CoA to an unstable enol intermediate which undergoes rearrangement to the β-ketodecanoyl CoA. Interestingly, although the mitochondrial crotonase can utilize the 2-acetylenic acid thioesters, this was not the case for the peroxisomal bifunctional hydratase which was markedly inhibited by varing concentrations of 2-decynoyl CoA.