Beta-oxidation of 5-hydroxydecanoate, a putative blocker of mitochondrial ATP-sensitive potassium channels

Abstract

5-Hydroxydecanoate (5-HD) inhibits ischaemic and pharmacological preconditioning of the heart. Since 5-HD is thought to inhibit specifically the putative mitochondrial ATP-sensitive K+ (KATP) channel, this channel has been inferred to be a mediator of preconditioning. However, it has recently been shown that 5-HD is a substrate for acyl-CoA synthetase, the mitochondrial enzyme which 'activates' fatty acids. Here, we tested whether activated 5-HD, 5-hydroxydecanoyl-CoA (5-HD-CoA), is a substrate for medium-chain acyl-CoA dehydrogenase (MCAD), the committed step of the mitochondrial beta-oxidation pathway. Using a molecular model, we predicted that the hydroxyl group on the acyl tail of 5-HD-CoA would not sterically hinder the active site of MCAD. Indeed, we found that 5-HD-CoA was a substrate for purified human liver MCAD with a Km of 12.8 +/- 0.6 microM and a kcat of 14.1 s-1. For comparison, with decanoyl-CoA (Km approximately 3 microM) as substrate, kcat was 6.4 s-1. 5-HD-CoA was also a substrate for purified pig kidney MCAD. We next tested whether the reaction product, 5-hydroxydecenoyl-CoA (5-HD-enoyl-CoA), was a substrate for enoyl-CoA hydratase, the second enzyme of the beta-oxidation pathway. Similar to decenoyl-CoA, purified 5-HD-enoyl-CoA was also a substrate for the hydratase reaction. In conclusion, we have shown that 5-HD is metabolised at least as far as the third enzyme of the beta-oxidation pathway. Our results open the possibility that beta-oxidation of 5-HD or metabolic intermediates of 5-HD may be responsible for the inhibitory effects of 5-HD on preconditioning of the heart.

Figure 1. Molecular model of MCAD-(S)-5-hydroxydecanoyl-CoA complex

A, structure of pig liver MCAD monomer in complex with modelled ligand (S)-5-HD-CoA. Amino acid residues within 4 Å of the ligand are shown (solid yellow structures). B, enlarged view of the MCAD-(S)-5-HD-CoA complex showing relation of the (S)-5-hydroxyl group to labelled residues in the fatty acyl binding pocket. Glu-376 is the active site base which abstracts the α-proton from the acyl-CoA substrate whereas the β-hydrogen is transferred to FAD (not depicted).

Figure 2. 5-Hydroxydecanoyl-CoA is a substrate for human liver and pig kidney MCAD

A and B, purified human liver MCAD (50 nm) was added (indicated by interruption in trace) to solution containing electron acceptor (FcPF₆) and saturating concentrations (43 μm) of either 5-HD-CoA (A) or decanoyl-CoA (B). A decrease in absorbance at 300 nm indicates enzyme activity. C and D, purified pig kidney MCAD (50 nm) was added to solution containing either 5-HD-CoA (C) or decanoyl-CoA (D), each at a concentration of 43 μm.

Figure 3. Kinetics of MCAD-catalysed dehydrogenation of 5-HD-CoA and metabolism of the product 5-hydroxydecenoyl-CoA

A, steady-state activity of human liver MCAD was determined at various concentrations of purified 5-HD-CoA. K_m was 12.8 ± 0.6 μm and V_max 23.6 μm min⁻¹. Data were fitted using the Michaelis-Menten equation (see Results). B, bovine liver enoyl-CoA hydratase was activated by addition of either decenoyl-CoA (dashed line) or purified 5-hydroxydecenoyl-CoA (continuous line). Enzyme activity was monitored by measuring absorbance at 263 nm.

Figure 4. Schematic diagrams of the metabolism of 5-HD and its uptake into the mitochondrial matrix

A, schematic summary showing activation and β-oxidation of 5-hydroxydecanoate. Medium-chain acyl-CoA dehydrogenase transfers electrons to the electron transport chain via electron transfer flavoprotein (ETF) and ETF oxidoreductase (ETF-QO). B, schematic diagram showing that, typical for medium-chain fatty acids, 5-HD can be activated via ACS (acyl-CoA synthetase) either on the mitochondrial outer membrane or in the matrix. Whether extramitochondrial 5-HD-CoA serves as a substrate for CPT-I (carnitine palmitoyltransferase-I) is not known.