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1 Institut für Normale and Pathologische Physiologie, Universität Marburg, Deutschhausstr. 2, 35037 Marburg, Germany
2 Universitätsklinikum Frankfurt, Institut für Biochemie I, 60590 Frankfurt am Main, Germany
3 Department of Biochemistry and Molecular Biology, North Dakota State University, Fargo, ND 58105, USA
4 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
5 Department of Biochemistry, University of Nebraska, Lincoln, NE 68588-0664, USA
6 Laboratory of Pharmacology, Katholieke Universiteit Leuven, Herestaat 49, B-3000, Leuven, Belgium
5-Hydroxydecanoate (5-HD) blocks pharmacological and ischaemic preconditioning, and has been postulated to be a specific inhibitor of mitochondrial ATP-sensitive K+ (KATP) channels. However, recent work has shown that 5-HD is activated to 5-hydroxydecanoyl-CoA (5-HD-CoA), which is a substrate for the first step of ß-oxidation. We have now analysed the complete ß-oxidation of 5-HD-CoA using specially synthesised (and purified) substrates and enzymes, as well as isolated rat liver and heart mitochondria, and compared it with the metabolism of the physiological substrate decanoyl-CoA. At the second step of ß-oxidation, catalysed by enoyl-CoA hydratase, enzyme kinetics were similar using either decenoyl-CoA or 5-hydroxydecenoyl-CoA as substrate. The last two steps were investigated using l-3-hydroxyacyl-CoA dehydrogenase (HAD) coupled to 3-ketoacyl-CoA thiolase. Vmax for the metabolite of 5-HD (3,5-dihydroxydecanoyl-CoA) was fivefold slower than for the corresponding metabolite of decanoate (L-3-hydroxydecanoyl-CoA). The slower kinetics were not due to accumulation of D-3-hydroxyoctanoyl-CoA since this enantiomer did not inhibit HAD. Molecular modelling of HAD complexed with 3,5-dihydroxydecanoyl-CoA suggested that the 5-hydroxyl group could decrease HAD turnover rate by interacting with critical side chains. Consistent with the kinetic data, 5-hydroxydecanoyl-CoA alone acted as a weak substrate in isolated mitochondria, whereas addition of 100 µM 5-HD-CoA inhibited the metabolism of decanoyl-CoA or lauryl-carnitine. In conclusion, 5-HD is activated, transported into mitochondria and metabolised via ß-oxidation, albeit with rate-limiting kinetics at the penultimate step. This creates a bottleneck for ß-oxidation of fatty acids. The complex metabolic effects of 5-HD invalidate the use of 5-HD as a blocker of mitochondrial KATP channels in studies of preconditioning.
(Received 15 August 2004;
accepted after revision 25 October 2004;
first published online 25 October 2004)
Corresponding author P. J. Hanley: Institut für Normale und Pathologische Physiologie, Universität Marburg, Deutschhausstrasse 2, 35037 Marburg, Germany. Email: hanley{at}mailer.uni-marburg.de
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