Carnitine palmitoyltransferase 2: New insights on the substrate specificity and implications for acylcarnitine profiling |
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Authors: | Sara Violante Lodewijk IJlst Henk van Lenthe Isabel Tavares de Almeida Ronald J Wanders Fátima V Ventura |
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Institution: | 1. Metabolism and Genetics Group, Research Institute for Medicines and Pharmaceutical Sciences, iMed.UL, Faculdade de Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal;2. Laboratory Genetic Metabolic Diseases, Department Clinical Chemistry and Pediatrics, University of Amsterdam, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands |
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Abstract: | Over the last years acylcarnitines have emerged as important biomarkers for the diagnosis of mitochondrial fatty acid β-oxidation (mFAO) and branched-chain amino acid oxidation disorders assuming they reflect the potentially toxic acyl-CoA species, accumulating intramitochondrially upstream of the enzyme block. However, the origin of these intermediates still remains poorly understood. A possibility exists that carnitine palmitoyltransferase 2 (CPT2), member of the carnitine shuttle, is involved in the intramitochondrial synthesis of acylcarnitines from accumulated acyl-CoA metabolites. To address this issue, the substrate specificity profile of CPT2 was herein investigated. Saccharomyces cerevisiae homogenates expressing human CPT2 were incubated with saturated and unsaturated C2–C26 acyl-CoAs and branched-chain amino acid oxidation intermediates. The produced acylcarnitines were quantified by ESI-MS/MS. We show that CPT2 is active with medium (C8–C12) and long-chain (C14–C18) acyl-CoA esters, whereas virtually no activity was found with short- and very long-chain acyl-CoAs or with branched-chain amino acid oxidation intermediates. Trans-2-enoyl-CoA intermediates were also found to be poor substrates for CPT2. Inhibition studies performed revealed that trans-2-C16:1-CoA may act as a competitive inhibitor of CPT2 (Ki of 18.8 μM). The results obtained clearly demonstrate that CPT2 is able to reverse its physiological mechanism for medium and long-chain acyl-CoAs contributing to the abnormal acylcarnitines profiles characteristic of most mFAO disorders. The finding that trans-2-enoyl-CoAs are poorly handled by CPT2 may explain the absence of trans-2-enoyl-carnitines in the profiles of mitochondrial trifunctional protein deficient patients, the only defect where they accumulate, and the discrepancy between the clinical features of this and other long-chain mFAO disorders such as very long-chain acyl-CoA dehydrogenase deficiency. |
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