Carnitine palmitoyltransferase 2: New insights on the substrate specificity and implications for acylcarnitine profiling.
Over the last years acylcarnitines have emerged as important biomarkers for the diagnosis of mitochondrial fatty acid beta-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 (K(i) of 18.8 microM). 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.
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.
Sara
S
Violante
Lodewijk
L
Ijlst
Henk
H
van Lenthe
Isabel Tavares
IT
de Almeida
Ronald J
RJ
Wanders
Fátima V
FV
Ventura
D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D002331:Carnitine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism / Q000493:pharmacokinetics; D002334:Carnitine O-Palmitoyltransferase / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D002384:Catalysis; D006801:Humans; D007700:Kinetics; D055442:Metabolome / Q000502:physiology; D030781:Organisms, Genetically Modified; D009994:Osmolar Concentration; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; C116917:acylcarnitine; D002334:Carnitine O-Palmitoyltransferase; D002331:Carnitine