title: Substrate specificity of human carnitine acetyltransferase: Implications for fatty acid and branched-chain amino acid metabolism.
abstract: Carnitine acyltransferases catalyze the reversible conversion of acyl-CoAs into acylcarnitine esters. This family includes the mitochondrial enzymes carnitine palmitoyltransferase 2 (CPT2) and carnitine acetyltransferase (CrAT). CPT2 is part of the carnitine shuttle that is necessary to import fatty acids into mitochondria and catalyzes the conversion of acylcarnitines into acyl-CoAs. In addition, when mitochondrial fatty acid β-oxidation is impaired, CPT2 is able to catalyze the reverse reaction and converts accumulating long- and medium-chain acyl-CoAs into acylcarnitines for export from the matrix to the cytosol. However, CPT2 is inactive with short-chain acyl-CoAs and intermediates of the branched-chain amino acid oxidation pathway (BCAAO). In order to explore the origin of short-chain and branched-chain acylcarnitines that may accumulate in various organic acidemias, we performed substrate specificity studies using purified recombinant human CrAT. Various saturated, unsaturated and branched-chain acyl-CoA esters were tested and the synthesized acylcarnitines were quantified by ESI-MS/MS. We show that CrAT converts short- and medium-chain acyl-CoAs (C2 to C10-CoA), whereas no activity was observed with long-chain species. Trans-2-enoyl-CoA intermediates were found to be poor substrates for this enzyme. Furthermore, CrAT turned out to be active towards some but not all the BCAAO intermediates tested and no activity was found with dicarboxylic acyl-CoA esters. This suggests the existence of another enzyme able to handle the acyl-CoAs that are not substrates for CrAT and CPT2, but for which the corresponding acylcarnitines are well recognized as diagnostic markers in inborn errors of metabolism.
authors.affiliation: Metabolism and Genetics Group, Research Institute for Medicines and Pharmaceutical Sciences, iMed.UL, Faculty of Pharmacy, University of Lisbon, Portugal.
authors.forename: Sara
authors.initials: S
authors.lastname: Violante
authors.forename: Lodewijk
authors.initials: L
authors.lastname: Ijlst
authors.forename: Jos
authors.initials: J
authors.lastname: Ruiter
authors.forename: Janet
authors.initials: J
authors.lastname: Koster
authors.forename: Henk
authors.initials: H
authors.lastname: van Lenthe
authors.forename: Marinus
authors.initials: M
authors.lastname: Duran
authors.forename: Isabel Tavares
authors.initials: IT
authors.lastname: de Almeida
authors.forename: Ronald J A
authors.initials: RJ
authors.lastname: Wanders
authors.forename: Sander M
authors.initials: SM
authors.lastname: Houten
authors.forename: Fátima V
authors.initials: FV
authors.lastname: Ventura
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000597:Amino Acids, Branched-Chain / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002332:Carnitine O-Acetyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002334:Carnitine O-Palmitoyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005227:Fatty Acids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D000214:Acyl Coenzyme A; D000597:Amino Acids, Branched-Chain; D005227:Fatty Acids; D002334:Carnitine O-Palmitoyltransferase; D002332:Carnitine O-Acetyltransferase