title: Identification of conserved amino acid residues in rat liver carnitine palmitoyltransferase I critical for malonyl-CoA inhibition. Mutation of methionine 593 abolishes malonyl-CoA inhibition.
abstract: Carnitine palmitoyltransferase (CPT) I, which catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine facilitating its transport through the mitochondrial membranes, is inhibited by malonyl-CoA. By using the SequenceSpace algorithm program to identify amino acids that participate in malonyl-CoA inhibition in all carnitine acyltransferases, we found 5 conserved amino acids (Thr(314), Asn(464), Ala(478), Met(593), and Cys(608), rat liver CPT I coordinates) common to inhibitable malonyl-CoA acyltransferases (carnitine octanoyltransferase and CPT I), and absent in noninhibitable malonyl-CoA acyltransferases (CPT II, carnitine acetyltransferase (CAT) and choline acetyltransferase (ChAT)). To determine the role of these amino acid residues in malonyl-CoA inhibition, we prepared the quintuple mutant CPT I T314S/N464D/A478G/M593S/C608A as well as five single mutants CPT I T314S, N464D, A478G, M593S, and C608A. In each case the CPT I amino acid selected was mutated to that present in the same homologous position in CPT II, CAT, and ChAT. Because mutant M593S nearly abolished the sensitivity to malonyl-CoA, two other Met(593) mutants were prepared: M593A and M593E. The catalytic efficiency (V(max)/K(m)) of CPT I in mutants A478G and C608A and all Met(593) mutants toward carnitine as substrate was clearly increased. In those CPT I proteins in which Met(593) had been mutated, the malonyl-CoA sensitivity was nearly abolished. Mutations in Ala(478), Cys(608), and Thr(314) to their homologous amino acid residues in CPT II, CAT, and ChAT caused various decreases in malonyl-CoA sensitivity. Ala(478) is located in the structural model of CPT I near the catalytic site and participates in the binding of malonyl-CoA in the low affinity site (Morillas, M., Gómez-Puertas, P., Rubi, B., Clotet, J., Ariño, J., Valencia, A., Hegardt, F. G., Serra, D., and Asins, G. (2002) J. Biol. Chem. 277, 11473-11480). Met(593) may participate in the interaction of malonyl-CoA in the second affinity site, whose location has not been reported.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Barcelona, School of Pharmacy, E-08028 Barcelona, Spain.
authors.forename: Montserrat
authors.initials: M
authors.lastname: Morillas
authors.forename: Paulino
authors.initials: P
authors.lastname: Gómez-Puertas
authors.forename: Assia
authors.initials: A
authors.lastname: Bentebibel
authors.forename: Eva
authors.initials: E
authors.lastname: Sellés
authors.forename: Nuria
authors.initials: N
authors.lastname: Casals
authors.forename: Alfonso
authors.initials: A
authors.lastname: Valencia
authors.forename: Fausto G
authors.initials: FG
authors.lastname: Hegardt
authors.forename: Guillermina
authors.initials: G
authors.lastname: Asins
authors.forename: Dolors
authors.initials: D
authors.lastname: Serra
mesh_terms: D000409:Alanine / Q000737:chemistry; D000465:Algorithms; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002333:Carnitine Acyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002334:Carnitine O-Palmitoyltransferase / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017354:Point Mutation; D011485:Protein Binding; D020033:Protein Isoforms; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000737:chemistry
chemical_list: D000596:Amino Acids; D020033:Protein Isoforms; D013912:Threonine; D008316:Malonyl Coenzyme A; D008715:Methionine; D002333:Carnitine Acyltransferases; D002334:Carnitine O-Palmitoyltransferase; D003545:Cysteine; D000409:Alanine