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

J Biol Chem. 2003 Mar 14;278(11):9058-63. doi: 10.1074/jbc.M209999200. Epub 2002 Dec 23.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alanine / chemistry
  • Algorithms
  • Amino Acid Sequence
  • Amino Acids / chemistry*
  • Animals
  • Binding Sites
  • Blotting, Western
  • Carnitine Acyltransferases / antagonists & inhibitors
  • Carnitine Acyltransferases / metabolism
  • Carnitine O-Palmitoyltransferase / chemistry*
  • Carnitine O-Palmitoyltransferase / metabolism
  • Catalysis
  • Cysteine / chemistry
  • Dose-Response Relationship, Drug
  • Humans
  • Inhibitory Concentration 50
  • Kinetics
  • Malonyl Coenzyme A / antagonists & inhibitors*
  • Malonyl Coenzyme A / chemistry*
  • Methionine / chemistry
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation*
  • Point Mutation
  • Protein Binding
  • Protein Isoforms
  • Rats
  • Saccharomyces cerevisiae / metabolism
  • Sequence Homology, Amino Acid
  • Threonine / chemistry


  • Amino Acids
  • Protein Isoforms
  • Threonine
  • Malonyl Coenzyme A
  • Methionine
  • Carnitine Acyltransferases
  • Carnitine O-Palmitoyltransferase
  • Cysteine
  • Alanine