Definition by functional and structural analysis of two malonyl-CoA sites in carnitine palmitoyltransferase 1A

J Biol Chem. 2007 Jun 22;282(25):18212-18224. doi: 10.1074/jbc.M700885200. Epub 2007 Apr 23.


Carnitine palmitoyltransferase 1 (CPT1) catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine in the presence of l-carnitine, thus facilitating the entry of fatty acids to mitochondria, in a process that is physiologically inhibited by malonyl-CoA. To examine the mechanism of CPT1 liver isoform (CPT1A) inhibition by malonyl-CoA, we constructed an in silico model of both its NH2- and COOH-terminal domains. Two malonyl-CoA binding sites were found. One of these, the "CoA site" or "A site," is involved in the interactions between NH2- and COOH-terminal domains and shares the acyl-CoA hemitunnel. The other, the "opposite-to-CoA site" or "O site," is on the opposite side of the enzyme, in the catalytic channel. The two sites share the carnitine-binding locus. To prevent the interaction between NH2- and COOH-terminal regions, we produced CPT1A E26K and K561E mutants. A double mutant E26K/K561E (swap), which was expected to conserve the interaction, was also produced. Inhibition assays showed a 12-fold decrease in the sensitivity (IC50) toward malonyl-CoA for CPT1A E26K and K561E single mutants, whereas swap mutant reverts to wild-type IC50 value. We conclude that structural interaction between both domains is critical for enzyme sensitivity to malonyl-CoA inhibition at the "A site." The location of the "O site" for malonyl-CoA binding was supported by inhibition assays of expressed R243T mutant. The model is also sustained by kinetic experiments that indicated linear mixed type malonyl-CoA inhibition for carnitine. Malonyl-CoA alters the affinity of carnitine, and there appears to be an exponential inverse relation between carnitine Km and malonyl-CoA IC50.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Carnitine / chemistry
  • Carnitine O-Palmitoyltransferase / chemistry*
  • Carnitine O-Palmitoyltransferase / genetics*
  • Humans
  • Inhibitory Concentration 50
  • Kinetics
  • Malonyl Coenzyme A / metabolism*
  • Molecular Conformation
  • Molecular Sequence Data
  • Mutation
  • Protein Binding
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae / metabolism
  • Sequence Homology, Amino Acid


  • Malonyl Coenzyme A
  • Carnitine O-Palmitoyltransferase
  • Carnitine