Monatshefte für Chemie / Chemical Monthly

, Volume 136, Issue 8, pp 1325–1340

Structure-Function Studies with the Mitochondrial Carnitine Palmitoyltransferases I and II


DOI: 10.1007/s00706-005-0334-7

Cite this article as:
Woldegiorgis, G., Dai, J. & Arvidson, D. Monatsh. Chem. (2005) 136: 1325. doi:10.1007/s00706-005-0334-7


Mitochondrial carnitine palmitoyltransferases I and II (CPTI and CPTII), together with the carnitine carrier, transport long-chain fatty acyl-CoAs from the cytosol to the mitochondrial matrix for β-oxidation. As an enzyme that catalyzes the rate-limiting step in fatty acid oxidation, CPTI is inhibited by malonyl-CoA, the first intermediate in fatty acid synthesis. Our development of a high level of expression for CPTI and CPTII in P. pastoris, a yeast with no endogenous CPT activity has enabled us to map the malonyl-CoA and substrate binding sites by mutational analysis. Using deletion and substitution mutants of L-CPTI expressed in P. pastoris, we have shown that Glu3 and His5 are necessary for malonyl-CoA inhibition and high-affinity binding of L-CPTI but not for catalysis. Similar studies of M-CPTI clearly establish that the N-terminal residues Glu3, Val19, Leu23, and Ser24 in M-CPTI are important for malonyl-CoA inhibition and binding, but not for catalysis. Furthermore, using chimeras between rat and pig L-CPTI, and deletion mutation analysis, we demonstrated that the differences in malonyl-CoA sensitivity observed between the pig and rat L-CPTI were due to differences in the interaction of the first 18 N-terminal amino acid residues with the C-terminal region of the respective enzymes. Consistent with this, the conserved C-terminal residues R601, E603, R606, and K560 were found to be important for L-CPTI activity, malonyl-CoA inhibition and binding, because mutation of these residues decreased malonyl-CoA sensitivity and enzyme activity. We also identified two conserved C-terminal residues in L-CPTI, D567, and E590, that when mutated to alanine cause a substantial increase in malonyl-CoA sensitivity, suggesting a structural basis for the differences in malonyl-CoA sensitivity between L-CPTI and M-CPTI. Our cysteine-scanning mutagenesis of M-CPTI revealed that a single Cys residue, Cys305, was essential for catalysis. In addition, deletion and substitution analysis of the extreme C-terminal region of M-CPTI, suggest that L764 may be important for proper folding and optimal activity. In summary, our structure-function studies with the mitochondrial carnitine palmitoyltransferases I and II have identified critical residues for inhibitor and substrate binding and catalysis.

Keywords. Structure-activity relationships; Enzymes; Fatty acids; Oxidations; Carnitine.

Copyright information

© Springer-Verlag/Wien 2005

Authors and Affiliations

  1. 1.Department of Environmental Science and Biomolecular SystemsOGI School of Science and Engineering, Oregon Health & Science UniversityBeavertonUSA
  2. 2.Department of Microbiology and Molecular GeneticsMichigan State UniversityEast LansingUSA