Identification of Conserved Amino Acid Residues in Rat Liver Carnitine Palmitoyltransferase I Critical for Malonyl-CoA Inhibition

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., Rubı́, 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.