Identification by Mutagenesis of Conserved Arginine and Glutamate Residues in the C-terminal Domain of Rat Liver Carnitine Palmitoyltransferase I That Are Important for Catalytic Activity and Malonyl-CoA Sensitivity

• Published in: The Journal of biological chemistry 2003-03, Vol.278 (13), p.11145-11149
• Main Authors: Treber, Michelle, Dai, Jia, Woldegiorgis, Gebre
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Arginine - metabolism; Base Sequence; Carnitine O-Palmitoyltransferase - chemistry; Carnitine O-Palmitoyltransferase - genetics; Carnitine O-Palmitoyltransferase - metabolism; Catalysis; DNA Primers; Glutamic Acid - metabolism; Humans; Kinetics; Liver - enzymology; Malonyl Coenzyme A - metabolism; Molecular Sequence Data; Mutagenesis; Pichia - genetics; Rats; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M210566200

Carnitine palmitoyltransferase I (CPTI) catalyzes the conversion of long chain fatty acyl-CoAs to acylcarnitines in the presence of l -carnitine. To determine the role of the conserved glutamate residue, Glu-603, on catalysis and malonyl-CoA sensitivity, we separately changed the residue to alanine, histidine, glutamine, and aspartate. Substitution of Glu-603 with alanine or histidine resulted in complete loss of L-CPTI activity. A change of Glu-603 to glutamine caused a significant decrease in catalytic activity and malonyl-CoA sensitivity. Substitution of Glu-603 with aspartate, a negatively charged amino acid with only one methyl group less than the glutamate residue in the wild type enzyme, resulted in partial loss in CPTI activity and a 15-fold decrease in malonyl-CoA sensitivity. The mutant L-CPTI with a replacement of the conserved Arg-601 or Arg-606 with alanine also showed over 40-fold decrease in malonyl-CoA sensitivity, suggesting that these two conserved residues may be important for substrate and inhibitor binding. Since a conservative substitution of Glu-603 to aspartate or glutamine resulted in partial loss of activity and malonyl-CoA sensitivity, it further suggests that the negative charge and the longer side chain of glutamate are essential for catalysis and malonyl-CoA sensitivity. We predict that this region of L-CPTI spanning these conserved C-terminal residues may be the region of the protein involved in binding the CoA moiety of palmitoyl-CoA and malonyl-CoA and/or the putative low affinity acyl-CoA/malonyl-CoA binding site.