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.
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.
Treber
Michelle
M
Department of Environmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health & Science University, Beaverton, Oregon 97006-8921.
Dai
Jia
J
Woldegiorgis
Gebre
G
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001483:Base Sequence; D002334:Carnitine O-Palmitoyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D017931:DNA Primers; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008316:Malonyl Coenzyme A / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010843:Pichia / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D011994:Recombinant Proteins; D018698:Glutamic Acid; D008316:Malonyl Coenzyme A; D001120:Arginine; D002334:Carnitine O-Palmitoyltransferase