Deletion of the conserved first 18 N-terminal amino acid residues in rat liver carnitine palmitoyltransferase I abolishes malonyl-CoA sensitivity and binding.
To assess the role of the 130 N-terminal amino acid residues of rat liver carnitine palmitoyltransferase I (L-CPTI) on malonyl-CoA sensitivity and binding, we constructed a series of mutants with deletions of the 18, 35, 52, 73, 83, or 129 most N-terminal amino acid residues. The deletion mutants were expressed in the yeast Pichia pastoris. We determined the effects of these mutations on L-CPTI activity, malonyl-CoA sensitivity, and binding in isolated mitochondria prepared from the yeast strains expressing the wild-type and deletion mutants. The mutant protein that lacked the first 18 N-terminal amino acid residues, Delta18, had activity and kinetic properties similar to wild-type L-CPTI, but it was almost completely insensitive to malonyl-CoA inhibition (I50 = 380 microM versus 2.0 microM). In addition, loss of malonyl-CoA sensitivity in Delta18 was accompanied by a 70-fold decrease in affinity for malonyl CoA (KD = 70 nM versus 1.1 nM) compared to wild-type L-CPTI. Deletion of the first 35, 52, 73, and 83 N-terminal amino acid residues had a similar effect on malonyl-CoA sensitivity as did the 18-residue deletion mutant, and there was a progressive reduction in the affinity for malonyl-CoA binding. By contrast, deletion of the first 129 N-terminal amino acid residues resulted in the synthesis of an inactive protein. To our knowledge, this is the first report to demonstrate a critical role for these perfectly conserved first 18 N-terminal amino acid residues of L-CPTI in malonyl-CoA sensitivity and binding.
Shi
J
J
Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland 97291-1000, USA.
Zhu
H
H
Arvidson
D N
DN
Cregg
J M
JM
Woldegiorgis
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002250:Carbon Radioisotopes; D002334:Carnitine O-Palmitoyltransferase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D017124:Conserved Sequence / Q000235:genetics*; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008316:Malonyl Coenzyme A / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics*; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D010957:Plasmids / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D017384:Sequence Deletion
D002250:Carbon Radioisotopes; D007527:Isoenzymes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D008316:Malonyl Coenzyme A; D002334:Carnitine O-Palmitoyltransferase