The first 28 N-terminal amino acid residues of human heart muscle carnitine palmitoyltransferase I are essential for malonyl CoA sensitivity and high-affinity binding.
Heart/skeletal muscle carnitine palmitoyltransferase I (M-CPTI) is 30-100-fold more sensitive to malonyl CoA inhibition than the liver isoform (L-CPTI). To determine the role of the N-terminal region of human heart M-CPTI on malonyl CoA sensitivity and binding, a series of deletion mutations were constructed ranging in size from 18 to 83 N-terminal residues. All of the deletions except Delta83 were active. Mitochondria from the yeast strains expressing Delta28 and Delta39 exhibited a 2.5-fold higher activity compared to the wild type, but were insensitive to malonyl CoA inhibition and had complete loss of high-affinity malonyl CoA binding. The high-affinity site (K(D1), B(max1)) for binding of malonyl CoA to M-CPTI was completely abolished in the Delta28, Delta39, Delta51, and Delta72 mutants, suggesting that the decrease in malonyl CoA sensitivity observed in these mutants was due to the loss of the high-affinity binding entity of the enzyme. Delta18 showed only a 4-fold loss in malonyl CoA sensitivity but had activity and high-affinity malonyl CoA binding similar to the wild type. Replacement of the N-terminal domain of L-CPTI with the N-terminal domain of M-CPTI does not change the malonyl CoA sensitivity of the chimeric L-CPTI, suggesting that the amino acid residues responsible for the differing sensitivity to malonyl CoA are not located in this N-terminal region. These results demonstrate that the N-terminal residues critical for activity and malonyl CoA sensitivity in M-CPTI are different from those of L-CPTI.
Shi
J
J
Department of Biochemistry, Oregon Graduate Institute of Science and Technology, Beaverton, Oregon 97006-8921, USA.
Zhu
H
H
Arvidson
D N
DN
Woldegiorgis
G
G
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002250:Carbon Radioisotopes; D002334:Carnitine O-Palmitoyltransferase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005822:Genetic Vectors; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000201:enzymology* / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010843:Pichia / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017384:Sequence Deletion
D000596:Amino Acids; D002250:Carbon Radioisotopes; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D008316:Malonyl Coenzyme A; D002334:Carnitine O-Palmitoyltransferase