A characteristic Glu17 residue of pig carnitine palmitoyltransferase 1 is responsible for the low Km for carnitine and the low sensitivity to malonyl-CoA inhibition of the enzyme.
Human carnitine palmitoyltransferase 1B (CPT1B) is a highly malonyl-CoA-sensitive enzyme (IC50=0.097 microm) and has a positive determinant (residues 18-28) of malonyl-CoA inhibition. By contrast, rat carnitine palmitoyltransferase 1A is less sensitive to malonyl-CoA inhibition (IC(50)=1.9 microm), and has both a positive (residues 1-18) and a negative (residues 18-28) determinant of its inhibition. Interestingly, pig CPT1B shows a low degree of malonyl-CoA sensitivity (IC(50)=0.804 microm). Here, we examined whether any additional molecular determinants affect malonyl-CoA inhibition of CPT1B. We show that the malonyl-CoA sensitivity of CPT1B is determined by the length (either 50 or 128 residues) of the N-terminal region constructed by recombining pig and human enzymes. We also show that the N-terminal region of pig CPT1B carries a single positive determinant of malonyl-CoA sensitivity, but that this is located between residues 1 and 18 of the N-terminal segment. Importantly, we found a single amino acid variation (D17E) relevant to malonyl-CoA sensitivity. Thus, Asp17 is specifically involved, under certain assay conditions, in the high malonyl-CoA sensitivity of the human enzyme, whereas the naturally occurring variation, Glu17, is responsible for both the low malonyl-CoA sensitivity and high carnitine affinity characteristics of the pig enzyme. This is the first demonstration that a single naturally occurring amino acid variation can alter CPT1B enzymatic properties.
Relat
Joana
J
Department of Biochemistry and Molecular Biology, School of Pharmacy and Institute of Biomedicine of Barcelona University (IBUB), Spain.
Pujol-Vidal
Magdalena
M
Haro
Diego
D
Marrero
Pedro F
PF
D019943:Amino Acid Substitution; D000818:Animals; D002331:Carnitine / Q000378:metabolism; D002334:Carnitine O-Palmitoyltransferase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018698:Glutamic Acid; D006801:Humans; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000494:pharmacology*; D008861:Microsomes / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D009474:Neurons / Q000201:enzymology; D017384:Sequence Deletion; D013552:Swine
D004791:Enzyme Inhibitors; D018698:Glutamic Acid; D008316:Malonyl Coenzyme A; D002334:Carnitine O-Palmitoyltransferase; D002331:Carnitine