Roles of the N- and C-terminal domains of carnitine palmitoyltransferase I isoforms in malonyl-CoA sensitivity of the enzymes: insights from expression of chimaeric proteins and mutation of conserved histidine residues.
The mitochondrial outer membrane enzyme carnitine palmitoyltransferase I (CPT I) plays a major role in the regulation of fatty acid entry into the mitochondrial matrix for beta-oxidation by virtue of its inhibition by malonyl-CoA. Two isoforms of CPT I, the liver type (L) and muscle type (M), have been identified, the latter being 100 times more sensitive to malonyl-CoA and having a much higher Km for the substrate carnitine. Here we have examined the roles of different regions of the CPT I molecules in their response to malonyl-CoA, etomoxir (an irreversible inhibitor) and carnitine. To this end, we analysed the properties of engineered rat CPT I constructs in which (a) the N-terminal domain of L-CPT I was deleted, (b) the N-terminal domains of L- and M-CPT I were switched, or (c) each of three conserved histidine residues located towards the N-terminus in L-CPT I was mutated. Several novel points emerged: (1) whereas the N-terminal domain is critical for a normal malonyl-CoA response, it does not itself account for the widely disparate sensitivities of the liver and muscle enzymes to the inhibitor; (2) His-5 and/or His-140 probably play a direct role in the malonyl-CoA response, but His-133 does not; (3) the truncated, chimaeric and point- mutant variants of the enzyme all bound the covalent, active-site- directed ligand, etomoxir; and (4) only the most radical alteration of L-CPT I, i.e. deletion of the N-terminal 82 residues, affected the response to carnitine. We conclude that the N-terminal domain of CPT I plays an essential, but permissive, role in the inhibition of the enzyme by malonyl-CoA. By contrast, the larger C-terminal region dictates the degree of sensitivity to malonyl-CoA, as well as the response to carnitine; it is also sufficient for etomoxir binding. Additionally, further weight is added to the notion that one or more histidine residues may be involved in the CPT I-malonyl-CoA interaction.
Swanson
S T
ST
Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9135, USA.
Foster
D W
DW
McGarry
J D
JD
Brown
N F
NF
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002334:Carnitine O-Palmitoyltransferase / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D017124:Conserved Sequence; D004852:Epoxy Compounds / Q000378:metabolism; D006639:Histidine; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism; D017354:Point Mutation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D004852:Epoxy Compounds; D007527:Isoenzymes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D006639:Histidine; D008316:Malonyl Coenzyme A; D002334:Carnitine O-Palmitoyltransferase; C054207:etomoxir