Functional characterization of mammalian mitochondrial carnitine palmitoyltransferases I and II expressed in the yeast Pichia pastoris.
Mitochondrial carnitine palmitoyltransferases I and II (CPTI and CPTII), together with the carnitine carrier, transport long-chain fatty acyl-CoA from the cytosol to the mitochondrial matrix for beta-oxidation. Recent progress in the expression of CPTI and CPTII cDNA clones in Pichia pastoris, a yeast with no endogenous CPT activity, has greatly facilitated the characterization of these important enzymes in fatty acid oxidation. It is now well established that yeast-expressed CPTI is a catalytically active, malonyl CoA-sensitive, distinct enzyme that is reversibly inactivated by detergents. CPTII is a catalytically active, malonyl CoA-insensitive, distinct enzyme that is detergent stable. Reconstitution studies with yeast-expressed CPTI have established for the first time that detergent inactivation of CPTI is reversible, suggesting that CPTI is active only in a membrane environment. By constructing a series of deletion mutants of the N-terminus of liver CPTI, we have mapped the residues essential for malonyl CoA inhibition and binding to the conserved first six N-terminal amino acid residues. Mutation of glutamic acid 3 to alanine abolished malonyl CoA inhibition and high affinity malonyl CoA binding, but not catalytic activity, whereas mutation of histidine 5 to alanine caused partial loss in malonyl CoA inhibition. Our mutagenesis studies demonstrate that glutamic acid 3 and histidine 5 are necessary for malonyl CoA inhibition and binding to liver CPTI, but not catalytic activity.
Woldegiorgis
G
G
Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Beaverton 97006-8921, USA.
Shi
J
J
Zhu
H
H
Arvidson
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DN
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002334:Carnitine O-Palmitoyltransferase / Q000378:metabolism / Q000502:physiology*; D005227:Fatty Acids / Q000378:metabolism*; D006801:Humans; D008316:Malonyl Coenzyme A / Q000037:antagonists & inhibitors*; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology* / Q000378:metabolism
D005227:Fatty Acids; D008316:Malonyl Coenzyme A; D002334:Carnitine O-Palmitoyltransferase