Functional characterization of mitochondrial carnitine palmitoyltransferases I and II expressed in the yeast Pichia pastoris.
The rate-limiting step in beta oxidation is the conversion of long-chain acyl-CoA to acylcarnitine, a reaction catalyzed by the outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPTI) and inhibited by malonyl-CoA. The acylcarnitine is then translocated across the inner mitochondrial membrane by the carnitine/acylcarnitine translocase and converted back to acyl-CoA by CPTII. Although CPTII has been examined in detail, studies on CPTI have been hampered by an inability to purify CPTI in an active form from CPTII. In particular, it has not been conclusively demonstrated that CPTI is even catalytically active, or whether sensitivity of CPTI to malonyl-CoA is an intrinsic property of the enzyme or is contained in a separate regulatory subunit that interacts with CPTI. To address these questions, the genes for CPTI and CPTII were separately expressed in Pichia pastoris, a yeast with no endogenous CPT activity. High levels of CPT activity were present in purified mitochondrial preparations from both CPTI- and CPTII-expressing strains. Furthermore, CPTI activity was highly sensitive to inhibition by malonyl-CoA while CPTII was not. Thus, CPT catalytic activity and malonyl-CoA sensitivity are contained within a single CPTI polypeptide in mammalian mitochondrial membranes. We describe the kinetic characteristics for the yeast-expressed CPTs, the first such report for a CPTI enzyme in the absence of CPTII. Yeast-expressed CPTI is inactivated by detergent solubilization. However, removal of the detergent in the presence of phospholipids resulted in the recovery of malonyl-CoA-sensitive CPTI activity, suggesting that CPTI requires a membranous environment. CPTI is thus reversibly inactivated by detergents.
de Vries
Y
Y
Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland 97291-1000, USA.
Arvidson
D N
DN
Waterham
H R
HR
Cregg
J M
JM
Woldegiorgis
G
G
D000214:Acyl Coenzyme A / Q000378:metabolism; D000818:Animals; D002334:Carnitine O-Palmitoyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D003576:Electron Transport Complex IV / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes; D008099:Liver / Q000201:enzymology; D008316:Malonyl Coenzyme A / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia; D051381:Rats
D000214:Acyl Coenzyme A; D018076:DNA, Complementary; D008081:Liposomes; D008316:Malonyl Coenzyme A; D003576:Electron Transport Complex IV; D002334:Carnitine O-Palmitoyltransferase