Cysteine-scanning mutagenesis of muscle carnitine palmitoyltransferase I reveals a single cysteine residue (Cys-305) is important for catalysis.
Carnitine palmitoyltransferase (CPT) I catalyzes the conversion of long-chain fatty acyl-CoAs to acyl carnitines in the presence of l-carnitine, a rate-limiting step in the transport of long-chain fatty acids from the cytoplasm to the mitochondrial matrix. To determine the role of the 15 cysteine residues in the heart/skeletal muscle isoform of CPTI (M-CPTI) on catalytic activity and malonyl-CoA sensitivity, we constructed a 6-residue N-terminal, a 9-residue C-terminal, and a 15-residue cysteineless M-CPTI by cysteine-scanning mutagenesis. Both the 9-residue C-terminal mutant enzyme and the complete 15-residue cysteineless mutant enzyme are inactive but that the 6-residue N-terminal cysteineless mutant enzyme had activity and malonyl-CoA sensitivity similar to those of wild-type M-CPTI. Mutation of each of the 9 C-terminal cysteines to alanine or serine identified a single residue, Cys-305, to be important for catalysis. Substitution of Cys-305 with Ala in the wild-type enzyme inactivated M-CPTI, and a single change of Ala-305 to Cys in the 9-residue C-terminal cysteineless mutant resulted in an 8-residue C-terminal cysteineless mutant enzyme that had activity and malonyl-CoA sensitivity similar to those of the wild type, suggesting that Cys-305 is the residue involved in catalysis. Sequence alignments of CPTI with the acyltransferase family of enzymes in the GenBank led to the identification of a putative catalytic triad in CPTI consisting of residues Cys-305, Asp-454, and His-473. Based on the mutagenesis and substrate labeling studies, we propose a mechanism for the acyltransferase activity of CPTI that uses a catalytic triad composed of Cys-305, His-473, and Asp-454 with Cys-305 serving as a probable nucleophile, thus acting as a site for covalent attachment of the acyl molecule and formation of a stable acyl-enzyme intermediate. This would in turn allow carnitine to act as a second nucleophile and complete the acyl transfer reaction.
Liu
Hongyan
H
Department of Environmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health & Science University, Beaverton, Oregon 97006-8921, USA.
Zheng
Guolu
G
Treber
Michelle
M
Dai
Jia
J
Woldegiorgis
Gebre
G
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002331:Carnitine / Q000737:chemistry; D002334:Carnitine O-Palmitoyltransferase / Q000737:chemistry* / Q000235:genetics*; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000737:chemistry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009206:Myocardium / Q000378:metabolism; D019308:Palmitic Acid; D010172:Palmitoylcarnitine / Q000737:chemistry; D010843:Pichia / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry
D017931:DNA Primers; D010172:Palmitoylcarnitine; D019308:Palmitic Acid; D012694:Serine; D008316:Malonyl Coenzyme A; D002334:Carnitine O-Palmitoyltransferase; D003545:Cysteine; D000409:Alanine; D002331:Carnitine