Cysteine-scanning Mutagenesis of Muscle Carnitine Palmitoyltransferase I Reveals a Single Cysteine Residue (Cys-305) Is Important for Catalysis

• Published in: The Journal of biological chemistry Vol. 280; no. 6; pp. 4524 - 4531
• Main Authors: Liu, HongYan, Zheng, Guolu, Treber, Michelle, Dai, Jia, Woldegiorgis, Gebre
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 11.02.2005; American Society for Biochemistry and Molecular Biology
• Subjects: Alanine - chemistry; Amino Acid Sequence; Animals; Binding Sites; Blotting, Western; Carnitine - chemistry; Carnitine O-Palmitoyltransferase - chemistry; Carnitine O-Palmitoyltransferase - genetics; Catalysis; Cysteine - chemistry; DNA Primers - chemistry; Humans; Kinetics; Malonyl Coenzyme A - chemistry; Models, Chemical; Molecular Sequence Data; Mutagenesis; Mutation; Myocardium - metabolism; Palmitic Acid; Palmitoylcarnitine - chemistry; Pichia - metabolism; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Serine - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M400893200

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.