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

Abstract

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 TM 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.Carnitine palmitoyltransferase (CPT) 1 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 (1, 2). Mammalian tissues express two isoforms of CPTI, a liver isoform (L-CPTI) and a heart/skeletal muscle isoform (M-CPTI), which are 62% identical in amino acid sequence (3-8). As an enzyme that catalyzes the first rate-limiting step in fatty acid oxidation, CPTI is regulated by its physiological inhibitor, malonyl-CoA, the first intermediate in fatty acid synthesis, suggesting a coordinated control of fatty acid synthesis and oxidation (1, 2). Previous studies by our laboratory and others have established that M-CPTI is more sensitive to malonyl-CoA inhibition than L-CPTI (3-8). The molecular/ structural basis for the differences in malonyl-CoA sensitivity between M-CPTI and L-CPTI was established recently by our demonstration that substitution of the conserved Cterminal L-CPTI residue Glu-590 with alanine increased...

Membrane Topology of Mouse Stearoyl-CoA Desaturase 1

“…This observation invited speculation that the thiol group in cysteines might be required in the formation of an acyl-S-enzyme before desaturation took place and be important for the overall stabilization of the protein (28). Moreover, cysteine-scanning mutagenesis study on muscle carnitine palmitoyltransferase I, which also utilizes acyl-CoAs as substrates, has revealed that a single cysteine is important for catalysis, probably serving as the site for the covalent attachment of the acylCoAs (51). In addition to that, Reiser and Raju (49) have also provided evidence that the inhibition of the desaturase by cyclopropene fatty acyl-CoAs is due to the irreversible binding of the sulfhydryl groups of the enzyme by the cyclopropene group.…”

The Crystal Structure of Carnitine Palmitoyltransferase 2 and Implications for Diabetes Treatment

“…Asp213 and Glu487 are affected by naturally occurring mutations and have been determined to be important for CPT-2 function by biochemical analyses (Zheng et al, 2002;Liu et al, 2005). Asp213 is located in a loop between b3 and a10 of CPT-2 and aligns with a cysteine conserved in all human CPT-1 isoforms.…”

Replacement of C305 in Heart/Muscle-Type Isozyme of Human Carnitine Palmitoyltransferase I with Aspartic Acid and Other Amino Acids

“…When a mutant C304W of L-CPTI observed with one of their patients was expressed in COS cells, it was not effectively expressed and showed no catalytic activity. In the other approach, Woldegiorgis and his colleagues carried out Cys-scanning mutagenesis to determine the roles of 15 Cys residues present in M-CPTI (Liu et al 2005). Using C305W 8 a CPTI activity observed with mitochondrial fraction prepared from each transfectants, as % of that observed with transfectant of wild type.…”

“…The only exceptionally acceptable amino acid as a substitute for C305 of M-CPTI was Asp. Liu et al (2005) carried out alignments of the amino acid sequence of CPTI with the acyltransferase family of enzymes and, based on the results of past mutation studies, identified conserved catalytically important residues C305, D454, and H473 for the acyltransferase activity of CPTI. Of these, D454 and H473 are conserved not only in CPTI and CPTII but also in the other acyltransferases.…”

“…However, different results were reported for mutation of C304 of human L-CPTI and its corresponding amino acid in human M-CPTI (C305). Replacement of C304 of L-CPTI with Trp (C304W) caused both reduced expression and loss of function (Brown et al 2001), but replacement of C305 in M-CPTI with Ala (C305A) caused only loss of function (Liu et al 2005). In order to understand why different results were obtained by replacement of this cysteine, in the present study we carried out additional expression studies using M-CPTI mutants.…”