Identification by Mutagenesis of Conserved Arginine and Tryptophan Residues in Rat Liver Carnitine Palmitoyltransferase I Important for Catalytic Activity

Abstract

Carnitine palmitoyltransferase I catalyzes the conversion of long-chain acyl-CoA to acylcarnitines in the presence of L-carnitine. To determine the role of the conserved arginine and tryptophan residues on catalytic activity in the liver isoform of carnitine palmitoyltransferase I (L-CPTI), we separately mutated five conserved arginines and two tryptophans to alanine. Substitution of arginine residues 388, 451, and 606 with alanine resulted in loss of 88, 82, and 93% of L-CPTI activity, respectively. Mutants R601A and R655A showed less than 2% of the wild type L-CPTI activity. A change of tryptophan 391 and 452 to alanine resulted in 50 and 93% loss in carnitine palmitoyltransferase activity, respectively. The mutations caused decreases in catalytic efficiency of 80 -98%. The residual activity in the mutant L-CPTIs was sensitive to malonyl-CoA inhibition. Mutants R388A, R451A, R606A, W391A, and W452A had no effect on the K m values for carnitine or palmitoyl-CoA. However, these mutations decreased the V max values for both substrates by 10 -40-fold, suggesting that the main effect of the mutations was to decrease the stability of the enzyme-substrate complex. We suggest that conserved arginine and tryptophan residues in L-CPTI contribute to the stabilization of the enzyme-substrate complex by charge neutralization and hydrophobic interactions. The predicted secondary structure of the 100-amino acid residue region of L-CPTI, containing arginines 388 and 451 and tryptophans 391 and 452, consists of four ␣-helices similar to the known three-dimensional structure of the acyl-CoA-binding protein. We predict that this 100-amino acid residue region constitutes the putative palmitoyl-CoA-binding site in L-CPTI.Transport of long-chain fatty acids from the cytoplasm to the mitochondrial matrix involves the conversion of their acyl-CoA derivatives to acylcarnitines, translocation across the inner mitochondrial membrane, and reconversion to acyl-CoA (1, 2). Carnitine palmitoyltransferase I (CPTI) 1 catalyzes the conversion of long-chain fatty acyl-CoAs to acylcarnitines in the presence of carnitine. Mammalian tissues express two isoforms of CPTI, a liver isoform (L-CPTI) and a heart/skeletal muscle isoform (M-CPTI), that are 62% identical in amino acid sequence (Refs. 3-7 and 9, GenBank™ accession number U62317)). As an enzyme that catalyzes the first rate-limiting step in ␤-oxidation, CPTI is tightly regulated by its physiological inhibitor, malonyl-CoA, the first intermediate in fatty acid synthesis, suggesting coordinated control of fatty acid oxidation and synthesis (1, 2). Because of its central role in fatty acid metabolism, a good understanding of the molecular mechanism of the regulation of the CPT system is an important first step in the development of treatments for diseases, such as myocardial ischemia and diabetes, and in human inherited CPT deficiency diseases (10 -12). We developed a novel high level expression system for human heart M-CPTI, rat L-CPTI, and CPTII in the yeast Pichia pastoris, an organism devoi...

Identification by Mutagenesis of Conserved Arginine and Glutamate Residues in the C-terminal Domain of Rat Liver Carnitine Palmitoyltransferase I That Are Important for Catalytic Activity and Malonyl-CoA Sensitivity

“…For M-CPTI, our mutagenesis studies demonstrate that in addition to Glu-3 and His-5, Val-19, Leu-23, and Ser-24 are necessary for malonyl-CoA inhibition and high affinity binding, in agreement with the differences in malonyl-CoA sensitivity observed between M-CPTI and L-CPTI (17). In addition, our site-directed mutagenesis studies of conserved residues in the C-terminal domain of L-CPTI demonstrated that conserved arginine and tryptophan residues are important for catalysis (18). In this report, our mutagenesis studies demonstrate for the first time that the conserved residues Arg-601, Glu-603, and Arg-606 in L-CPTI are important for catalytic activity and malonyl-CoA sensitivity.…”

“…Substitution of Glu-603 with glutamine resulted in a 92% loss in L-CPTI activity and a 14-fold decrease in malonyl-CoA sensitivity (Table II). We previously reported that mutation of the highly conserved arginine residues Arg-601 and Arg-606 to alanine resulted in Ͼ98 and 93% loss in L-CPTI activity, respectively (18). The loss in activity observed with the R601A and R606A mutants was also accompanied by decreased sensitivity to malonyl-CoA inhibition (18).…”

“…1), Glu-590 and Glu-603. The conserved Glu-603 residue is flanked by two highly conserved arginine residues, Arg-601 and Arg-606, that we previously demonstrated were important for L-CPTI activity and malonyl-CoA sensitivity (18).…”

“…Generation of Mutations and Expression in P. pastorisConstruction of plasmids carrying substitution mutations E590A, E603A, E603H, E603Q, and E603D was performed as described under "Experimental Procedures," and for R601A and R606A as described previously (18). P. pastoris was chosen as an expression system for L-CPTI and the mutants, because it does not have endogenous CPT activity (6,(12)(13)(14)(15)(16).…”

“…Bacterial colonies obtained upon transformation of the mutagenesis reactions were screened by PCR using the primer pair f-GWW3-r-E603QCK for Gln and f-GWW3-r-E603HCK for His and Asp. The R601A and R606A mutants were constructed as described previously (18). The mutations were confirmed by DNA sequencing.…”