A Single Amino Acid Change (Substitution of the Conserved Glu-590 with Alanine) in the C-terminal Domain of Rat Liver Carnitine Palmitoyltransferase I Increases its Malonyl-CoA Sensitivity Close to That Observed with the Muscle Isoform of the Enzyme

Abstract

Carnitine palmitoyltransferase I (CPTI) catalyzes the conversion of long-chain fatty acyl-CoAs to acylcarnitines in the presence of L-carnitine. To determine the role of the highly conserved C-terminal glutamate residue, Glu-590, on catalysis and malonyl-CoA sensitivity, we separately changed the residue to alanine, lysine, glutamine, and aspartate. Substitution of Glu-590 with aspartate, a negatively charged amino acid with only one methyl group less than the glutamate residue in the wild-type enzyme, resulted in complete loss in the activity of the liver isoform of CPTI (L-CPTI). A change of Glu-590 to alanine, glutamine, and lysine caused a significant 9-to 16-fold increase in malonyl-CoA sensitivity but only a partial decrease in catalytic activity. Substitution of Glu-590 with neutral uncharged residues (alanine and glutamine) and/or a basic positively charged residue (lysine) significantly increased L-CPTI malonylCoA sensitivity to the level observed with the muscle isoform of the enzyme, suggesting the importance of neutral and/or positive charges in the switch of the kinetic properties of L-CPTI to the muscle isoform of CPTI. Since a conservative substitution of Glu-590 to aspartate but not glutamine resulted in complete loss in activity, we suggest that the longer side chain of glutamate is essential for catalysis and malonyl-CoA sensitivity. This is the first demonstration whereby a single residue mutation in the C-terminal region of the liver isoform of CPTI resulted in a change of its kinetic properties close to that observed with the muscle isoform of the enzyme and provides the rationale for the high malonyl-CoA sensitivity of muscle CPTI compared with the liver isoform of the enzyme. Carnitine palmitoyltransferase I (CPTI)1 catalyzes the conversion of long-chain fatty acyl-CoAs to acylcarnitines in the presence of L-carnitine, the first step in the transport of longchain fatty acids from the cytoplasm to the mitochondria matrix, a rate-limiting step in ␤-oxidation (1, 2). Mammalian tissues express two isoforms of CPTI, a liver isoform (L-CPTI) and a muscle isoform (M-CPTI), that are 62% identical in amino acid sequence (3-8). As an enzyme that catalyzes the first rate-limiting step in ␤-oxidation, CPTI is regulated by its physiological inhibitor, malonyl-CoA (1, 2), the first intermediate in fatty acid synthesis, suggesting a coordinated control of fatty acid oxidation and synthesis. Previous studies by our laboratory and others have demonstrated that the muscle isoform of CPTI, M-CPTI, is significantly more sensitive to malonyl-CoA inhibition than the liver isoform, but the molecular/ structural basis for the differences in malonyl-CoA sensitivity between M-CPTI and L-CPTI remain to be established (3-8).Because of its central role in fatty acid metabolism, understanding 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 CPTI deficiency diseases (9 -11...

Structural insight into function and regulation of carnitine palmitoyltransferase

“…The importance of this region is confirmed by the finding that the intrinsically less malonylCoA-sensitive isoform L-CPT-1 displays a marked increase in inhibition by malonyl-CoA on introduction of a single point mutation, Glu590Ala [77]. Both Glu590 and Met593 are part of strand b13, which lines the entrance to the active site via the CoA site.…”

Molecular characterization of a gilthead sea bream (Sparus aurata) muscle tissue cDNA for carnitine palmitoyltransferase 1B (CPT1B)

“…Thus, in the rat liver enzyme, residues Glu-590, Arg-601, Glu-603, and Arg-606 are involved both in catalysis and malonyl-CoA sensitivity as mutations on these sites affect both properties , Napal et al, 2003. The above residues are conserved in the sea bream CPT1 (Glu-588, Arg-599, Glu-601, and Arg-604, respectively in Fig.…”

C75 [4-Methylene-2-octyl-5-oxo-tetrahydro-furan-3-carboxylic Acid] Activates Carnitine Palmitoyltransferase-1 in Isolated Mitochondria and Intact Cells without Displacement of Bound Malonyl CoA

“…Between the cytosolic domains are two transmembrane regions and a short intramitochondrial loop (Fraser et al, 1997;Zammit, 1999). Malonyl CoA binding and inhibition of both CPT-1␣ and CPT-1␤ can be disrupted by deletions or point mutations in both the short N terminus and the C-terminal catalytic domain 1 (Cohen et al, 1998;Swanson et al, 1998;Jackson et al, 2000;Shi et al, 2000;Napal et al, 2003). Interestingly, Zammit and colleagues have shown that deletions within the N-terminal region can actually increase sensitivity of CPT-1␣ to inhibition by malonyl CoA (Jackson et al, 2000).…”