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

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 conserved glutamate residue, Glu-603, on catalysis and malonyl-CoA sensitivity, we separately changed the residue to alanine, histidine, glutamine, and aspartate. Substitution of Glu-603 with alanine or histidine resulted in complete loss of L-CPTI activity. A change of Glu-603 to glutamine caused a significant decrease in catalytic activity and malonyl-CoA sensitivity. Substitution of Glu-603 with aspartate, a negatively charged amino acid with only one methyl group less than the glutamate residue in the wild type enzyme, resulted in partial loss in CPTI activity and a 15-fold decrease in malonyl-CoA sensitivity. The mutant L-CPTI with a replacement of the conserved Arg-601 or Arg-606 with alanine also showed over 40-fold decrease in malonyl-CoA sensitivity, suggesting that these two conserved residues may be important for substrate and inhibitor binding. Since a conservative substitution of Glu-603 to aspartate or glutamine resulted in partial loss of activity and malonyl-CoA sensitivity, it further suggests that the negative charge and the longer side chain of glutamate are essential for catalysis and malonyl-CoA sensitivity. We predict that this region of L-CPTI spanning these conserved C-terminal residues may be the region of the protein involved in binding the CoA moiety of palmitoyl-CoA and malonyl-CoA and/or the putative low affinity acyl-CoA/malonyl-CoA binding site.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 long chain fatty acids from the cytoplasm to the mitochondrial 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 coordinated control of fatty acid oxidation and synthesis. 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).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 devoid of endogenous CPT activity (6,(12)(13)(14). Furthermore, by using this system, we have shown that CPTI and CPTII are active distinct enzymes and that L-CPTI and M-CPTI are distinct malonyl-CoA-sensitive CPTs that are reversibly inactivated by detergents. Recent site-directed mutagenesis studies from our laboratory have demonstrated t...

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

“…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 malonylCoA inhibition and high affinity binding, in agreement with the differences in malonyl-CoA sensitivity observed between M-CPTI and L-CPTI (17,18). In addition, our site-directed mutagenesis studies of conserved residues in the C-terminal domain of L-CPTI demonstrated that conserved arginine and glutamate residues are important for catalytic activity and malonyl-CoA sensitivity (19,23). Others have reported that the deletion of N-terminal residues 19 -30 containing Ser-24 and Gln-30 or substitution of Ser-24 and Gln-30 with alanine in L-CPTI increased malonyl-CoA sensitivity but their effect was entirely dependent on the presence of Glu-3 because mutation of Glu-3 to alanine was found to override the effects of both the deletion and the combined effects of S24A and Q30A on malonyl-CoA sensitivity (28,29), confirming our previous report that Glu-3 in the extreme N-terminal region of L-CPTI is the main determinant of malonyl-CoA sensitivity (16).…”

“…1). We have previously demonstrated that the conserved Glu-603 residue together with the two adjacent highly conserved arginine residues, Arg-601 and Arg-606, are important for L-CPTI activity and malonyl-CoA sensitivity (19,23).…”

“…Glu-603 is a conserved residue within the family of CPT enzymes, whereas other acyltransferases have aspartate at this position. We have recently demonstrated that a change of Glu-603 to alanine or histidine resulted in complete loss in L-CPTI activity (23), whereas a change to glutamine caused a significant loss in activity and malonyl-CoA sensitivity. In contrast with the complete loss of activity observed with the E590D mutant, substitution of Glu-603 with aspartate resulted in only partial loss in CPTI activity but a significant loss in malonyl-CoA sensitivity.…”

Functional and Structural Basis of Carnitine Palmitoyltransferase 1A Deficiency

“…Moreover, this illustrates that residues located far from the catalytic residues in the primary amino acid sequence can in fact be crucial elements of the catalytic core. This also explains why deletion and/or mutation within the C terminus of CPT1A so dramatically affected initial protein folding and/or catalytic activity (19,20).…”

“…The N-terminal domain (1-147 residues) was shown to be essential for mitochondrial import and for maintenance of a folded active and malonyl-CoA-sensitive conformation (12)(13)(14). Functional analysis of natural and/or engineered mutations in CPT1A strongly contributed to understanding the catalytic and regulatory mechanisms implied in the acyltransferase family (15)(16)(17)(18)(19)(20). The recent three-dimensional structural models of the mouse and human carnitine acetyltransferase (CAT) provided critical insights into the molecular basis for fatty acyl chain transfer (21,22).…”