Identification by Mutagenesis of a Conserved Glutamate (Glu487) Residue Important for Catalytic Activity in Rat Liver Carnitine Palmitoyltransferase II

Zheng, Guolu; Dai, Jia Le; Woldegiorgis, Gebre

doi:10.1074/jbc.m202914200

Cited by 6 publications

(6 citation statements)

References 27 publications

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“…Glu-603 may thus be required for L-CPTI stability and positioning of the imidazole ring of His-473 for efficient catalysis and inhibition, thus facilitating productive interaction with the substrates and the inhibitor (23). Mutation of the corresponding conserved residue in CPTII, Glu-500, to alanine resulted in 50% loss in activity (24).…”

Section: Resultsmentioning

confidence: 99%

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

Treber

Dai

Woldegiorgis

2003

Journal of Biological Chemistry

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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...

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Section: Resultsmentioning

confidence: 99%

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

Treber

Dai

Woldegiorgis

2003

Journal of Biological Chemistry

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“…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. A change of the highly conserved glutamate residue corresponding to Glu-590 in CPTII, Glu-487 to aspartate inactivated the enzyme (25), suggesting the importance of this highly conserved residue in CPTI and CPTII in maintaining the active site conformation of the two enzymes. Substitution of the highly conserved Glu-590 with alanine did not have a major effect on catalytic activity but caused a significant increase in L-CPTI malonyl-CoA sensitivity, indicating the opposing roles played by the conserved C-terminal glutamate residues in L-CPTI on activity and malonyl-CoA sensitivity.…”

Section: Discussionmentioning

confidence: 99%

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

Napal

Dai

Treber

et al. 2003

Journal of Biological Chemistry

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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...

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“…The glycine residue Gly600, at a position where bulkier residues are found in CrAT (Met564) and CrOT (Gln552), allows for binding of LCFA carnitine derivatives to CPT-2, thereby determining substrate specificity. Glu487 and Glu500 of CPT-2, which are conserved throughout the carnitine acyltransferases, have been implicated in substrate binding and catalysis by means of mutational analysis (Zheng et al, 2002). The crystal structure of rCPT-2 reveals that Glu487 is indeed located in the part of the active site tunnel that accommodates the (modeled) CoA.…”

Section: Binding Mode Of St1326mentioning

confidence: 99%

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

et al. 2006

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Carnitine palmitoyltransferases 1 and 2 (CPTs) facilitate the import of long-chain fatty acids into mitochondria. Modulation of the catalytic activity of the CPT system is currently under investigation for the development of novel drugs against diabetes mellitus. We report here the 1.6 A resolution structure of the full-length mitochondrial membrane protein CPT-2. The structure of CPT-2 in complex with the generic CPT inhibitor ST1326 ([R]-N-[tetradecylcarbamoyl]-aminocarnitine), a substrate analog mimicking palmitoylcarnitine and currently in clinical trials for diabetes mellitus treatment, was solved at 2.5 A resolution. These structures of CPT-2 provide insight into the function of residues involved in substrate binding and determination of substrate specificity, thereby facilitating the rational design of antidiabetic drugs. We identify a sequence insertion found in CPT-2 that mediates membrane localization. Mapping of mutations described for CPT-2 deficiency, a hereditary disorder of lipid metabolism, implies effects on substrate recognition and structural integrity of CPT-2.

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Identification by Mutagenesis of a Conserved Glutamate (Glu487) Residue Important for Catalytic Activity in Rat Liver Carnitine Palmitoyltransferase II

Cited by 6 publications

References 27 publications

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

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

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

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

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