Carnitine palmitoyltransferase. Activation by palmitoyl-CoA and inactivation by malonyl-CoA

Bremer, Jon; Woldegiorgis, Gebre; Schalinske, Kevin L.; Shrago, Earl

doi:10.1016/0005-2760(85)90247-4

Cited by 80 publications

(48 citation statements)

References 19 publications

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“…CPT activity was assayed radiochemically with either L-[methyl-3 H]carnitine hydrochloride or L-[methyl-14 C]carnitine hydrochloride in the direction of palmitoylcarnitine formation (Bremer et al, 1985). The assay buffer contains 50 mM KCl, 5 mM Tris-HCl, 1 mM Mg 2 Cl, 0.1% fatty acid-free BSA, 1 mM KCN, 0.1 mM ATP, and 0.5 M palmitoylCoA, pH 6.0.…”

Section: Methodsmentioning

confidence: 99%

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

Yang

Kays²,

Skillman³

et al. 2004

J Pharmacol Exp Ther

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Carnitine palmitoyltransferase 1␤ (CPT-1␤) is a key regulator of the ␤ oxidation of long-chain fatty acids in skeletal muscle and therefore a potential therapeutic target for diseases associated with defects in lipid metabolism such as obesity and type 2 diabetes. C75 [4-methylene-2-octyl-5-oxo-tetrahydro-furan-3-carboxylic acid] is an ␣-methylene-butyrolactone that has been characterized as both an inhibitor of fatty acid synthase and more recently, an activator of CPT-1 (Thupari et al., 2002). Using human CPT-1␤ expressed in the yeast Pichia pastoris, we demonstrate that C75 can activate the skeletal muscle isoform of CPT-1 and overcome inactivation of the enzyme by malonyl CoA, an important physiological repressor of CPT-1, and the malonyl CoA mimetic Ro25-0187 [{5-[2-(naphthalen-2-yloxy)-ethoxy]-thiophen-2-yl}-oxo-acetic acid]. We also show that C75 can activate CPT-1 in intact hepatocytes to levels similar to those achieved with inhibition of acetyl-CoA carboxylase, the enzyme that produces malonyl CoA. Finally, we demonstrate that concentrations of C75 sufficient for activation of CPT-1 do not displace bound malonyl CoA. We conclude that CPT-1 is an activator of human CPT-1␤ and other CPT-1 isoforms but that it does not activate CPT-1 through antagonism of malonyl CoA binding.

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Section: Methodsmentioning

confidence: 99%

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

Yang

Kays²,

Skillman³

et al. 2004

J Pharmacol Exp Ther

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“…CPT Assay-CPT activity was assayed by the forward exchange method using L-[methyl-3 H]carnitine (12,20). The K m value for palmitoyl-CoA was determined by varying the palmitoyl-CoA concentration from 2.8 to 225 M at a fixed molar ratio (6.1:1) of palmitoyl-CoA to albumin as described previously (16).…”

mentioning

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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“…3 H]carnitine (12,21). The K m value for palmitoyl-CoA was determined by varying the palmitoyl-CoA concentration from 2.8 to 225 M at a fixed molar ratio (6.1:1) of palmitoyl-CoA to albumin as described previously (16).…”

mentioning

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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Carnitine palmitoyltransferase. Activation by palmitoyl-CoA and inactivation by malonyl-CoA

Cited by 80 publications

References 19 publications

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

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

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

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