RESUMEN
Although the rate limiting step in mitochondrial fatty acid oxidation, catalyzed by carnitine palmitoyl transferase I (CPTI), utilizes long-chain fatty acyl-CoAs (LCFA-CoA) as a substrate, how LCFA-CoA is transferred to CPTI remains elusive. Based on secondary structural predictions and conserved tryptophan residues, the cytoplasmic C-terminal domain was hypothesized to be the LCFA-CoA binding site and important for interaction with cytoplasmic LCFA-CoA binding/transport proteins to provide a potential route for LCFA-CoA transfer. To begin to address this question, the cytoplasmic C-terminal region of liver CPTI (L-CPTI) was recombinantly expressed and purified. Data herein showed for the first time that the L-CPTI C-terminal 89 residues were sufficient for high affinity binding of LCFA-CoA (K (d) = 2-10 nM) and direct interaction with several cytoplasmic LCFA-CoA binding proteins (K (d) < 10 nM), leading to enhanced CPTI activity. Furthermore, alanine substitutions for tryptophan in L-CPTI (W391A and W452A) altered secondary structure, decreased binding affinity for LCFA-CoA, and almost completely abolished L-CPTI activity, suggesting that these amino acids may be important for ligand stabilization necessary for L-CPTI activity. Moreover, while decreased activity of the W452A mutant could be explained by decreased binding of lipid binding proteins, W391 itself seems to be important for activity. These data suggest that both interactions with lipid binding proteins and the peptide itself are important for optimal enzyme activity.
Asunto(s)
Acilcoenzima A/química , Carnitina O-Palmitoiltransferasa/química , Mitocondrias/enzimología , Fragmentos de Péptidos/química , Proteínas Recombinantes/química , Animales , Sitios de Unión , Unión Competitiva , Dicroismo Circular , Pruebas de Enzimas , Transferencia Resonante de Energía de Fluorescencia , Humanos , Ratones , Unión Proteica , Estructura Secundaria de Proteína , Ratas , Espectrometría de FluorescenciaRESUMEN
Transgenic mice carrying the human heart muscle carnitine palmitoyltransferase I (M-CPTI) gene fused to a CAT reporter gene were generated to study the regulation of M-CPTI gene expression. When the mice were fasted for 48 h, CAT activity and mRNA levels increased by more than 2-fold in heart and skeletal muscle, but not liver or kidney. In the diabetic transgenic mice, there was a 2- to 3-fold increase in CAT activity and CAT mRNA levels in heart and skeletal muscle which upon insulin administration reverted to that observed with the control insulin sufficient transgenic mice. Feeding a high fat diet increased CAT activity and mRNA levels by 2- to 4-fold in heart and skeletal muscle of the transgenic mice compared to the control transgenic mice on regular diet. Overall, the M-CPTI promoter was found to be necessary for the tissue-specific hormonal and dietary regulation of the gene expression.
Asunto(s)
Carnitina O-Palmitoiltransferasa/metabolismo , Diabetes Mellitus Experimental/metabolismo , Grasas de la Dieta/metabolismo , Ayuno/metabolismo , Regulación Enzimológica de la Expresión Génica , Hormonas/metabolismo , Músculo Esquelético/metabolismo , Animales , Femenino , Masculino , Ratones , Ratones Transgénicos , Especificidad de Órganos , Distribución TisularRESUMEN
BACKGROUND: Transport rates of long-chain free fatty acids into mitochondria via carnitine palmitoyltransferase I relative to overall oxidative rates in hypertrophied hearts remain poorly understood. Furthermore, the extent of glucose oxidation, despite increased glycolysis in hypertrophy, remains controversial. The present study explores potential compensatory mechanisms to sustain tricarboxylic acid cycle flux that resolve the apparent discrepancy of reduced fatty acid oxidation without increased glucose oxidation through pyruvate dehydrogenase complex in the energy-poor, hypertrophied heart. METHODS AND RESULTS: We studied flux through the oxidative metabolism of intact adult rat hearts subjected to 10 weeks of pressure overload (hypertrophied; n=9) or sham operation (sham; n=8) using dynamic 13C-nuclear magnetic resonance. Isolated hearts were perfused with [2,4,6,8,10,12,14,16-(13)C8] palmitate (0.4 mmol/L) plus glucose (5 mmol/L) in a 14.1-T nuclear magnetic resonance magnet. At similar tricarboxylic acid cycle rates, flux through carnitine palmitoyltransferase I was 23% lower in hypertrophied (P<0.04) compared with sham hearts and corresponded to a shift toward increased expression of the L-carnitine palmitoyltransferase I isoform. Glucose oxidation via pyruvate dehydrogenase complex did not compensate for reduced palmitate oxidation rates. However, hypertrophied rats displayed an 83% increase in anaplerotic flux into the tricarboxylic acid cycle (P<0.03) that was supported by glycolytic pyruvate, coincident with increased mRNA transcript levels for malic enzyme. CONCLUSIONS: In cardiac hypertrophy, fatty acid oxidation rates are reduced, whereas compensatory increases in anaplerosis maintain tricarboxylic acid cycle flux and account for a greater portion of glucose oxidation than previously recognized. The shift away from acetyl coenzyme A production toward carbon influx via anaplerosis bypasses energy, yielding reactions contributing to a less energy-efficient heart.
Asunto(s)
Cardiomegalia/metabolismo , Carnitina O-Palmitoiltransferasa/metabolismo , Metabolismo Energético , Transducción de Señal , Animales , Ciclo del Ácido Cítrico , Glucosa/metabolismo , Pruebas de Función Cardíaca , Masculino , Técnicas de Cultivo de Órganos , Oxidación-Reducción , Ácido Palmítico/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/fisiologíaRESUMEN
Carnitine palmitoyltransferase (CPT) I catalyzes the conversion of long-chain fatty acyl-CoAs to acyl carnitines in the presence of l-carnitine, a rate-limiting step in the transport of long-chain fatty acids from the cytoplasm to the mitochondrial matrix. To determine the role of the 15 cysteine residues in the heart/skeletal muscle isoform of CPTI (M-CPTI) on catalytic activity and malonyl-CoA sensitivity, we constructed a 6-residue N-terminal, a 9-residue C-terminal, and a 15-residue cysteineless M-CPTI by cysteine-scanning mutagenesis. Both the 9-residue C-terminal mutant enzyme and the complete 15-residue cysteineless mutant enzyme are inactive but that the 6-residue N-terminal cysteineless mutant enzyme had activity and malonyl-CoA sensitivity similar to those of wild-type M-CPTI. Mutation of each of the 9 C-terminal cysteines to alanine or serine identified a single residue, Cys-305, to be important for catalysis. Substitution of Cys-305 with Ala in the wild-type enzyme inactivated M-CPTI, and a single change of Ala-305 to Cys in the 9-residue C-terminal cysteineless mutant resulted in an 8-residue C-terminal cysteineless mutant enzyme that had activity and malonyl-CoA sensitivity similar to those of the wild type, suggesting that Cys-305 is the residue involved in catalysis. Sequence alignments of CPTI with the acyltransferase family of enzymes in the GenBank led to the identification of a putative catalytic triad in CPTI consisting of residues Cys-305, Asp-454, and His-473. Based on the mutagenesis and substrate labeling studies, we propose a mechanism for the acyltransferase activity of CPTI that uses a catalytic triad composed of Cys-305, His-473, and Asp-454 with Cys-305 serving as a probable nucleophile, thus acting as a site for covalent attachment of the acyl molecule and formation of a stable acyl-enzyme intermediate. This would in turn allow carnitine to act as a second nucleophile and complete the acyl transfer reaction.
Asunto(s)
Carnitina O-Palmitoiltransferasa/química , Carnitina O-Palmitoiltransferasa/genética , Cisteína/química , Alanina/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Western Blotting , Carnitina/química , Catálisis , Cartilla de ADN/química , Humanos , Cinética , Malonil Coenzima A/química , Modelos Químicos , Datos de Secuencia Molecular , Mutagénesis , Mutación , Miocardio/metabolismo , Ácido Palmítico , Palmitoilcarnitina/química , Pichia/metabolismo , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Serina/químicaRESUMEN
Carnitine palmitoyltransferase I (CPT-I) and II (CPT-II) enzymes are components of the carnitine palmitoyltransferase shuttle system which allows entry of long-chain fatty acids into the mitochondrial matrix for subsequent oxidation. This system is tightly regulated by malonyl-CoA levels since this metabolite is a strong reversible inhibitor of the CPT-I enzyme. There are two distinct CPT-I isotypes (CPT-Ialpha and CPT-Ibeta), that exhibit different sensitivity to malonyl-CoA inhibition. Because of its ability to inhibit fatty acid synthase, C75 is able to increase malonyl-CoA intracellular levels. Paradoxically it also activates long-chain fatty acid oxidation. To identify the exact target of C75 within the CPT system, we expressed individually the different components of the system in the yeast Pichia pastoris. We show here that C75 acts on recombinant CPT-Ialpha, but also on the other CPT-I isotype (CPT-Ibeta) and the malonyl-CoA insensitive component of the CPT system, CPT-II.
Asunto(s)
4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacología , Carnitina O-Palmitoiltransferasa/efectos de los fármacos , Carnitina O-Palmitoiltransferasa/metabolismo , Malonil Coenzima A/metabolismo , Pichia/efectos de los fármacos , Pichia/enzimología , Animales , Carnitina O-Palmitoiltransferasa/genética , Activación Enzimática/efectos de los fármacos , Humanos , Pichia/genética , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPTI) catalyzes the initial and regulatory step in the beta-oxidation of long-chain fatty acids. There are two well-characterized isotypes of CPTI: CPTIalpha (also known as L-CPTI) and CPTIbeta (also known as M-CPTI) that in human and rat encode for enzymes with very different kinetic properties and sensitivity to malonyl-CoA inhibition. Kinetic hallmarks of the CPTIalpha are high affinity for carnitine and low sensitivity to malonyl-CoA inhibition, while the opposite characteristics, low affinity for carnitine and high sensitivity to malonyl-CoA, are intrinsic to the CPTIbeta isotype. We have isolated the pig CPTIbeta cDNA which encodes for a protein of 772 amino acids that shares extensive sequence identity with human (88%), rat (85%), and mouse (86%) CPTIbeta, while the degree of homology with the CPTIalpha from human (61%), rat (62%), and mouse (60%) is much lower. However, when expressed in the yeast Pichia pastoris, pig CPTIbeta shows kinetic characteristics similar to those of the CPTIalpha isotype. Thus, the pig CPTIbeta, unlike the corresponding human or rat enzyme, has a high affinity for carnitine (K(m) = 197 microM) and low sensitive to malonyl-CoA inhibition (IC(50) = 906 nM). Therefore, the recombinant pig CPTIbeta has unique kinetic characteristics, which makes it a useful model to study the structure-function relationship of the CPTI enzymes.
Asunto(s)
Carnitina O-Palmitoiltransferasa/antagonistas & inhibidores , Carnitina O-Palmitoiltransferasa/química , Carnitina/química , Inhibidores Enzimáticos/química , Malonil Coenzima A/química , Mitocondrias Hepáticas/enzimología , Mitocondrias Musculares/enzimología , Secuencia de Aminoácidos , Animales , Carnitina O-Palmitoiltransferasa/biosíntesis , Carnitina O-Palmitoiltransferasa/genética , Clonación Molecular , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/biosíntesis , Isoenzimas/química , Isoenzimas/genética , Cinética , Ratones , Datos de Secuencia Molecular , Ratas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , PorcinosRESUMEN
Although it is hypothesized that long-chain fatty acyl CoAs (LCFA-CoAs) and long-chain fatty acids (LCFAs) regulate transcription in the nucleus, little is known regarding factors that determine the distribution of these ligands to nuclei of living cells. Immunofluorescence colocalization showed that liver fatty acid-binding protein (L-FABP; binds LCFA-CoA as well as LCFA) significantly colocalized with PPARalpha in nuclei of transfected L-cell fibroblasts. Colocalization with a DNA binding dye (SYTO59) revealed that, within the nucleus of control L-cells, the nonhydrolyzable fluorescent LCFA-CoA (BODIPY-C16-S-S-CoA) was distributed primarily in a punctate pattern throughout the nucleoplasm, while nonmetabolizable fluorescent LCFAs (BODIPY-C16 and BODIPY-C12) were localized primarily near the nuclear envelope membranes. L-FABP overexpression selectively increased the targeting of BODIPY-C16-S-S-CoA by 1.9- and 2.7-fold into the nuclear membrane and nucleoplasm, respectively. L-FABP also increased the targeting of fluorescent LCFAs (especially long-chain-length BODIPY-C16) by 1.7-fold to the nuclear membrane and 7.4-fold into the nucleoplasm. A cis-parinaric acid displacement assay showed that L-FABP bound BODIPY-C12 and BODIPY-C16 with K(i)s of 10.1 +/- 2.5 and 20.7 +/- 1.5 nM, respectively, in the same range as naturally occurring LCFAs. Finally, solid-phase extraction and HPLC analysis revealed that, depending on the fatty acid content of the culture medium, L-FABP expression also increased the cellular LCFA-CoA pool size and altered the LCFA-CoA acyl chain composition. Thus, L-FABP may function as a carrier for selectively enhancing the distribution of LCFA-CoA, as well as LCFA, to nuclei for potential interaction with nuclear receptors.
Asunto(s)
Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Ácidos Grasos/metabolismo , Hígado/metabolismo , Proteínas del Tejido Nervioso , Peroxisomas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Acilcoenzima A/análisis , Acilcoenzima A/metabolismo , Animales , Biomarcadores/química , Compuestos de Boro/metabolismo , Proteínas Portadoras/biosíntesis , Núcleo Celular/química , Citoplasma/metabolismo , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Ácidos Grasos/análisis , Fibroblastos/química , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Colorantes Fluorescentes/metabolismo , Hidrólisis , Células L , Ligandos , Ratones , Membrana Nuclear/metabolismo , Unión Proteica , TransfecciónRESUMEN
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 malonyl-CoA 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.
Asunto(s)
Carnitina O-Palmitoiltransferasa/química , Ácido Glutámico/química , Hígado/enzimología , Malonil Coenzima A/metabolismo , Músculos/enzimología , Alanina/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Western Blotting , Carnitina/química , Carnitina/farmacología , Pollos , Relación Dosis-Respuesta a Droga , Glutamina/química , Humanos , Immunoblotting , Cinética , Lisina/química , Ratones , Datos de Secuencia Molecular , Mutación , Palmitoil Coenzima A/farmacología , Pichia/metabolismo , Reacción en Cadena de la Polimerasa , Isoformas de Proteínas , Estructura Terciaria de Proteína , Ratas , Homología de Secuencia de Aminoácido , PorcinosRESUMEN
The muscle isoform of carnitine palmitoyltransferase I (M-CPTI) is 30- to 100-fold more sensitive to malonyl CoA inhibition than the liver isoform (L-CPTI). We have previously shown that deletion of the first 28 N-terminal amino acid residues in M-CPTI abolished malonyl CoA inhibition and high-affinity binding [Biochemistry 39 (2000) 712-717]. To determine the role of specific residues within the first 28 N-terminal amino acids of human heart M-CPTI on malonyl CoA sensitivity and binding, we constructed a series of substitution mutations and a mutant M-CPTI composed of deletion 18 combined with substitution mutations V19A, L23A, and S24A. All mutants had CPT activity similar to that of the wild type. A change of Glu3 to Ala resulted in a 60-fold decrease in malonyl CoA sensitivity and loss of high-affinity malonyl CoA binding. A change of His5 to Ala in M-CPTI resulted in only a 2-fold decrease in malonyl CoA sensitivity and a significant loss in the low- but not high-affinity malonyl CoA binding. Deletion of the first 18 N-terminal residues combined with substitution mutations V19A, L23A, and S24A resulted in a mutant M-CPTI with an over 140-fold decrease in malonyl CoA sensitivity and a significant loss in both high- and low-affinity malonyl CoA binding. This was further confirmed by a combined four-residue substitution of Glu3, Val19, Leu23, and Ser24 with alanine. Our site-directed mutagenesis studies demonstrate that Glu3, Val19, Leu23, and Ser24 in M-CPTI are important for malonyl CoA inhibition and binding, but not for catalysis.
Asunto(s)
Carnitina O-Palmitoiltransferasa/química , Carnitina O-Palmitoiltransferasa/metabolismo , Miocardio/enzimología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Carnitina O-Palmitoiltransferasa/antagonistas & inhibidores , Carnitina O-Palmitoiltransferasa/genética , ADN/genética , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas In Vitro , Cinética , Malonil Coenzima A/metabolismo , Malonil Coenzima A/farmacología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación de SecuenciaRESUMEN
Muscle carnitine palmitoyltransferase I (M-CPTI) catalyzes the conversion of long-chain fatty acyl-CoAs to acylcarnitines in the presence of L-carnitine. To determine the role of the C-terminal region of M-CPTI in enzyme activity, we constructed a series of deletion and substitution mutants. The mutants were expressed in the yeast Pichia pastoris, and the effect of the mutations on M-CPTI activity and malonyl-CoA sensitivity was determined in isolated mitochondria prepared from the yeast strains expressing the wild-type and deletion mutants. Deletion of the last 210, 113, 44, 20, 10, and 9 C-terminal amino-acid residues resulted in an inactive M-CPTI, but deletion of the last 8, 7, 6, and 3 C-terminal residues had no effect on activity, demonstrating that leucine-764 (L764) is essential for catalysis. Substitution of L764 with alanine caused a 40% loss in catalytic activity, but replacement of L764 with arginine resulted in an 84% loss of activity; substitution of L764 with valine had no effect on catalytic activity. The catalytic efficiency for the L764R mutant decreased by 80% for both substrates. Secondary structure prediction of the M-CPTI sequence identified a 21-amino-acid residue, 744-764, predicted to fold into a coiled-coil alpha-helix in the extreme C-terminal region of M-CPTI that may be important for native folding and activity. In summary, our data demonstrate that deletion of L764 or substitution with arginine inactivates the enzyme, suggesting that L764 may be important for proper folding of M-CPTI and optimal activity.
Asunto(s)
Carnitina O-Palmitoiltransferasa/química , Carnitina O-Palmitoiltransferasa/metabolismo , Leucina/fisiología , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Secuencia de Aminoácidos , Carnitina O-Palmitoiltransferasa/genética , Humanos , Cinética , Leucina/genética , Malonil Coenzima A/metabolismo , Datos de Secuencia Molecular , Proteínas Musculares/genética , Pichia/genética , Mutación Puntual , Alineación de Secuencia , Eliminación de SecuenciaRESUMEN
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.
Asunto(s)
Arginina/metabolismo , Carnitina O-Palmitoiltransferasa/metabolismo , Ácido Glutámico/metabolismo , Hígado/enzimología , Malonil Coenzima A/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Carnitina O-Palmitoiltransferasa/química , Carnitina O-Palmitoiltransferasa/genética , Catálisis , Cartilla de ADN , Humanos , Cinética , Datos de Secuencia Molecular , Mutagénesis , Pichia/genética , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Mammalian mitochondrial membranes express two active but distinct carnitine palmitoyltransferases: carnitine palmitoyltransferase I (CPTI), which is malonyl coA-sensitive and detergent-labile; and carnitine palmitoyltransferase II (CPTII), which is malonyl coA-insensitive and detergent-stable. To determine the role of the highly conserved C-terminal acidic residues glutamate 487 (Glu(487)) and glutamate 500 (Glu(500)) on catalytic activity in rat liver CPTII, we separately mutated these residues to alanine, aspartate, or lysine, and the effect of the mutations on CPTII activity was determined in the Escherichia coli-expressed mutants. Substitution of Glu(487) with alanine, aspartate, or lysine resulted in almost complete loss in CPTII activity. Because a conservative substitution mutation of this residue, Glu(487) with aspartate (E487D), resulted in a 97% loss in activity, we predicted that Glu(487) would be at the active-site pocket of CPTII. The substantial loss in CPTII activity observed with the E487K mutant, along with the previously reported loss in activity observed in a child with a CPTII deficiency disease, establishes that Glu(487) is crucial for maintaining the configuration of the liver isoform of the CPTII active site. Substitution of the conserved Glu(500) in CPTII with alanine or aspartate reduced the V(max) for both substrates, suggesting that Glu(500) may be important in stabilization of the enzyme-substrate complex. A conservative substitution of Glu(500) to aspartate resulted in a significant decrease in the V(max) for the substrates. Thus, Glu(500) may play a role in substrate binding and catalysis. Our site-directed mutagenesis studies demonstrate that Glu(487) in the liver isoform of CPTII is essential for catalysis.
Asunto(s)
Carnitina O-Palmitoiltransferasa/química , Hígado/enzimología , Secuencia de Aminoácidos , Animales , Carnitina O-Palmitoiltransferasa/metabolismo , Catálisis , Secuencia Conservada , Escherichia coli/genética , Glutamina , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Ratas , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
Pig and rat liver carnitine palmitoyltransferase I (L-CPTI) share common K(m) values for palmitoyl-CoA and carnitine. However, they differ widely in their sensitivity to malonyl-CoA inhibition. Thus, pig l-CPTI has an IC(50) for malonyl-CoA of 141 nm, while that of rat L-CPTI is 2 microm. Using chimeras between rat L-CPTI and pig L-CPTI, we show that the entire C-terminal region behaves as a single domain, which dictates the overall malonyl-CoA sensitivity of this enzyme. The degree of malonyl-CoA sensitivity is determined by the structure adopted by this domain. Using deletion mutation analysis, we show that malonyl-CoA sensitivity also depends on the interaction of this single domain with the first 18 N-terminal amino acid residues. We conclude that pig and rat L-CPTI have different malonyl-CoA sensitivity, because the first 18 N-terminal amino acid residues interact differently with the C-terminal domain. This is the first study that describes how interactions between the C- and N-terminal regions can determine the malonyl-CoA sensitivity of L-CPTI enzymes using active C-terminal chimeras.