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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.

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.

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.

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.

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.

Fatty acid translocase (FAT)/CD36 is a long chain fatty acid transporter present at the plasma membrane, as well as in intracellular pools of skeletal muscle. In this study, we assessed the unexpected presence of FAT/CD36 in both subsarcolemmal and intermyofibril fractions of highly purified mitochondria. Functional assessments demonstrated that the mitochondria could bind (14)C-labeled palmitate, but could only oxidize it in the presence of carnitine. However, the addition of sulfo-N-succinimidyl oleate, a known inhibitor of FAT/CD36, resulted in an 87 and 85% reduction of palmitate oxidation in subsarcolemmal and intermyofibril fractions, respectively. Further studies revealed that maximal carnitine palmitoyltransferase I (CPTI) activity in vitro was inhibited by succinimidyl oleate (42 and 48% reduction). Interestingly, CPTI immunoprecipitated with FAT/CD36, indicating a physical pairing. Tissue differences in mitochondrial FAT/CD36 protein follow the same pattern as the capacity for fatty acid oxidation (heart >> red muscle > white muscle). Additionally, chronic stimulation of hindlimb muscles (7 days) increased FAT/CD36 expression and also resulted in a concomitant increase in mitochondrial FAT/CD36 content (46 and 47% increase). Interestingly, with acute electrical stimulation of hindlimb muscles (30 min), FAT/CD36 expression was not altered, but there was an increase in the mitochondrial content of FAT/CD36 compared with the non-stimulated control limb (35 and 37% increase). Together, these data suggest a role for FAT/CD36 in mitochondrial long chain fatty acid uptake and demonstrate system flexibility to match FAT/CD36 mitochondrial content with an increased capacity for fatty acid oxidation, possibly involving translocation of FAT/CD36 to the mitochondria.

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.

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.

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.

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.

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.

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.

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.

The outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPTI) catalyzes the initial and regulatory step in the beta-oxidation of fatty acids. The genes for the two isoforms of CPTI-liver (L-CPTI) and muscle (M-CPTI) have been cloned and expressed, and the genes encode for enzymes with very different kinetic properties and sensitivity to malonyl-CoA inhibition. Pig L-CPTI encodes for a 772 amino acid protein that shares 86 and 62% identity, respectively, with rat L- and M-CPTI. When expressed in Pichia pastoris, the pig L-CPTI enzyme shows kinetic characteristics (carnitine, K(m) = 126 microM; palmitoyl-CoA, K(m) = 35 microM) similar to human or rat L-CPTI. However, the pig enzyme, unlike the rat liver enzyme, shows a much higher sensitivity to malonyl-CoA inhibition (IC(50) = 141 nM) that is characteristic of human or rat M-CPTI enzymes. Therefore, pig L-CPTI behaves like a natural chimera of the L- and M-CPTI isotypes, which makes it a useful model to study the structure--function relationships of the CPTI enzymes.

Carnitine palmitoyltransferase I catalyzes the conversion of long-chain acyl-CoA to acylcarnitines in the presence of l-carnitine. To determine the role of the conserved arginine and tryptophan residues on catalytic activity in the liver isoform of carnitine palmitoyltransferase I (L-CPTI), we separately mutated five conserved arginines and two tryptophans to alanine. Substitution of arginine residues 388, 451, and 606 with alanine resulted in loss of 88, 82, and 93% of L-CPTI activity, respectively. Mutants R601A and R655A showed less than 2% of the wild type L-CPTI activity. A change of tryptophan 391 and 452 to alanine resulted in 50 and 93% loss in carnitine palmitoyltransferase activity, respectively. The mutations caused decreases in catalytic efficiency of 80-98%. The residual activity in the mutant L-CPTIs was sensitive to malonyl-CoA inhibition. Mutants R388A, R451A, R606A, W391A, and W452A had no effect on the K(m) values for carnitine or palmitoyl-CoA. However, these mutations decreased the V(max) values for both substrates by 10-40-fold, suggesting that the main effect of the mutations was to decrease the stability of the enzyme-substrate complex. We suggest that conserved arginine and tryptophan residues in L-CPTI contribute to the stabilization of the enzyme-substrate complex by charge neutralization and hydrophobic interactions. The predicted secondary structure of the 100-amino acid residue region of L-CPTI, containing arginines 388 and 451 and tryptophans 391 and 452, consists of four alpha-helices similar to the known three-dimensional structure of the acyl-CoA-binding protein. We predict that this 100-amino acid residue region constitutes the putative palmitoyl-CoA-binding site in L-CPTI.

Animals, including humans, express two isoforms of acetyl-CoA carboxylase (EC ), ACC1 (M(r) = 265 kDa) and ACC2 (M(r) = 280 kDa). The predicted amino acid sequence of ACC2 contains an additional 136 aa relative to ACC1, 114 of which constitute the unique N-terminal sequence of ACC2. The hydropathic profiles of the two ACC isoforms generally are comparable, except for the unique N-terminal sequence in ACC2. The sequence of amino acid residues 1-20 of ACC2 is highly hydrophobic, suggesting that it is a leader sequence that targets ACC2 for insertion into membranes. The subcellular localization of ACC2 in mammalian cells was determined by performing immunofluorescence microscopic analysis using affinity-purified anti-ACC2-specific antibodies and transient expression of the green fluorescent protein fused to the C terminus of the N-terminal sequences of ACC1 and ACC2. These analyses demonstrated that ACC1 is a cytosolic protein and that ACC2 was associated with the mitochondria, a finding that was confirmed further by the immunocolocalization of a known human mitochondria-specific protein and the carnitine palmitoyltransferase 1. Based on analyses of the fusion proteins of ACC-green fluorescent protein, we concluded that the N-terminal sequences of ACC2 are responsible for mitochondrial targeting of ACC2. The association of ACC2 with the mitochondria is consistent with the hypothesis that ACC2 is involved in the regulation of mitochondrial fatty acid oxidation through the inhibition of carnitine palmitoyltransferase 1 by its product malonyl-CoA.

Heart/skeletal muscle carnitine palmitoyltransferase I (M-CPTI) is 30-100-fold more sensitive to malonyl CoA inhibition than the liver isoform (L-CPTI). To determine the role of the N-terminal region of human heart M-CPTI on malonyl CoA sensitivity and binding, a series of deletion mutations were constructed ranging in size from 18 to 83 N-terminal residues. All of the deletions except Delta83 were active. Mitochondria from the yeast strains expressing Delta28 and Delta39 exhibited a 2.5-fold higher activity compared to the wild type, but were insensitive to malonyl CoA inhibition and had complete loss of high-affinity malonyl CoA binding. The high-affinity site (K(D1), B(max1)) for binding of malonyl CoA to M-CPTI was completely abolished in the Delta28, Delta39, Delta51, and Delta72 mutants, suggesting that the decrease in malonyl CoA sensitivity observed in these mutants was due to the loss of the high-affinity binding entity of the enzyme. Delta18 showed only a 4-fold loss in malonyl CoA sensitivity but had activity and high-affinity malonyl CoA binding similar to the wild type. Replacement of the N-terminal domain of L-CPTI with the N-terminal domain of M-CPTI does not change the malonyl CoA sensitivity of the chimeric L-CPTI, suggesting that the amino acid residues responsible for the differing sensitivity to malonyl CoA are not located in this N-terminal region. These results demonstrate that the N-terminal residues critical for activity and malonyl CoA sensitivity in M-CPTI are different from those of L-CPTI.

Mitochondrial carnitine palmitoyltransferases I and II (CPTI and CPTII), together with the carnitine carrier, transport long-chain fatty acyl-CoA from the cytosol to the mitochondrial matrix for beta-oxidation. Recent progress in the expression of CPTI and CPTII cDNA clones in Pichia pastoris, a yeast with no endogenous CPT activity, has greatly facilitated the characterization of these important enzymes in fatty acid oxidation. It is now well established that yeast-expressed CPTI is a catalytically active, malonyl CoA-sensitive, distinct enzyme that is reversibly inactivated by detergents. CPTII is a catalytically active, malonyl CoA-insensitive, distinct enzyme that is detergent stable. Reconstitution studies with yeast-expressed CPTI have established for the first time that detergent inactivation of CPTI is reversible, suggesting that CPTI is active only in a membrane environment. By constructing a series of deletion mutants of the N-terminus of liver CPTI, we have mapped the residues essential for malonyl CoA inhibition and binding to the conserved first six N-terminal amino acid residues. Mutation of glutamic acid 3 to alanine abolished malonyl CoA inhibition and high affinity malonyl CoA binding, but not catalytic activity, whereas mutation of histidine 5 to alanine caused partial loss in malonyl CoA inhibition. Our mutagenesis studies demonstrate that glutamic acid 3 and histidine 5 are necessary for malonyl CoA inhibition and binding to liver CPTI, but not catalytic activity.

To assess the role of the 130 N-terminal amino acid residues of rat liver carnitine palmitoyltransferase I (L-CPTI) on malonyl-CoA sensitivity and binding, we constructed a series of mutants with deletions of the 18, 35, 52, 73, 83, or 129 most N-terminal amino acid residues. The deletion mutants were expressed in the yeast Pichia pastoris. We determined the effects of these mutations on L-CPTI activity, malonyl-CoA sensitivity, and binding in isolated mitochondria prepared from the yeast strains expressing the wild-type and deletion mutants. The mutant protein that lacked the first 18 N-terminal amino acid residues, Delta18, had activity and kinetic properties similar to wild-type L-CPTI, but it was almost completely insensitive to malonyl-CoA inhibition (I50 = 380 microM versus 2.0 microM). In addition, loss of malonyl-CoA sensitivity in Delta18 was accompanied by a 70-fold decrease in affinity for malonyl CoA (KD = 70 nM versus 1.1 nM) compared to wild-type L-CPTI. Deletion of the first 35, 52, 73, and 83 N-terminal amino acid residues had a similar effect on malonyl-CoA sensitivity as did the 18-residue deletion mutant, and there was a progressive reduction in the affinity for malonyl-CoA binding. By contrast, deletion of the first 129 N-terminal amino acid residues resulted in the synthesis of an inactive protein. To our knowledge, this is the first report to demonstrate a critical role for these perfectly conserved first 18 N-terminal amino acid residues of L-CPTI in malonyl-CoA sensitivity and binding.

Long-chain fatty acids are the primary source of energy production in the heart. Carnitine palmitoyltransferase I (CPT-I) catalyzes the first reaction in the transport of long-chain fatty acids from the cytoplasm to the mitochondrion, a rate-limiting step in beta-oxidation. In this study, we report the functional expression of the human heart/skeletal muscle isoform of CPT-I (M-CPT-I) in the yeast Pichia pastoris. Screening of a human heart cDNA library with cDNA fragments encoding the rat heart M-CPT-I resulted in the isolation of a single full-length human heart M-CPT-I cDNA clone. The clone has an open reading frame of 2316 bp with a 5' untranslated region of 38 bp and a 256-bp 3' untranslated region with the poly(A)+ addition sequence AATAAA. The predicted protein has 772 amino acids and a molecular mass of 88 kDa. Northern blot analysis of mRNAs from different human tissues using the human M-CPT-I cDNA as a probe revealed an abundant transcript of approximately 3.1 kb that was only present in human heart and skeletal muscle tissue. Expression of the human M-CPT-I cDNA in P. pastoris, a yeast with no endogenous CPT activity, produced an 80-kDa protein that was located in the mitochondria. Isolated mitochondria from the M-CPT-I expression strain exhibited a malonyl-coenzyme A (CoA)-sensitive CPT activity that was detergent labile. The I50 for malonyl-CoA inhibition of the yeast-expressed M-CPT-I was 69 nM, and the Kms for carnitine and palmitoyl-CoA were 666 and 42 microM, respectively. The I50 for malonyl-CoA inhibition of the heart enzyme is 30 times lower than that of the yeast-expressed liver CPT-I, and the Km for carnitine is more than 20 times higher than that of the liver CPT-I. This is the first report of the expression of a heart CPT-I in a system devoid of endogenous CPT activity and the functional characterization of a human heart M-CPT-I in the absence of the liver isoform and CPT-II., (Copyright 1997 Academic Press. Copyright 1997Academic Press)

The human heart muscle carnitine palmitoyltransferase I (M-CPTI) gene was expressed at high levels from a strain of the methylotrophic yeast Pichia pastoris containing approximately 24 copies of the expression vector. Levels of M-CPTI were more than ten-fold higher than previously reported by our group with a single-copy strain (Arch. Biochem. Biophys., in press) and were sufficient to perform reconstitution studies on the membrane protein, a key step in purification and structural analysis of the enzyme. Solubilization of yeast mitochondria containing M-CPTI in 5% Triton X-100 abolished M-CPTI activity. The detergent-inactivated M-CPTI was then reconstituted by removal of the detergent in the presence of phospholipids. The reconstituted proteoliposomes exhibited M-CPTI activity of 2.4 nmol palmitoylcarnitine formed/mg protein/min, a recovery of 23% of the activity present in the starting mitochondrial preparation. The malonyl-CoA sensitivity of the reconstituted reactivated M-CPTI was 88%. This is the first demonstration of direct reactivation of malonyl-CoA-sensitive M-CPTI activity from solubilized materials from any organism. Previously, M-CPTI was presumed to be irreversibly inactivated by detergents., (Copyright 1997 Academic Press.)

The rate-limiting step in beta oxidation is the conversion of long-chain acyl-CoA to acylcarnitine, a reaction catalyzed by the outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPTI) and inhibited by malonyl-CoA. The acylcarnitine is then translocated across the inner mitochondrial membrane by the carnitine/acylcarnitine translocase and converted back to acyl-CoA by CPTII. Although CPTII has been examined in detail, studies on CPTI have been hampered by an inability to purify CPTI in an active form from CPTII. In particular, it has not been conclusively demonstrated that CPTI is even catalytically active, or whether sensitivity of CPTI to malonyl-CoA is an intrinsic property of the enzyme or is contained in a separate regulatory subunit that interacts with CPTI. To address these questions, the genes for CPTI and CPTII were separately expressed in Pichia pastoris, a yeast with no endogenous CPT activity. High levels of CPT activity were present in purified mitochondrial preparations from both CPTI- and CPTII-expressing strains. Furthermore, CPTI activity was highly sensitive to inhibition by malonyl-CoA while CPTII was not. Thus, CPT catalytic activity and malonyl-CoA sensitivity are contained within a single CPTI polypeptide in mammalian mitochondrial membranes. We describe the kinetic characteristics for the yeast-expressed CPTs, the first such report for a CPTI enzyme in the absence of CPTII. Yeast-expressed CPTI is inactivated by detergent solubilization. However, removal of the detergent in the presence of phospholipids resulted in the recovery of malonyl-CoA-sensitive CPTI activity, suggesting that CPTI requires a membranous environment. CPTI is thus reversibly inactivated by detergents.

A long-chain fatty acyl CoA photolabel, 2-azido [32P]palmitoyl CoA, was synthesized and its covalent interaction with mitochondrial membrane proteins examined. On binding of 2-azido [32P]palmitoyl CoA to beef heart mitochondria, two polypeptides were primarily labeled, the 30 kDa ADP/ATP carrier and a 41 kDa protein of unknown identity. Carboxyatractyloside and palmitoyl CoA completely protected against labeling of the 30 kDa protein indicating that it was the ADP/ATP carrier. With inverted submitochondrial particles, only the 30 kDa polypeptide was labeled by 2-azido [32P]palmitoyl CoA. The labeling was inhibited by bongkrekic acid and palmitoyl CoA but not carboxyatractyloside, providing evidence that the ADP/ATP carrier was covalently bound from the matrix side of the membrane. In brown adipose tissue mitochondria, 2-azido [32P]palmitoyl CoA photolabeled the ADP/ATP carrier and the 32 kDa uncoupling protein with some minor labeling of 36 and 68 kDa polypeptides. The results indicated that this physiological photolabeling reagent with the azido group on the CoA portion of the molecule interacts like 2-azido ADP with nucleotide binding sites of a number of important enzymes in cell metabolism. Moreover, the evidence strongly supports the hypothesis that long chain fatty acyl CoA esters are natural ligands for key nucleotide binding proteins.

An immunoaffinity column against the 86-kDa malonyl-CoA-binding protein of beef heart mitochondria was prepared, and the properties of the eluates were compared to those of eluates of an anti-carnitine palmitoyltransferase immunoaffinity column. Both eluates contain seven to eight major proteins with a malonyl-CoA-binding capacity of approximately 5 nmol/mg of protein; in contrast, the eluates from a preimmune IgG column did not contain any of the major proteins. The eluates from both immunoaffinity columns conferred malonyl-CoA sensitivity to purified rat heart mitochondrial carnitine palmitoyltransferase (CPTi/CPT-II). Addition of phospholipids increased the degree of malonyl-CoA inhibition. Doubling the amount of column eluate approximately doubled the malonyl-CoA sensitivity when added to a fixed amount of CPT; i.e., the inhibition increased from 32 to 67%. These results show that CPTi/CPT-II is capable of exhibiting malonyl-CoA sensitivity in the presence of malonyl-CoA-binding proteins. The results do not support the concept that the 86-kDa malonyl-CoA-binding protein is detergent-inactivated carnitine palmitoyltransferase I;rather, they suggest that it is a regulatory subunit of a carnitine palmitoyltransferase complex.

Solubilization of rat liver mitochondria in 5% Triton X-100 followed by chromatography on a hydroxylapatite column resulted in the identification of malonyl-CoA binding protein(s) distinct from a major carnitine palmitoyltransferase activity peak. Further purification of the malonyl-CoA binding protein(s) on an acyl-CoA affinity column followed by sodium dodecyl sulfate gel electrophoresis indicated proteins with Mr mass of 90 and 45-33 kDa. A purified liver malonyl-CoA binding fraction, which was devoid of carnitine palmitoyltransferase, and a soluble malonyl-CoA-insensitive carnitine palmitoyltransferase were reconstituted by dialysis in a liposome system. The enzyme activity in the reconstituted system was decreased by 50% in the presence of 100 microM malonyl-CoA. Rat liver mitochondria carnitine palmitoyltransferase may be composed of an easily dissociable catalytic unit and a malonyl-CoA sensitivity conferring regulatory component.