Inhibition of carnitine acetyltransferase by bile acids: implications for carnitine analysis

Carnitine acetyltransferase is used in a radioenzymatic assay to measure the concentration of carnitine. While determining the concentration of carnitine in rat bile, we found that the apparent concentration increased as bile was diluted (6.7 +/- 1.0 and 66.6 +/- 9.4 nmol/ml in undiluted and 20-fold diluted bile, respectively). The present study was designed to investigate whether a component of bile inhibited carnitine acetyltransferase. Inhibition was evaluated by measuring carnitine concentration in bile or by determining the recovery of a known amount of carnitine in the presence of bile. Inhibitory activity was extractable in organic solvents, stable to heat and base treatments, resistant to trypsin and lipase digestions, and removable by cholestyramine, a bile acid-binding resin. These results suggested that the inhibitory activity was associated with bile acids. Direct evidence was obtained by showing a reduced detectability of carnitine in the presence of individual bile acids. Chenodeoxycholic acid was the most potent inhibitor. Inhibition was unrelated to the detergent properties of bile acids. Kinetic studies revealed that carnitine acetyltransferase was inhibited competitively by chenodeoxycholic acid with a Ki of 520 microM. Bile acids also interfered in the quantitation of carnitine in cholestatic plasma. Carnitine concentration in such plasma was underestimated (17.5 +/- 2.1 mmol/ml). Reduction of bile acid concentration by a 20-fold dilution of cholestatic plasma resulted in a 3-fold higher carnitine concentration (54.6 +/- 9.0 nmol/ml). Results demonstrate that, because of the inhibition of carnitine acetyltransferase by bile acids, the radioenzymatic assay will underestimate carnitine concentration in bile or in cholestatic plasma. Accurate measurement requires either the removal of bile acids or a marked reduction in their concentration.     

Redesign of carnitine acetyltransferase specificity by protein engineering

In eukaryotes, L-carnitine is involved in energy metabolism by facilitating beta-oxidation of fatty acids. Carnitine acetyltransferases (CrAT) catalyze the reversible conversion of acetyl-CoA and carnitine to acetylcarnitine and free CoA. To redesign the specificity of rat CrAT toward its substrates, we mutated Met564. The M564G mutated CrAT showed higher activity toward longer chain acyl-CoAs: activity toward myristoyl-CoA was 1250-fold higher than that of the wild-type CrAT, and lower activity toward its natural substrate, acetyl-CoA. Kinetic constants of the mutant CrAT showed modification in favor of longer acyl-CoAs as substrates. In the reverse case, mutation of the orthologous glycine (Gly553) to methionine in carnitine octanoyltransferase (COT) decreased activity toward its natural substrates, medium- and long-chain acyl-CoAs, and increased activity toward short-chain acyl-CoAs. Another CrAT mutant, M564A, was prepared and tested in the same way, with similar results. We conclude that Met564 blocks the entry of medium- and long-chain acyl-CoAs to the catalytic site of CrAT. Three-dimensional models of wild-type and mutated CrAT and COT support this hypothesis. We show for the first time that a single amino acid is able to determine the substrate specificity of CrAT and COT.     

Active-site-directed inhibition of carnitine acetyltransferase

Methoxycarbonyl-CoA disulfide has been used as an active-site-directed inhibitor of carnitine acetyltransferase. Stoichiometric addition of methoxycarbonyl-CoA disulfide to carnitine acetyltransferase showed the modification of one sulfhydryl group with concomitant loss of about 80% enzyme activity. The rate of modification of this sulfhydryl group is an order of magnitude faster than that of the remaining sulfhydryl groups in the enzyme. Methoxycarbonyl-CoA disulfide inactivation is biphasic: k₁ = 1.09 × 10²m^?1s^?1, k₂ = 1.1 × 10¹m^?1s^?1. This modification, Enz-SS-CoA is covalent; it can be reversed with either dithioerythritol or thiocholine. Acetyl-carnitine and acetyl-CoA protected the enzyme against methoxycarbonyl-CoA disulfide inactivation; however, carnitine did not. These results indicate the presence of a sulfhydryl group in carnitine acetyltransferase at the site of acetyl group transfer. Titration of carnitine acetyltransferase with nonspecific sulfhydryl reagents, DTNB, and ?-nitrophenoxycarbonyl methyl disulfide, revealed that four sulfhydryl groups were preferentially modified by these reagents. The results also show that seven other sulfhydryl groups are available for modification.     

pH-dependence of carnitine acetyltransferase activity

1. The pH-dependence of the kinetic constants of the carnitine acetyltransferase reaction has been investigated with the enzyme from pigeon breast muscle. 2. Michaelis constants for (-)-carnitine and acetyl-(-)-carnitine vary in a similar fashion in the pH range 6.0-9.0. A single ionizing group on the enzyme with an apparent pK7.2 is required in the basic form for binding of these substrates. 3. Binding of CoASH or acetyl-CoA raises the apparent pK of an ionizing group on the enzyme from 7.85 to 8.25. This group is probably not directly involved in forming the enzyme-substrate complex, but its microscopic environment is presumably altered. Another group in either the substrate or the free enzyme, with an apparent pK6.4, is needed in the basic form for optimum binding of CoA substrates. 4. This last group has been unequivocally identified as the 3'-phosphate of CoA, by showing that the K(m) of carnitine acetyltransferase for the substrate acetyl-3'-dephospho-CoA is independent of pH in the range 6.0-7.8. 5. V'(max.), the maximum velocity of the catalysed reaction between acetyl-CoA and (-)-carnitine, is constant between pH6.0 and 8.8. 6. The significance of these results in terms of a previously postulated reaction scheme for this enzyme is discussed.     

Induction of carnitine acetyltransferase by clofibrate in rat liver.

Administration of the anti-hypercholesterolaemic drug clofibrate to the rat increases the activity of carnitine acetyltransferase (acetyl-CoA-carnitine O-acetyltransferase, EC 2.3.1.7) in liver and kidney. The drug-mediated increase in enzyme activity in hepatic mitochondria shows a time lag during which the activity increases in the microsomal and peroxisomal fractions. The enzyme induced in the particulate fractions is identical with one normally present in mitochondria. The increase in enzyme activity is prevented by inhibitors of RNA and general protein synthesis. Mitochondrial protein-synthetic machinery does not appear to be involved in the process. Immunoprecipitation shows increased concentration of the enzyme protein in hepatic mitochondria isolated from drug-treated animals. In these animals, the rate of synthesis of the enzyme is increased 7-fold.     

Active-site probes of carnitine acyltransferases. Inhibition of carnitine acetyltransferase by hemiacetylcarnitinium, a reaction intermediate analogue

Hemiacetylcarnitinium (2S,6R:2R,65)-6-carboxymethyl-2-hydroxy-2,4,4- trimethylmorpholinium) chloride is a relatively potent competitive inhibitor (Ki = 0.89 mM) of pigeon breast carnitine acetyltransferase (CAT) and of the crude rat liver CAT (Ki = 4.72 mM) but is neither an inhibitor nor an effective substrate for purified rat liver carnitine palmitoyltransferase (CPT). It does not inhibit state 3 oxygen consumption in isolated hepatic mitochondria using palmitoyl-CoA or palmitoylcarnitine as substrates. This compound is a reaction intermediate analogue of the proposed tetrahedral intermediate for acetyl transfer between acetylcarnitine and CoASH. Because the hemiketal carbon is chiral, a suggestion is made that one of the enantiomers has the same relative configuration as the proposed tetrahedral intermediate.     

Inhibition of carnitine acetyltransferase by mildronate,a regulator of energy metabolism

Carnitine acetyltransferase (CrAT; EC 2.3.1.7) catalyzes the reversible transfer of acetyl groups between acetyl-coenzyme A (acetyl-CoA) and L-carnitine; it also regulates the cellular pool of CoA and the availability of activated acetyl groups. In this study, biochemical measurements, saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy, and molecular docking were applied to give insights into the CrAT binding of a synthetic inhibitor, the cardioprotective drug mildronate (3-(2,2,2-trimethylhydrazinium)-propionate). The obtained results show that mildronate inhibits CrAT in a competitive manner through binding to the carnitine binding site, not the acetyl-CoA binding site. The bound conformation of mildronate closely resembles that of carnitine except for the orientation of the trimethylammonium group, which in the mildronate molecule is exposed to the solvent. The dissociation constant of the mildronate CrAT complex is approximately 0.1?mM, and the K_i is 1.6?mM. The results suggest that the cardioprotective effect of mildronate might be partially mediated by CrAT inhibition and concomitant regulation of cellular energy metabolism pathways.     

The substrate specificity of carnitine acetyltransferase

1. A study of the acyl group specificity of the carnitine acetyltransferase reaction [acyl-(-)carnitine+CoASH right harpoon over left harpoon (-)-carnitine+acyl-CoA] has been made with the enzyme from pigeon breast muscle. Acyl groups containing up to 10 carbon atoms are transferred and detailed kinetic investigations with a range of acyl-CoA and acylcarnitine substrates are reported. 2. Acyl-CoA derivatives with 12 or more carbon atoms in the acyl group are potent reversible inhibitors of carnitine acetyltransferase, competing with acetyl-CoA. Lauroyl- and myristoyl-CoA show a mixed inhibition with respect to (-)-carnitine, but palmitoyl-CoA competes strictly with this substrate also. Palmitoyl-dl-carnitine shows none of these effects. 3. Ammonium palmitate inhibits the enzyme competitively with respect to (-)-carnitine and non-competitively with respect to acetyl-CoA. 4. It is suggested that a hydrophobic site exists on the carnitine acetyltransferase molecule. The hydrocarbon chain of an acyl-CoA derivative containing eight or more carbon atoms in the acyl group may interact with this, which results in enhanced acyl-CoA binding. Competition occurs between ligands bound to this hydrophobic site and the carnitine binding site. 5. The possible physiological significance of long-chain acyl-CoA inhibition of this enzyme is discussed.     

Characterization of a soluble carnitine acetyltransferase from Candida tropicalis

Carnitine acetyltransferase was purified from the cytoplasmic fraction of Candida tropicalis grown on alkanes in continuous culture. By ion-exchange chromatography the enzyme was resolved in two fractions with the same specific activity of 80 U/mg. The molecular mass of both enzyme forms, determined by non-denaturing gradient gel electrophoresis, was 540 kDa. After SDS electrophoresis only one band of 64 kDa was detected indicating that both enzymes are oligomers each containing eight subunits. Isoelectric focusing in agarose under non-denaturing conditions demonstrated the presence of at least four different charged species in the pH range between 5.6 and 6.7. After isoelectric focusing in 9 M urea/1% Nonidet P-40 gels, both enzyme forms were resolved into four bands. Peptide mapping, performed by cyanogen bromide cleavage of polypeptides separated by denaturing isoelectric focusing followed by second-dimension SDS electrophoresis, revealed a very high degree of homology between these polypeptides. The presence of the octameric form of carnitine acetyltransferase already in the starting material was demonstrated by non-denaturing gradient gel electrophoresis and immunoblotting. Antibodies against carnitine acetyltransferase from C. tropicalis ATCC 32113 formed precipitation lines with extracts from several Candida species but not with extracts of Candida utilis, Candida ethanothermophilum and an another strain of C. tropicalis.     

Inhibition of carnitine acetyltransferase by metabolites of 4-pentenoic acid

The inhibition of carnitine acetyltransferase (EC 2.3.1.7) by metabolites of 4-pentenoic acid was studied. 3-Keto-4-pentenoyl-CoA, a beta-oxidation metabolite of 4-pentenoic acid, was found to be an effective inhibitor of the enzyme in the presence, but not in the absence of L-carnitine. Since acetyl-CoA protects the enzyme against this inhibition, 3-keto-4-pentenoyl-CoA seems to be an active site-directed inhibitor. 3-Keto-4-pentenoyl-CoA, which is a substrate of carnitine acetyltransferase, causes the irreversible inactivation of the enzyme. All observations together lead to the suggestion that 3-keto-4-pentenoyl-CoA is a mechanism-based inhibitor of carnitine acetyltransferase.     

Mutagenesis of specific amino acids converts carnitine acetyltransferase into carnitine palmitoyltransferase

Carnitine acyltransferases catalyze the exchange of acyl groups between carnitine and CoA. The members of the family can be classified on the basis of their acyl-CoA selectivity. Carnitine acetyltransferases (CrATs) are very active toward short-chain acyl-CoAs but not toward medium- or long-chain acyl-CoAs. Previously, we identified an amino acid residue (Met(564) in rat CrAT) that was critical to fatty acyl-chain-length specificity. M564G-mutated CrAT behaved as if its natural substrates were medium-chain acyl-CoAs, similar to that of carnitine octanoyltransferase (COT). To extend the specificity of rat CrAT to other substrates, we have performed new mutations. Using in silico molecular modeling procedures, we have now identified a second putative amino acid involved in acyl-CoA specificity (Asp(356) in rat CrAT). The double CrAT mutant D356A/M564G showed 6-fold higher activity toward palmitoyl-CoA than that of the single CrAT mutant M564G and a new activity toward stearoyl-CoA. We show that by performing two amino acid replacements a CrAT can be converted into a pseudo carnitine palmitoyltransferase (CPT) in terms of substrate specificity. To change CrAT specificity from carnitine to choline, we also prepared a mutant CrAT that incorporates four amino acid substitutions (A106M/T465V/T467N/R518N). The quadruple mutant shifted the catalytic discrimination between l-carnitine and choline in favor of the latter substrate and showed a 9-fold increase in catalytic efficiency toward choline compared with that of the wild-type. Molecular in silico docking supports kinetic data for the positioning of substrates in the catalytic site of CrAT mutants.     

Metabolic roles of carnitine expressed through the carnitine acetyltransferase system in Candida pintolopesii

• 1.1. The carnitine-responsive mutant yeast, Candida pintolopesii ATCC 26014 and the wild type strain (ATCC 22987) were used to investigate the role of carnitine and the carnitine acetyltransferase system.
• 2.2. [³H]l-Carnitine, supplied to the cells, was incorporated into acetylcamitine and [¹⁴C]pantothenate was incorporated into CoA and its derivatives.
• 3.3. Both bioautography and quantitative assays indicated that the relative amounts of CoA and acetylCoA were very different in the mutant and wild type cells.
• 4.4. The wild type yeast maintained an acetylCoA/CoA ratio of 0.33 ± 0.09 indicating that most of the CoA in the cell is in the free CoA form. Carnitine was not required to establish this ratio nor did its presence lower it further.
• 5.5. In contrast, the mutant cells contained a high acetylCoA/CoA ratio (12.8 ± 3.0).
• 6.6. In the mutant cells, carnitine lowered the ratio by decreasing the intracellular acetylCoA concentration and releasing free CoA.
• 7.7. These data indicated that wild type yeast possess an effective mechanism that is not related to the CAT system for regulating the acetylCoA/CoA ratio.
• 8.8. This mechanism appears to be lacking in the mutant. The CAT system decreased the acetylCoA/CoA ratio in the mutant cells but not to the value which is found in the wild type strain.
• 9.9. In both stains of Candida pintolopesii, in the presence of carnitine, an acetylcamitine pool can be created whose concentration exceeds that of acetylCoA.
• 10.10. The intracellular apparent equilibrium constant (K_app) for carnitine acetyltransferase for wild type Candida pintolopesii ATCC 22987 was 0.73 ± 0.12, close to the established value of 0.6, indicating that the CAT system ran close to equilibrium.
• 11.11. The K_app for the CAT system of the carnitine-responsive mutant yeast was 7.7 ± 1.7 indicating that this reaction was not at equilibrium.

     

Seminal carnitine and acetylcarnitine content and carnitine acetyltransferase activity in young Maremmano stallions

The reproductive characteristics and seminal carnitine and acetylcarnitine content as well as carnitine acetyltransferase activity of young Maremmano stallions (n=25) are reported. The stallions were subjected to semen collection in November and January; in each trial two ejaculates were collected 1h apart. The total motile morphologically normal spermatozoa (TMMNS) and the progressively motile spermatozoa at collection and during storage at +4 degrees C were evaluated. Seminal L-carnitine (LC), acetylcarnitine (AC), pyruvate and lactate were measured using spectrophotometric methods, whereas carnitine acetyltransferase activity was measured by radioenzymatic methods. Since there were no major significant differences in seminal and biochemical characteristics between the November and January trials, data were also pooled for the first and second ejaculates. Significant differences (P<0.001) were observed between the first and second ejaculates for sperm count (0.249+/-0.025 versus 0.133+/-0.014x10(9)/ml), total number spermatozoa by ejaculate (12.81+/-1.23 versus 6.36+/-0.77x10(9)), progressively motile spermatozoa (48.6+/-3.0 versus 52.6+/-3.0%) and TMMNS (3.35+/-0.50 versus 2.02+/-0.37x10(9)). In the raw semen the LC and AC were significantly higher in the first ejaculate than in the second (P<0.001), whereas, pyruvate and pyruvate/lactate ratio were higher in the second ejaculate (P<0.05). Seminal plasma AC and LC concentrations resulted higher in the first ejaculate (P<0.001). The pyruvate/lactate ratio was higher in the second ejaculate (P<0.05). Both raw semen and seminal plasma LC and AC concentrations were positively correlated with spermatozoa concentration (P<0.01); in raw semen AC was also correlated to TMMNS (P<0.01). Lactate levels of raw semen was correlated to progressively motile spermatozoa after storage (P<0.01). In the second ejaculate, significant correlations were also observed among AC/LC ratio in raw semen and progressively motile spermatozoa after 48 and 72h of refrigeration. Furthermore, AC levels were correlated to lactate concentration. The positive correlation between LC, AC and spermatozoa concentration, and between AC and TMMNS indicated carnitine as potential semen quality marker. Moreover, the correlation between AC/LC ratio and progressive spermatozoa motility after refrigeration, suggests that carnitine may contribute towards improving the maintenance of spermatozoa viability during in vitro storage.     

Stimulation of carnitine acetyltransferase in PC12 cells by nerve growth factor: Relationship to choline acetyltransferase stimulation

The activity of carnitine acetyltransferase (acetyl-CoA:L-carnitine O-acetyltransferase) was found to be at least 50-fold higher than that of choline acetyltransferase in PC12 cells. Nerve growth factor stimulated both enzymes in a parallel manner with respect to concentration of NGF and culture time. The stimulation of both enzymes was completely inhibited by 10 M 6-thioguanine, an inhibitor of protein kinase N. Results are discussed with reference to the hypothesis that the two enzymes may be functionally related in neuronal cells.     

Biosynthesis and turnover of carnitine acetyltransferase in rat liver

Male Wistar rats were fed on a diet with and without di(2-ethylhexyl)phthalate (DEHP) for 2 weeks. Carnitine acetyltransferase in the liver was increased about 100-fold by administration of DEHP. The results of in vivo experiments showed that the incorporation of L-[4,5-3H]leucine into the enzyme was 12-fold higher and the half-life of the labeled enzyme was elongated by a factor 4.6. The results of in vitro translation experiments with total hepatic RNA in a rabbit reticulocytelysate system and the results concerning the synthesis of the enzyme in isolated hepatocytes indicate that the translatable mRNA for the enzyme was increased upon administration of DEHP and that the enzyme is synthesized as a precursor (Mw = 69,000) larger than the mature enzyme (Mw = 67,500). RNA in the free polysomes directed the synthesis of the enzyme precursor five times more actively than RNA in membrane-bound polysomes.     

The sidedness of carnitine acetyltransferase and carnitine octanoyltransferase of rat liver endoplasmic reticulum

The location of carnitine acetyltransferase and carnitine octanoyltransferase on the inner and outer surfaces of rat liver microsomes was investigated. Latency of mannose-6-phosphate phosphatase showed that the microsomes were 90–94% sealed. All of the octanoyltransferase is associated with the cytosolic face, while the acetyltransferase is distributed between the cytosolic face (68–73%) and the lumen face (27–32%) of the endoplasmic reticulum membrane. Small amounts of trypsin inhibit the carnitine octanoyltransferase equally in either sealed or permeable microsomes but the acetyltransferase of sealed microsomes is stimulated. Large amounts of trypsin inhibit all transferase activities by about 60%, except for acetyltransferase of sealed microsomes. Other studies show that 0.1% Triton X-100 partially inhibits carnitine octanoyltransferase of microsomes but does not inhibit the acetyltransferase or any of the mitochondrial carnitine acyltransferase.     

Facile alkylation of methionine by benzyl bromide and demonstration of fumarase inactivation accompanied by alkylation of a methionine residue.

Benzyl bromide is a selective alkylator of sulfur nucleophiles including methionine and cysteine. Only the mercaptide ion is a more efficient nucleophile than is the sulfur ether of methionine. Alkylation rates relative to methionine are 200: less than or equal to 0.03: less than or equal to 0.03: less than or equal to 0.02 for GS-, histidine, tryptophan, and GSH, respectively. Alkylation of methionine by benzyl bromide is more than 50 times faster than alkylation by iodoacetate. Fumarase is readily inactivated by exposure to benzyl bromide at pH 6.6 to 6.8 accompanied by alkylation of close to 1 methionine residue/subunit. Fumarase fully inactivated by exposure to benzyl bromide shows no detected alkylation of amino acid residues other than methionine. The rate of inactivation of fumarase by benzyl bromide is decreased about 4-fold by the presence of excess substrates. Denaturation of fumarase in 6 M urea at pH 6.5 exposes additional methionine as well as cysteine residues to alkylation.