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1.
Cordente AG López-Viñas E Vázquez MI Gómez-Puertas P Asins G Serra D Hegardt FG 《Biochemistry》2006,45(19):6133-6141
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. 相似文献
2.
Carnitine acyltransferases catalyze the reversible exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids in the mitochondria for energy production, and are attractive targets for drug discovery against diabetes and obesity. To help define in molecular detail the catalytic mechanism of these enzymes, we report here the high resolution crystal structure of wild-type murine carnitine acetyltransferase (CrAT) in a ternary complex with its substrates acetyl-CoA and carnitine, and the structure of the S554A/M564G double mutant in a ternary complex with the substrates CoA and hexanoylcarnitine. Detailed analyses suggest that these structures may be good mimics for the Michaelis complexes for the forward and reverse reactions of the enzyme, representing the first time that such complexes of CrAT have been studied in molecular detail. The structural information provides significant new insights into the catalytic mechanism of CrAT and possibly carnitine acyltransferases in general. 相似文献
3.
Sara Violante Lodewijk IJlst Jos Ruiter Janet Koster Henk van Lenthe Marinus Duran Isabel Tavares de Almeida Ronald J.A. Wanders Sander M. Houten Fátima V. Ventura 《生物化学与生物物理学报:疾病的分子基础》2013,1832(6):773-779
Carnitine acyltransferases catalyze the reversible conversion of acyl-CoAs into acylcarnitine esters. This family includes the mitochondrial enzymes carnitine palmitoyltransferase 2 (CPT2) and carnitine acetyltransferase (CrAT). CPT2 is part of the carnitine shuttle that is necessary to import fatty acids into mitochondria and catalyzes the conversion of acylcarnitines into acyl-CoAs. In addition, when mitochondrial fatty acid β-oxidation is impaired, CPT2 is able to catalyze the reverse reaction and converts accumulating long- and medium-chain acyl-CoAs into acylcarnitines for export from the matrix to the cytosol. However, CPT2 is inactive with short-chain acyl-CoAs and intermediates of the branched-chain amino acid oxidation pathway (BCAAO). In order to explore the origin of short-chain and branched-chain acylcarnitines that may accumulate in various organic acidemias, we performed substrate specificity studies using purified recombinant human CrAT. Various saturated, unsaturated and branched-chain acyl-CoA esters were tested and the synthesized acylcarnitines were quantified by ESI-MS/MS. We show that CrAT converts short- and medium-chain acyl-CoAs (C2 to C10-CoA), whereas no activity was observed with long-chain species. Trans-2-enoyl-CoA intermediates were found to be poor substrates for this enzyme. Furthermore, CrAT turned out to be active towards some but not all the BCAAO intermediates tested and no activity was found with dicarboxylic acyl-CoA esters. This suggests the existence of another enzyme able to handle the acyl-CoAs that are not substrates for CrAT and CPT2, but for which the corresponding acylcarnitines are well recognized as diagnostic markers in inborn errors of metabolism. 相似文献
4.
Carnitine acyltransferases catalyze the exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids, and are attractive targets for drug discovery against diabetes and obesity. These enzymes are classified based on their substrate selectivity for short-chain, medium-chain, or long-chain fatty acids. Structural information on carnitine acetyltransferase suggests that residues Met-564 and Phe-565 may be important determinants of substrate selectivity with the side chain of Met-564 located in the putative binding pocket for acyl groups. Both residues are replaced by glycine in carnitine palmitoyltransferases. To assess the functional relevance of this structural observation, we have replaced these two residues with small amino acids by mutagenesis, characterized the substrate preference of the mutants, and determined the crystal structures of two of these mutants. Kinetic studies confirm that the M564G or M564A mutation is sufficient to increase the activity of the enzyme toward medium-chain substrates with hexanoyl-CoA being the preferred substrate for the M564G mutant. The crystal structures of the M564G mutant, both alone and in complex with carnitine, reveal a deep binding pocket that can accommodate the larger acyl group. We have determined the crystal structure of the F565A mutant in a ternary complex with both the carnitine and CoA substrates at a 1.8-A resolution. The F565A mutation has minor effects on the structure or the substrate preference of the enzyme. 相似文献
5.
Sarah E. Seiler Ola J. Martin Robert C. Noland Dorothy H. Slentz Karen L. DeBalsi Olga R. Ilkayeva Jie An Christopher B. Newgard Timothy R. Koves Deborah M. Muoio 《Journal of lipid research》2014,55(4):635-644
Carnitine acetyltransferase (CrAT) is a mitochondrial matrix enzyme that catalyzes the interconversion of acetyl-CoA and acetylcarnitine. Emerging evidence suggests that this enzyme functions as a positive regulator of total body glucose tolerance and muscle activity of pyruvate dehydrogenase (PDH), a mitochondrial enzyme complex that promotes glucose oxidation and is feedback inhibited by acetyl-CoA. Here, we used tandem mass spectrometry-based metabolic profiling to identify a negative relationship between CrAT activity and muscle content of lipid intermediates. CrAT specific activity was diminished in muscles from obese and diabetic rodents despite increased protein abundance. This reduction in enzyme activity was accompanied by muscle accumulation of long-chain acylcarnitines (LCACs) and acyl-CoAs and a decline in the acetylcarnitine/acetyl-CoA ratio. In vitro assays demonstrated that palmitoyl-CoA acts as a direct mixed-model inhibitor of CrAT. Similarly, in primary human myocytes grown in culture, nutritional and genetic manipulations that promoted mitochondrial influx of fatty acids resulted in accumulation of LCACs but a pronounced decrease of CrAT-derived short-chain acylcarnitines. These results suggest that lipid-induced antagonism of CrAT might contribute to decreased PDH activity and glucose disposal in the context of obesity and diabetes. 相似文献
6.
Purification and properties of carnitine octanoyltransferase and carnitine palmitoyltransferase from rat liver 总被引:18,自引:0,他引:18
The activities of carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) in rat liver were markedly increased by administration of di(2-ethyl-hexyl)phthalate. COT and CPT were purified from the enzyme-induced rat liver. COT was a 66,000-dalton polypeptide. The molecular weight of native CPT was 280,000--320,000 daltons, and the enzyme consisted of 69,200-dalton polypeptides. CAT, COT, and CPT were immunologically different. COT exhibited activity with all of the substrates tested (acyl-CoA's and acylcarnitines of saturated fatty acids having carbon chain lengths of C2--C20), though maximum activity was observed with hexanoyl derivatives. CPT exhibited catalytic activity with medium- and long-chain acyl derivatives. 2-Bromo-palmitoyl-CoA inactivated COT but not CPT. Malonyl-CoA inhibited CPT but not COT. CPT was confined to mitochondria, whereas COT was found in peroxisomes and the soluble compartment but not in mitochondria. 相似文献
7.
Montserrat Morillas Paulino Gómez-Puertas Blanca Rubí Josep Clotet Joaquín Ari?o Alfonso Valencia Fausto G Hegardt Dolors Serra Guillermina Asins 《The Journal of biological chemistry》2002,277(13):11473-11480
Carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) I, which facilitate the transport of medium- and long-chain fatty acids through the peroxisomal and mitochondrial membranes, are physiologically inhibited by malonyl-CoA. Using an "in silico" macromolecular docking approach, we built a model in which malonyl-CoA could be attached near the catalytic core. This disrupts the positioning of the acyl-CoA substrate in the channel in the model reported for both proteins (Morillas, M., Gómez-Puertas, P., Roca, R., Serra, D., Asins, G., Valencia, A., and Hegardt, F. G. (2001) J. Biol. Chem. 276, 45001-45008). The putative malonyl-CoA domain contained His(340), implicated together with His(131) in COT malonyl-CoA sensitivity (Morillas, M., Clotet, J., Rubi, B., Serra, D., Asins, G., Ari?o, J., and Hegardt F. G. (2000) FEBS Lett. 466, 183-186). When we mutated COT His(131) the IC(50) increased, and malonyl-CoA competed with the substrate decanoyl-CoA. Mutation of COT Ala(332), present in the domain 8 amino acids away from His(340), decreased the malonyl-CoA sensitivity of COT. The homologous histidine and alanine residues of L-CPT I, His(277), His(483), and Ala(478) were also mutated, which decreased malonyl-CoA sensitivity. Natural mutation of Pro(479), which is also located in the malonyl-CoA predicted site, to Leu in a patient with human L-CPT I hereditary deficiency, modified malonyl-CoA sensitivity. We conclude that this malonyl-CoA domain is present in both COT and L-CPT I proteins and might be the site at which malonyl-CoA interacts with the substrate acyl-CoA. Other malonyl-CoA non-inhibitable members of the family, CPT II and carnitine acetyltransferase, do not contain this domain. 相似文献
8.
The activities of peroxisomal and mitochondrial beta-oxidation and carnitine acyltransferases changed during the process of development from embryo to adult chicken, and the highest activities of peroxisomal beta-oxidation, palmitoyl-CoA oxidase, and carnitine acetyltransferase were found at the hatching stage of the embryo. The profiles of these alterations were in agreement with those of the contents of triglycerides and free fatty acids in the liver. The highest activities of mitochondrial beta-oxidation and palmitoyl-CoA dehydrogenase were observed at the earlier stages of the embryo; then the activities decreased gradually from embryo to adult chicken. The ratio of activities of carnitine acetyltransferase in peroxisomes and mitochondria (peroxisomes/mitochondria) increased from 0.54 to 0.82 during the development from embryo to adult chicken. The ratio of activities of carnitine palmitoyltransferase decreased from 0.82 to 0.25 during the development. The affinity of fatty acyl-CoA dehydrogenase toward the medium-chain acyl-CoAs (C6 and C8) was high in the embryo and decreased with development, whereas the substrate specificity of fatty acyl-CoA oxidase did not change. The substrate specificity of mitochondrial carnitine acyltransferases did not change with development. The affinity of peroxisomal carnitine acyltransferases toward the long-chain acyl-CoAs (C10 to C16) was high in the embryo, but low in adult chicken. 相似文献
9.
K Carpenter R J Pollitt B Middleton 《Biochemical and biophysical research communications》1992,183(2):443-448
We have purified to homogeneity the long-chain specific 3-hydroxyacyl-CoA dehydrogenase from mitochondrial membranes of human infant liver. The enzyme is composed of non-identical subunits of 71 kDa and 47 kDa within a native structure of 230 kDa. The pure enzyme is active with 3-ketohexanoyl-CoA and gives maximum activity with 3-ketoacyl-CoA substrates of C10 to C16 acyl-chain length but is inactive with acetoacetyl-CoA. In addition to 3-hydroxyacyl-CoA dehydrogenase activity, the enzyme possesses 2-enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase activities which cannot be separated from the dehydrogenase. None of these enzymes show activity with C4 substrates but all are active with C6 and longer acyl-chain length substrates. They are thus distinct from any described previously. This human liver mitochondrial membrane-bound enzyme catalyses the conversion of medium- and long-chain 2-enoyl-CoA compounds to: 1) 3-ketoacyl-CoA in the presence of NAD alone and 2) to acetyl-CoA (plus the corresponding acyl-CoA derivatives) in the presence of NAD and CoASH. It is therefore a multifunctional enzyme, resembling the beta-oxidation enzyme of E. coli, but unique in its membrane location and substrate specificity. We propose that its existence explains the repeated failure to detect any intermediates of mitochondrial beta-oxidation. 相似文献
10.
During the glyoxysomal β-oxidation of long-chain acyl-CoAs, short-chain intermediates accumulate transiently (Kleiter and
Gerhardt 1998, Planta 206: 125–130). The studies reported here address the underlying factors. The studies concentrated upon
the aspects of (i) chain length specificity and (ii) metabolic regulation of the glyoxysomal β-oxidation of sunflower (Helianthus annuus L.) cotyledons. (i) Concentration-rate curves of the β-oxidation of acyl-CoAs of various chain lengths showed that the β-oxidation
activity towards long-chain acyl-CoAs was higher than that towards short-chain acyl-CoAs at substrate concentrations <20 μM.
At substrate concentrations >20 μM, long-chain acyl-CoAs were β-oxidized more slowly than short-chain acyl-CoAs because the
β-oxidation of long-chain acyl-CoAs is subject to substrate inhibition which had already started at 5–10 μM substrate concentration
and results from an inhibition of the multifunctional protein (MFP) of the β-oxidation reaction sequence. However, low concentrations
of free long-chain acyl-CoAs are rather likely to exist within the glyoxysomes due to the acyl-CoA-binding capacity of proteins.
Consequently, the β-oxidation rate towards a parent long-chain acyl-CoA will prevail over that towards the short-chain intermediates.
(ii) Low concentrations (≤5 μM) of a long-chain acyl-CoA exerted an inhibitory effect on the β-oxidation rate of butyryl-CoA.
Reversibility of the inhibition was observed as well as metabolization of the inhibiting long-chain acyl-CoA. Regarding the
activities of the individual β-oxidation enzymes towards their C4 substrates in the presence of a long-chain acyl-CoA, the MFP activity exhibited strong inhibition. This inhibition appears
not to be due to the detergent-like physical properties of long-chain acyl-CoAs. The results of the studies, which are consistent
with the observation that short-chain intermediates accumulate transiently during complete degradation of a long-chain acyl-CoA,
suggest that the substrate concentration-dependent chain-length specificity of the β-oxidation and a metabolic regulation
at the level of MFP are factors determining this transient accumulation.
Received: 2 February 1999 / Accepted: 14 April 1999 相似文献
11.
S Ferdinandusse J Mulders L IJlst S Denis G Dacremont H R Waterham R J Wanders 《Biochemical and biophysical research communications》1999,263(1):213-218
To study the putative role of human carnitine octanoyltransferase (COT) in the beta-oxidation of branched-chain fatty acids, we identified and cloned the cDNA encoding human COT and expressed it in the yeast Saccharomyces cerevisiae. Enzyme activity measurements showed that COT efficiently converts one of the end products of the peroxisomal beta-oxidation of pristanic acid, 4, 8-dimethylnonanoyl-CoA, to its corresponding carnitine ester. Production of the carnitine ester of this branched/medium-chain acyl-CoA within the peroxisome is required for its transport to the mitochondrion where further beta-oxidation occurs. In contrast, 4, 8-dimethylnonanoyl-CoA is not a substrate for carnitine acetyltransferase, another acyltransferase localized in peroxisomes, which catalyzes the formation of carnitine esters of the other products of pristanic acid beta-oxidation, namely acetyl-CoA and propionyl-CoA. Our results shed new light on the function of COT in fatty acid metabolism and point to a crucial role of COT in the beta-oxidation of branched-chain fatty acids. 相似文献
12.
Quantitation of the effect of L-carnitine on the levels of acid-soluble short-chain acyl-CoA and CoASH in rat heart and liver mitochondria 总被引:4,自引:0,他引:4
W Lysiak K Lilly F DiLisa P P Toth L L Bieber 《The Journal of biological chemistry》1988,263(3):1151-1156
The steady state levels of mitochondrial acyl-CoAs produced during the oxidation of pyruvate, alpha-ketoisovalerate, alpha-ketoisocaproate, and octanoate during state 3 and state 4 respiration by rat heart and liver mitochondria were determined. Addition of carnitine lowered the amounts of individual short-chain acyl-CoAs and increased CoASH in a manner that was both tissue- and substrate-dependent. The largest effects were on acetyl-CoA derived from pyruvate in heart mitochondria using either state 3 or state 4 oxidative conditions. Carnitine greatly reduced the amounts of propionyl-CoA derived from alpha-ketoisovalerate, while smaller effects were obtained on the branched-chain acyl-CoA levels, consistent with the latter acyl moieties being poorer substrates for carnitine acetyltransferase and also poorer substrates for the carnitine/acylcarnitine translocase. The levels of acetyl-CoA in heart and liver mitochondria oxidizing octanoate during state 3 respiration were lower than those obtained with pyruvate. The rate of acetylcarnitine efflux from heart mitochondria during state 3 (with pyruvate or octanoate as substrate, in the presence or absence of malate with 0.2 mM carnitine) shows a linear response to the acetyl-CoA/CoASH ratio generated in the absence of carnitine. This relationship is different for liver mitochondria. These data demonstrate that carnitine can modulate the aliphatic short-chain acyl-CoA/CoA ratio in heart and liver mitochondria and indicate that the degree of modulation varies with the aliphatic acyl moiety. 相似文献
13.
Carnitine-acyltransferase activity assayed with acetyl-CoA, octanoyl-CoA, or palmitoyl-CoA is associated with the mitochondrial but not with the peroxisomes of mung-bean hypocotyls. Using mitochondria as an enzyme source, a half-maximal reaction rate is obtained with a palmitoyl-CoA concentration approximately twice that required with acetyl-CoA. In the presence of a saturating acetyl-CoA concentration the carnitine-acyltransferase activity is not enhanced by palmitoyl-CoA as additional substrate. However, palmitoylcarnitine is formed in addition to acetylcarnitine, and the formation of acetylcarnitine is competitively inhibited by palmitoyl-CoA. It is concluded that the mitochondria of mung-bean hypocotyls possess a carnitine acyltransferase of broad substrate specificity with respect to the chainlength of the acyl-CoA and that the demonstration of a carnitine-palmitoyltransferase activity in plant mitochondria does not indicate the presence of a specific carnitine long-chain acyltransferase. 相似文献
14.
Sara Violante Lodewijk IJlst Henk van Lenthe Isabel Tavares de Almeida Ronald J. Wanders Fátima V. Ventura 《生物化学与生物物理学报:疾病的分子基础》2010,1802(9):728-732
Over the last years acylcarnitines have emerged as important biomarkers for the diagnosis of mitochondrial fatty acid β-oxidation (mFAO) and branched-chain amino acid oxidation disorders assuming they reflect the potentially toxic acyl-CoA species, accumulating intramitochondrially upstream of the enzyme block. However, the origin of these intermediates still remains poorly understood. A possibility exists that carnitine palmitoyltransferase 2 (CPT2), member of the carnitine shuttle, is involved in the intramitochondrial synthesis of acylcarnitines from accumulated acyl-CoA metabolites. To address this issue, the substrate specificity profile of CPT2 was herein investigated. Saccharomyces cerevisiae homogenates expressing human CPT2 were incubated with saturated and unsaturated C2–C26 acyl-CoAs and branched-chain amino acid oxidation intermediates. The produced acylcarnitines were quantified by ESI-MS/MS. We show that CPT2 is active with medium (C8–C12) and long-chain (C14–C18) acyl-CoA esters, whereas virtually no activity was found with short- and very long-chain acyl-CoAs or with branched-chain amino acid oxidation intermediates. Trans-2-enoyl-CoA intermediates were also found to be poor substrates for CPT2. Inhibition studies performed revealed that trans-2-C16:1-CoA may act as a competitive inhibitor of CPT2 (Ki of 18.8 μM). The results obtained clearly demonstrate that CPT2 is able to reverse its physiological mechanism for medium and long-chain acyl-CoAs contributing to the abnormal acylcarnitines profiles characteristic of most mFAO disorders. The finding that trans-2-enoyl-CoAs are poorly handled by CPT2 may explain the absence of trans-2-enoyl-carnitines in the profiles of mitochondrial trifunctional protein deficient patients, the only defect where they accumulate, and the discrepancy between the clinical features of this and other long-chain mFAO disorders such as very long-chain acyl-CoA dehydrogenase deficiency. 相似文献
15.
Luo YS Nicaud JM Van Veldhoven PP Chardot T 《Archives of biochemistry and biophysics》2002,407(1):32-38
One of the acyl-CoA oxidases from the yeast Yarrowia lipolytica, acyl-CoA oxidase 2 (Aox2p), has been expressed in Escherichia coli as an active, N-terminally tagged (His)(6) fusion protein. The specific activity of the purified enzyme, containing FAD, was 19.7 micromolmin(-1)mg(-1) using myristoyl-CoA as substrate. Using substrates with different chain lengths and different substituents, its kinetic properties were further analyzed. Straight-chain acyl-CoAs, with a chain length of 10-14C, are well oxidized, reflecting the properties of Aox2p as deduced from in vivo studies. Acyl-CoAs containing more than 14C were also desaturated, if their concentration was below 25 microM or if proteins capable of binding these CoA-esters, such as albumin or beta-casein, were added to the assay. These long-chain acyl-CoAs, although poor substrates, acted as competitors for the short- and medium-chain substrates. Compared to palmitoyl-CoA, activity toward hexadecadioyl-CoA, containing a omega-carboxy group, was similar. Taken together, these data suggest that micelles of long-chain acyl-CoAs are able to bind and inhibit Aox2p. The enzyme was also active toward acyl-CoA-esters containing a 2-methyl group, but only the 2S isomer was recognized. 相似文献
16.
Computer-based approaches identified PTE2 as a candidate human peroxisomal acyl-CoA thioesterase gene. The PTE2 gene product is highly similar to the rat cytosolic and mitochondrial thioesterases, CTE1 and MTE1, respectively, and terminates in a tripeptide sequence, serine-lysine-valine(COOH), that resembles the consensus sequence for type-1 peroxisomal targeting signals. PTE2 was targeted to peroxisomes and recombinant PTE2 showed intrinsic acyl-CoA thioesterase activity with a pH optimum of 8.5. A comparison of PTE2 and PTE1 thioesterase activities across multiple acyl-CoA substrates indicated that while PTE1 was most active on medium-chain acyl-CoAs, with little activity on long-chain acyl-CoAs, PTE2 displayed high activity on medium- and long-chain acyl-CoAs. The identification of PTE2 therefore offers an explanation for the observed long-chain acyl-CoA thioesterase activity of mammalian peroxisomes. 相似文献
17.
Carnitine octanoyltransferase of mouse liver peroxisomes: properties and effect of hypolipidemic drugs 总被引:2,自引:0,他引:2
Carnitine octanoyltransferase (COT) in 500g supernatant fluids from mouse liver has a specific activity at least twice that of carnitine acetyltransferase (CAT) or carnitine palmitoyltransferase (CPT). When mice are fed diets containing the lipid-lowering drugs, clofibrate or nafenopin, the specific activity of COT increases 4- and 11-fold, respectively. Liver homogenates from mice fed a control diet, and diets containing clofibrate, nafenopin, or Wy-14,643 were fractionated by sucrose gradient centrifugation, and the subcellular distribution of carnitine acyltransferases was determined. In the controls, peroxisomes contained about 70% of the total COT. The specific activity of COT in the peroxisomal peak was 12-fold greater than either CAT or CPT, and 20-fold greater than the COT activity in the mitochondrial fraction. Treatment with hypolipidemic drugs increased the specific activity of peroxisomal COT 2- to 3-fold and CAT 6- to 12-fold, while mitochondrial COT increased 5- to 11-fold and CAT 19- to 54-fold. COT was purified to homogeneity from livers of mice treated with Wy-14,643. It had an apparent Mr of 60,000 by Sephadex G-100 and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and a maximum activity for octanoyl-CoA with acetyl-CoA and palmitoyl-CoA having activities of 2 and 10%, respectively, when 100 microM acyl-CoA substrates were used. The Km's for 1-carnitine, octanoyl-CoA, palmitoyl-CoA, and acetyl-CoA were 130, 15, 69, and 155 microM, respectively, in the forward direction; and in the reverse direction were 110, 100, 104, and 783 microM for CoASH, octanoylcarnitine, palmitoylcarnitine, and acetylcarnitine, respectively. With Vmax conditions, acetyl-CoA and palmitoyl-CoA had activities of 8 and 26% of the activity for octanoyl-CoA, and acetylcarnitine and palmitoylcarnitine had activities of 7 and 22%, respectively, of the activity for octanoylcarnitine. It is concluded that COT is a separate enzyme present in large amounts in the matrix of mouse liver peroxisomes, with kinetic properties that greatly favor medium-chain acylcarnitine formation. 相似文献
18.
K Izai Y Uchida T Orii S Yamamoto T Hashimoto 《The Journal of biological chemistry》1992,267(2):1027-1033
Freeze-thawed rat liver mitochondria were extensively washed with potassium phosphate, pH 7.5, and the residue was extracted with 10 mM potassium phosphate, pH 7.5, 1% (w/v) sodium cholate, 0.5 M KCl. The four beta-oxidation enzyme activities of the washes and the last extract were assayed with substrates of various carbon chain lengths. Our data suggest that the last extract contains a novel acyl-CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase. A novel acyl-CoA dehydrogenase was purified. The molecular masses of the native enzyme and the subunit were estimated to be 150 and 71 kDa, respectively. One mole of enzyme contained 2 mole of FAD. These properties and immunochemical properties of the enzyme differed from those of three other acyl-CoA dehydrogenases: short-, medium-, and long-chain acyl-CoA dehydrogenases. Carbon chain length specificity of the enzyme differed from that of other acyl-CoA dehydrogenases. The enzyme was active toward CoA esters of long- and very-long-chain fatty acids, but not toward those of medium- and short-chain fatty acids. The specific enzyme activity was greater than 10 times that of long-chain acyl-CoA dehydrogenase when palmitoyl-CoA was used as substrate. We propose the name "very-long-chain acyl-CoA dehydrogenase" for this enzyme. 相似文献
19.
《Journal of enzyme inhibition and medicinal chemistry》2013,28(6):1269-1275
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 Ki 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. 相似文献
20.
Hepatic peroxisomes and mitochondria from 20-day-old chick embryo were separated by sucrose density gradient centrifugation and the characteristics of carnitine acyltransferases in these organelles were studied. The carnitine acyltransferase activities in peroxisomes were increased markedly by the treatment of chick embryo with clofibrate, while those in mitochondria did not change. In the liver of clofibrate-treated chick embryo, approximately 50% of total liver carnitine palmitoyltransferase (CPT) activity was present in the peroxisomal fraction. Peroxisomal CPT activity was easily solubilized, in contrast with mitochondrial CPT. The solubilized protein solutions from isolated peroxisomes and mitochondria were separately chromatographed on a column of Blue Sepharose CL-6B after the gel filtration on Sephadex G-25. Peroxisomal CPT was completely bound to a Blue Sepharose CL-6B column and was eluted below 0.25 M KCl, whereas mitochondrial CPT was not retained on the column. The substrate specificity profile of peroxisomal CPT with long-chain acyl-CoAs (C8 to C18) was similar to that of mitochondrial CPT, and the apparent Km value of peroxisomal CPT for palmitoyl-CoA was 5.2 microM, being similar to that of mitochondrial CPT. It is concluded that carnitine long-chain acyltransferase, which is different from mitochondrial CPT and is induced by clofibrate treatment, is present in peroxisomes of chick embryo liver. 相似文献