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The Peroxisome Proliferator-induced Cytosolic Type I Acyl-CoA Thioesterase (CTE-I) Is a Serine-Histidine-Aspartic Acid α/β Hydrolase*

  • Kaisa Huhtinen
    Affiliations
    Department of Medical Laboratory Sciences and Technology, Division of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, SE-141 86 Stockholm, Sweden
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  • James O'Byrne
    Affiliations
    Department of Medical Laboratory Sciences and Technology, Division of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, SE-141 86 Stockholm, Sweden
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  • Per J.G. Lindquist
    Affiliations
    Department of Medical Laboratory Sciences and Technology, Division of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, SE-141 86 Stockholm, Sweden
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  • Juan A. Contreras
    Affiliations
    Department of Medical Laboratory Sciences and Technology, Division of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, SE-141 86 Stockholm, Sweden
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  • Stefan E.H. Alexson
    Correspondence
    To whom correspondence should be addressed: Division of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, SE-141 86 Stockholm, Sweden. Tel.: 46-8-585-812-74; Fax: 46-8-585-812-60
    Affiliations
    Department of Medical Laboratory Sciences and Technology, Division of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, SE-141 86 Stockholm, Sweden
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  • Author Footnotes
    * This work was supported by grants from the Swedish Natural Science Research Council, the Swedish Medical Research Council, and the Swedish Research Council for Engineering Sciences.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:November 02, 2001DOI:https://doi.org/10.1074/jbc.M109040200
      Long-chain acyl-CoA thioesterases hydrolyze long-chain acyl-CoAs to the corresponding free fatty acid and CoASH and may therefore play important roles in regulation of lipid metabolism. We have recently cloned four members of a highly conserved acyl-CoA thioesterase multigene family expressed in cytosol (CTE-I), mitochondria (MTE-I), and peroxisomes (PTE-Ia and -Ib), all of which are regulated via the peroxisome proliferator-activated receptor α (Hunt, M. C., Nousiainen, S. E. B., Huttunen, M. K., Orii, K. E., Svensson, L. T., and Alexson, S. E. H. (1999) J. Biol. Chem. 274, 34317–34326). Sequence comparison revealed the presence of putative active-site serine motifs (GXSXG) in all four acyl-CoA thioesterases. In the present study we have expressed CTE-I in Escherichia coli and characterized the recombinant protein with respect to sensitivity to various amino acid reactive compounds. The recombinant CTE-I was inhibited by phenylmethylsulfonyl fluoride and diethyl pyrocarbonate, suggesting the involvement of serine and histidine residues for the activity. Extensive sequence analysis pinpointed Ser232, Asp324, and His358 as the likely components of a catalytic triad, and site-directed mutagenesis verified the importance of these residues for the catalytic activity. A S232C mutant retained about 2% of the wild type activity and incubation with 14C-palmitoyl-CoA strongly labeled this mutant protein, in contrast to wild-type enzyme, indicating that deacylation of the acyl-enzyme intermediate becomes rate-limiting in this mutant protein. These data are discussed in relation to the structure/function of acyl-CoA thioesterasesversus acyltransferases. Furthermore, kinetic characterization of recombinant CTE-I showed that this enzyme appears to be a true acyl-CoA thioesterase being highly specific for C12–C20 acyl-CoAs.
      PPAR
      peroxisome proliferator-activated receptor
      CTE-I
      cytosolic acyl-CoA thioesterase I
      MTE-I
      mitochondrial acyl-CoA thioesterase I
      PTE-Ia and -Ib
      peroxisomal acyl-CoA thioesterase Ia and Ib
      BAAT
      bile acid-CoA:amino acid N-acyltransferase
      BSA
      bovine serum albumin
      pCMB
      p-chloromercuribenzoic acid
      DTNB
      5,5′-dithiobis-(2-nitrobenzoic acid)
      DFP
      diisopropyl fluorophosphate
      DTT
      dithiothreitol
      BNPP
      bis-(4-nitrophenyl)phosphate
      SCD1
      stearoyl-CoA desaturase 1
      Long-chain fatty acids entering cells are rapidly esterified to the corresponding CoA esters prior to degradation via β-oxidation in mitochondria or peroxisomes or esterification into triacylglycerol, phospholipids, cholesterol esters, or proteins (for review, see Ref.
      • Waku K.
      ). However, long-chain acyl-CoAs have also been shown to exert a number of regulatory functions such as allosteric regulators of various enzyme activities and ion channels, membrane fusion, cell signaling, etc. (
      • Færgeman N.J.
      • Knudsen J.
      ,
      • Bränström R.
      • Leibiger I.B.
      • Leibiger B.
      • Corkey B.E.
      • Berggren P.-O.
      • Larsson O.
      ,
      • Færgeman N.J.
      • Ballegaard T.
      • Knudsen J.
      • Black P.N.
      • DiRusso C.
      ). Recently free fatty acids were shown to activate and bind as ligands to the peroxisome proliferator-activated receptors (PPARs)1 (
      • Göttlicher M.
      • Widmark E.
      • Li Q.
      • Gustafsson J.-Å.
      ,
      • Forman B.M.
      • Chen J.
      • Evans R.M.
      ,
      • Kliewer S.A.
      • Sundseth S.S.
      • Jones S.A.
      • Brown P.J.
      • Wisely G.B.
      • Koble C.S.
      • Devchand P.
      • Wahli W.
      • Willson T.M.
      • Lenhard J.M.
      • Lehmann J.M.
      ), and long-chain acyl-CoAs bind as ligands to the hepatocyte nuclear factor 4α (HNF-4α) (
      • Hertz R.
      • Magenheim J.
      • Berman I.
      • Bar-Tana J.
      ). Interestingly, long-chain acyl-CoA esters act as antagonists for the PPARs (
      • Elholm M.
      • Dam I.
      • Jørgensen C.
      • Krogsdam A.-M.
      • Holst D.
      • Kratchmarova I.
      • Göttlicher M.
      • Gustafsson J.-Å.
      • Berge R.
      • Flatmark T.
      • Knudsen J.
      • Mandrup S.
      • Kristiansen K.
      ,
      • Murakami K.
      • Ide T.
      • Nakazawa T.
      • Okazaki T.
      • Machizuki T.
      • Kadowaki T.
      ), suggesting that free fatty acid/acyl-CoA levels may be important regulators of gene transcription. Thus, the diversity of cellular processes involving activated fatty acyl-CoA esters underscores the central role of acyl-CoA thioesters in lipid metabolism.
      Acyl-CoA thioesterases are enzymes that catalyze the hydrolysis of acyl-CoA thioesters to the corresponding free fatty acid and CoASH, and are therefore likely to regulate lipid metabolism as well as a number of cellular processes (for review, see Ref.
      • Hunt M.C.
      • Alexson S.E.H.
      ). This enzyme activity is widely distributed among organisms and shows multiple subcellular localizations. The enzyme activity is found in peroxisomes, mitochondria, cytosol, and endoplasmic reticulum and can be induced in liver by feeding peroxisome proliferators (
      • Miyazawa S.
      • Furuta S.
      • Hashimoto T.
      ,
      • Berge R.K.
      • Skrede S.
      • Farstad M.
      ,
      • Wilcke M.
      • Alexson S.E.H.
      ,
      • Svensson L.T.
      • Wilcke M.
      • Alexson S.E.H.
      ). These peroxisome proliferators (e.g. clofibrate) are potent modulators of lipid metabolism, and treatment of rodents with these compounds results in hypolipidemic effects and induction of fatty acid-catabolizing enzymes. The acyl-CoA thioesterase activity induced in mouse liver cytosol by treatment with these peroxisome proliferators is mainly because of induction of an ∼45-kDa protein called CTE-I (
      • Kawashima Y.
      • Katoh H.
      • Kozuka H.
      ,
      • Lindquist P.J.G.
      • Svensson L.T.
      • Alexson S.E.H.
      ). CTE-I is closely related to an acyl-CoA thioesterase expressed in mitochondria (MTE-I) (
      • Svensson L.T.
      • Wilcke M.
      • Alexson S.E.H.
      ,
      • Svensson L.T.
      • Alexson S.E.H.
      • Hiltunen J.K.
      ). We and others (
      • Lindquist P.J.G.
      • Svensson L.T.
      • Alexson S.E.H.
      ,
      • Svensson L.T.
      • Alexson S.E.H.
      • Hiltunen J.K.
      ,
      • Yamada J.
      • Matsumoto I.
      • Furihata T.
      • Sakuma M.
      • Suga T.
      ,
      • Yamada J.
      • Furihata T.
      • Iida N.
      • Watanabe T.
      • Hosokawa M.
      • Satoh T.
      • Someya A.
      • Nagaoka I.
      • Suga T.
      ,
      • Svensson L.T.
      • Engberg S.T.
      • Aoyama T.
      • Usuda N.
      • Alexson S.E.H.
      • Hashimoto T.
      ) have recently isolated and cloned type I long-chain acyl-CoA thioesterases from rat and mouse liver mitochondria and cytosol. Cloning of the corresponding genes from mouse revealed a unique, highly conserved gene family encoding acyl-CoA thioesterases with putative expression in cytosol (CTE-I), mitochondria (MTE-I), and peroxisomes (PTE-Ia and -Ib), all of which can be regulated by feeding peroxisome proliferators and by fasting (
      • Hunt M.C.
      • Nousiainen S.E.B.
      • Huttunen M.K.
      • Orii K.E.
      • Svensson L.T.
      • Alexson S.E.H.
      ,
      • Hunt M.C.
      • Lindquist P.J.G.
      • Peters J.M.
      • Gonzalez F.J.
      • Diczfalusy U.
      • Alexson S.E.H.
      ). Based on sequence similarity, these enzymes belong to a unique family of proteins that show sequence homology only to a bile acid-CoA:amino acid N-acyltransferase (BAAT), which catalyzes the conjugation of bile acids to glycine or taurine. Still, however, the functions of these acyl-CoA thioesterases are largely unknown.
      So far, the best characterized thioesterases are thioesterases I and II, which are involved in chain termination of fatty acid synthesis by cleavage of the newly synthesized fatty acid from the fatty acid synthase complex (
      • Lin C.Y.
      • Smith S.
      ,
      • Mikkelsen J.
      • Witkowski A.
      • Smith S.
      ). These thioesterases also hydrolyze acyl-CoAs and, similar to serine proteases, lipases, and cholinesterases, they have been proposed to contain a Ser-His-Asp/Glu catalytic triad, which catalyzes the ester hydrolysis. A nucleophilic serine is thought to be part of a charge-relay system along with a conserved histidine and an aspartic or glutamic acid residue (
      • Pazirandeh M.
      • Chirala S.S.
      • Wakil S.J.
      ,
      • Witkowski A.
      • Naggert J.
      • Wessa B.
      • Smith S.
      ,
      • Witkowski A.
      • Naggert J.
      • Witkowska H.E.
      • Randhawa Z.I.
      • Smith S.
      ). Previous site-directed mutagenesis experiments of thioesterase II have identified two of the residues that are involved in catalysis: a serine residue found within a so-called esterase consensus sequence, GXSXG (
      • Derewenda Z.S.
      • Sharp A.M.
      ), and a histidine residue present within a GXH motif (
      • Witkowski A.
      • Naggert J.
      • Wessa B.
      • Smith S.
      ,
      • Witkowski A.
      • Naggert J.
      • Witkowska H.E.
      • Randhawa Z.I.
      • Smith S.
      ). The GXSXG motif seems to be conserved among esterases, and in mouse CTE-I Ser232 is found within a GXSXG motif. To gain further insight into the catalytic mechanism of the type I acyl-CoA thioesterases, we have characterized recombinant CTE-I with respect to chemical modifiers and used site-directed mutagenesis to identify the catalytic amino acid residues. Our results demonstrate that CTE-I is a member of the α/β hydrolase superfamily of esterases and that Ser232, Asp324, and His358 constitute the catalytic triad. Interestingly, the active-site serine is apparently replaced by a cysteine in the BAAT enzyme, which may mediate the different activities of these enzymes.

      RESULTS

      We recently cloned four highly conserved members of a peroxisome proliferator-induced acyl-CoA thioesterase gene family (
      • Hunt M.C.
      • Nousiainen S.E.B.
      • Huttunen M.K.
      • Orii K.E.
      • Svensson L.T.
      • Alexson S.E.H.
      ). These genes encode putative mitochondrial, peroxisomal, and cytosolic thioesterases based on the presence or absence of targeting signals. The encoded proteins show high homology, and sequence comparisons show that they are closely related only to BAAT, an enzyme involved in conjugation of bile acids (
      • Falany C.N.
      • Johnson S.
      • Barnes S.
      • Diasio R.B.
      ,
      • Falany C.N.
      • Fortinberry H.
      • Leiter E.H.
      • Barnes S.
      ). Sequence analysis identified a putative active-site serine within a consensus motif (Gly-Xaa-Ser/Cys-Xaa-Gly) frequently found in the active site of α/β hydrolases (
      • Ollis D.
      • Cheah E.
      • Cygler M.
      • Dijkstra B.
      • Frolow F.
      • Franken S.
      • Harel M.
      • Remington S.
      • Silman I.
      • Schrag J.
      • Sussmann J.
      • Verscheuren K.
      • Goldman A.
      ). This superfamily of proteins is one of the largest known and includes esterases, lipases, transferases, thioesterases, haloperoxidases, lyases, etc. All of the enzymes in the α/β hydrolase superfamily share a common fold and harbor a catalytic triad formed by Ser/Cys, His, Asp/Glu. There is only one crystal structure available for a mammalian thioesterase, the palmitoyl protein thioesterase 1, shown to belong to the α/β hydrolase family (
      • Bellizzi J.J.
      • Widom J.
      • Kemp C.
      • Lu J.-Y.
      • Das A.K.
      • Hofmann S.L.
      • Clardy J.
      ). The presence of the GXSXG consensus motif suggests that also the type I thioesterases may conform to the paradigm of the α/β hydrolase fold. Furthermore, biochemical and site-directed mutagenic analyses have provided evidence for the involvement of a serine and a histidine residue in the catalytic triad of thioesterase II, an enzyme involved in termination of fatty acid synthesis (
      • Witkowski A.
      • Naggert J.
      • Wessa B.
      • Smith S.
      ,
      • Witkowski A.
      • Naggert J.
      • Witkowska H.E.
      • Randhawa Z.I.
      • Smith S.
      ). When mouse type I thioesterase sequences were aligned to the mouse BAAT sequence (Fig.1), a reduced number of conserved carboxylic acid, serine, and histidine residues was identified that may constitute a catalytic triad in type I thioesterases and BAAT, although Ser232 aligned with a cysteine residue in BAAT. Fasta3 searches of the GenBankTM detected a number of proteins sharing significant overall sequence homology with mouse CTE-I (Fig.1). The alignment further suggested Ser232, Asp324, and His358 as a tentative triad, because these are the only residues of this type conserved in all of the sequences found. When each of the sequences shown in Fig. 1 was used to search for homologous proteins in the three-dimensional structure data bank, one of the Caenorhabditis elegansproteins (T16563) showed a distant but unequivocal homology with a bacterial dienelactone hydrolase of known three-dimensional structure (Protein Data Bank code 1DIN). This hydrolytic enzyme belongs to the α/β hydrolase superfamily, and therefore its evolutionary relationship to CTE-I confirms that this thioesterase also shares the α/β hydrolase fold. The sequence homology between 1DIN and CTE-I is too low to permit homology modeling, but a secondary structure prediction of CTE-I allowed us to identify some of the central elements of the fold in CTE-I. A partial secondary structure guided alignment of1DIN and CTE-I is shown in Fig. 2. A reduced number of conserved residues can be detected in strategic positions of the structure. Among these, Ser232, Asp324, and His358 in CTE-I aligned to the corresponding residues in the catalytic triad of 1DIN, further supporting the theory that these residues constitute the catalytic triad of CTE-I.
      Figure thumbnail gr1
      Figure 1Multiple sequence alignment of mouse CTE-Iand related proteins. A multiple sequence alignment was performed using Fasta3, and only proteins showing an overall sequence identity to CTE-I of more than 20% were included in the alignment. The percentage of sequence identity to mouse CTE-I is indicated at the beginning of each sequence, and the putative residues of the catalytic triad (Ser/Cys, Asp, and His) are indicated. 1, mouse CTE-I (GenBankTM accession No. O55137); 2,mouse MTE-I (Q9QYR9); 3, mouse PTE-Ia (Q9QYR7);4, mouse PTE-Ib (Q9QYR4); 5, mouse BAAT (NP031545); 6–9, four hypothetical proteins from C. elegans (O62086, O01862, O45003,T16563) (
      • Murakami K.
      • Ide T.
      • Nakazawa T.
      • Okazaki T.
      • Machizuki T.
      • Kadowaki T.
      ) putative peptidase from Bacillus subtilis(O34493).
      Figure thumbnail gr2
      Figure 2Secondary structure-guided partial alignment of mouse CTE-I and 1DIN. β-Strands (underlined) and α-helices (double underlined) in the structure of 1DIN(Protein Data Bank code) are indicated. Secondary structure elements predicted for CTE-I with the PHD PredictProtein program are indicated in the same manner. The asterisks denote identities, anddots indicate conservative substitutions. The nomenclature of these structurally conserved elements follows the recommendations for the α/β hydrolase superfamily (
      • Ollis D.
      • Cheah E.
      • Cygler M.
      • Dijkstra B.
      • Frolow F.
      • Franken S.
      • Harel M.
      • Remington S.
      • Silman I.
      • Schrag J.
      • Sussmann J.
      • Verscheuren K.
      • Goldman A.
      ). The residues in the triad of 1DIN are indicated by arrows.

      Effects of Chemical Modifiers on Mouse CTE-I Thioesterase Activity

      The finding of a putative active-site serine was surprising, as previous characterizations of purified type I acyl-CoA thioesterases showed that the activity is not inhibited by DFP or BNPP, common serine esterase inhibitors, but is very sensitive topCMB, a cysteine-reactive compound (
      • Svensson L.T.
      • Alexson S.E.H.
      • Hiltunen J.K.
      ,
      • Yamada J.
      • Matsumoto I.
      • Furihata T.
      • Sakuma M.
      • Suga T.
      ). To further investigate amino acids involved in the active site, recombinant mouse CTE-I protein was produced to characterize the enzyme with respect to sensitivity to common amino acid modifying reagents. The recombinant protein was found to be insensitive to the serine reactive agents DFP and BNPP when tested at concentrations up to 1 mm (Fig. 3A). However, phenylmethylsulfonyl fluoride, another serine-reactive agent, inhibited the activity with an IC50 ≈ 0.5 mm(Fig. 3A). The histidine-reacting reagent diethyl pyrocarbonate was also found to abolish CTE-I thioesterase activity (Fig. 3A). These results are consistent with the presence of a Ser-His-Asp/Glu catalytic triad. In addition, the cysteine reducing reagent DTT had no effect on CTE-I thioesterase activity, whereaspCMB and DTNB were found to be potent inhibitors (Fig.3B), suggesting that a cysteine residue may be located near the active site.
      Figure thumbnail gr3
      Figure 3Effects of chemical amino acid modifiers on mouse CTE-I thioesterase activity. Wild-type CTE-I was expressed in E. coli, and protein extracts were prepared as described under “Experimental Procedures.” The protein extracts were incubated with the indicated compounds, and activity was measured with 25 μm myristoyl-CoA. A, effects of serine- and histidine-reactive reagents. The myristoyl-CoA thioesterase activity of mouse CTE-I was measured in the presence of various concentrations of diethyl pyrocarbonate (DEPC), phenylmethylsulfonyl fluoride (PMSF), DFP, and BNPP.B, effects of cysteine reactive reagents. The myristoyl-CoA thioesterase activity of mouse CTE-I was measured in the presence of various concentrations of pCMB, DTT, and DTNB.

      Site-directed Mutagenesis and Expression of Mouse CTE-I

      As discussed above, multiple sequence alignments strongly suggested that Ser232, Asp324, and His358 constituted the active-site amino acids. Therefore, PCR-based site-directed mutagenesis was performed as described under “Experimental Procedures” to verify the identity of these amino acids as being part of the active site. Sequencing of all mutant plasmids verified the introduction of the correct mutations (shown in Table I), and wild-type and mutant plasmids were expressed in E. coli. Acyl-CoA thioesterase activity was measured in crude extracts using myristoyl-CoA, which is the best substrate for CTE-I. The specific activity of CTE-I wild-type enzyme in the crude extract was about 140 nmol/min/mg protein, as compared with ∼17 nmol/min/mg protein in non-expressing bacterial extracts (data not shown). Mutation of Ser232 to Ala, His358 to Gln, and Asp324 to Ala abolished the activity, suggesting that Ser232, Asp324, and His358 indeed constitute the active-site amino acids, whereas none of the other mutations had any major effect on activity (data not shown). Expressed His-tagged recombinant wild-type CTE-I, S232C, S232A, D324A, and H358Q were purified and characterized kinetically. Expression of the recombinant proteins were verified by SDS-PAGE (Fig. 4), and these proteins were used for further kinetic characterization. TheVmax of wild-type CTE-I was about 1.2 μmol/min/mg protein when measured with palmitoyl-CoA, with a calculated Km of about 2.6 μm (TableII). However, the specific activities of the S232A, D324A, and H358Q were about 0.1 nmol/min/mg protein (when measured with 10 μm palmitoyl-CoA), which is about four orders of magnitude lower than the activity of the wild-type CTE-I. The activities of the mutants were at the base-line detection level for the assay, and more detailed kinetic studies could therefore not be performed. Interestingly, the S232C mutant retained about 2% of the wild-type activity, with a calculated Km of about 0.6 μm, suggesting that this mutant is still active as an acyl-CoA thioesterase, albeit with profoundly decreased activity. A possible explanation for the lower activity of the S232C mutant may be that the deacylation step becomes rate-limiting and that this mutant therefore becomes acylated as the acyl-enzyme intermediate. To test this possibility, we carried out experiments to compare acylation of the wild-type CTE-I and the various mutants.
      Figure thumbnail gr4
      Figure 4SDS-PAGE analysis of expressed and affinity-purified wild-type and mutant CTE-I proteins used for kinetic characterization. Wild-type and single amino acid-specific mutants of CTE-I, as indicated, were expressed in E. coli after which each protein was purified using affinity chromatography as described under “Experimental Procedures.” 2–8 μg of protein was electrophoresed in 10% polyacrylamide gels and stained with Coomassie Brilliant Blue. Wt, wild type.
      Table IIComparison of enzyme kinetics of recombinantly expressed CTE-I and mutant proteins
      CTE-I enzymeVmaxKmActivity
      nmol/min/mg proteinμm% wild type
      Wild-type CTE-I1,2002.6100
      S232C230.61.9
      S232A≈0.1NA≈0.01
      D324A≈0.1NA≈0.01
      H358Q≈0.1NA≈0.01
      Wild-type and various mutants of CTE-I were expressed and purified as described under “Experimental Procedures.” The purified proteins were analyzed for acyl-CoA thioesterase activity using palmitoyl-CoA as substrate. The activities of the S232A, D324A, and H358Q mutants were at the base-line detection level, and therefore proper kinetic characterization could not be carried out. NA, not attainable.

      Acylation of CTE-I Mutants

      Acylation of CTE-I was analyzed as covalent incorporation of labeled palmitate after incubation with14C-palmitoyl-CoA. Neither wild-type CTE-I nor the S232A mutant contained any detectable incorporation of palmitate (Fig.5). However, the S232C mutant, which showed low palmitoyl-CoA thioesterase activity (see above) was strongly labeled with radioactive palmitate, and the H358Q and D324A mutants were also labeled, albeit much more weakly. Quantitation of the acylation (normalized to protein amount) showed that the H358Q D324A mutants contained about 14 and 10% radioactivity, respectively, of the labeling seen with the S232C mutant, whereas the wild-type CTE-I and the S232A mutants contained <2.6% (indistinguishable from background) of the radioactivity found in the S232C mutant. The incorporated fatty acids were covalently bound, as the labeling could be removed completely by treatment with neutral hydroxylamine (data not shown).
      Figure thumbnail gr5
      Figure 5Acylation of wild-type and various mutants ofCTE-I. Wild-type (Wt) and mutated CTE-I cDNAs, as indicated, were expressed in E. coli, and bacterial extracts were prepared and incubated with 14C-palmitoyl-CoA. Aliquots were subjected to SDS-PAGE and blotted onto nitrocellulose membranes, and incorporation of 14C-palmitate was analyzed using a phosphorimaging device (upper panel). Aliquots were also analyzed by Western blotting, quantitated by scanning, and used to calculate the relative incorporation of radioactivity (lower panel).

      Acyl-CoA Substrate Specificity of CTE-I

      Previous studies on CTE-I (corresponding to ACH2 of Yamada et al.(
      • Yamada J.
      • Matsumoto I.
      • Furihata T.
      • Sakuma M.
      • Suga T.
      )) showed that the enzyme is inhibited at higher substrate concentrations. We therefore tested the addition of BSA to the incubations, which at a molar ratio of 1:4.5 showed a strong protective effect against substrate inhibition (data not shown). Therefore, albumin was added at a constant molar ratio of albumin to substrate when the acyl-CoA chain length specificity was tested with acyl-CoAs longer than lauroyl-CoA (C12-CoA). When measured at 10 μm substrate, CTE-I is active on saturated acyl-CoAs of 12–20 carbon atoms, with only very low activity with decanoyl-CoA as substrate (Fig. 6). Introduction of one or two double bonds decreased the activity to about half or less compared with the corresponding saturated acyl-CoA. However, the activity is negligible with arachidonoyl-CoA (C20:4), about 35-fold lower than the activity with arachidoyl-CoA (C20:0). A summary of Vmax andKm values with various acyl-CoAs is shown in TableIII. The highestVmax values were obtained with C12–C18 saturated and monounsaturated acyl-CoAs with Km values below 4.5 μm. We also tested a number of other acyl-CoA substrates (all measured at 10 μm) of varying structures; two methyl-branched chain acyl-CoAs, 4,8-dimethylnonanoyl-CoA and 2-methylstearoyl-CoA, which were both poor substrates. Also the β-oxidation intermediates 2-trans-decenoyl-CoA and 3-hydroxypalmitoyl-CoA were much poorer substrates than the corresponding saturated acyl-CoAs. Clofibroyl-CoA thioesterase activity has been reported to be localized mainly in cytosol and to be induced by clofibrate treatment in rats (
      • Berge R.K.
      • Stensland E.
      • Aarsland A.
      • Tsegai G.
      • Osmundsen H.
      • Aarsæter N.
      • Gjellesvik D.R.
      ). However, CTE-I showed almost no activity with clofibroyl-CoA, which excludes CTE-I as the enzyme responsible for the activity seen in rodent liver. In addition, because of the sequence similarity between CTE-I and the BAAT enzyme (which uses choloyl-CoA and chenodeoxycholoyl-CoA as substrates) we tested the activity of CTE-I with these bile acid intermediates. Somewhat surprisingly we did not detect any activity with choloyl-CoA or chenodeoxycholoyl-CoA. Furthermore, CTE-I showed no phospholipase A2 or diacylglycerol lipase activity.
      P. Larsson, personal communication.
      Taken together, the kinetic characterization suggests that CTE-I functions as a highly specific long-chain acyl-CoA thioesterase with very low activity for “bulky” substrates.
      Figure thumbnail gr6
      Figure 6Kinetic characterization of recombinantCTE-I. Wild-type CTE-I was expressed in E. coli and purified as described under “Experimental Procedures.” Acyl-CoA chain length specificity of CTE-I, measured with the indicated substrate at 10 μm. Stippled bars, saturated acyl-CoAs; filled bars, monounsaturated acyl-CoAs;patterned bar, linoleoyl-CoA; open bar (indicated by an arrow), arachidonoyl-CoA (C20:4).
      Table IIIKinetic characterization of wild-type CTE-I
      Acyl-CoAKmVmaxμmol/min/mg Protein
      μmμmol/min/mg protein
      C10:015.20.192
      C12:02.60.780
      C14:03.51.68
      C14:11.50.745
      C16:02.61.20
      C16:11.10.621
      C18:04.30.692
      C18:1 (cis)2.40.176
      C18:1(trans)1.80.188
      C18:24.50.316
      C20:00.50.245
      C20:43.00.007
      4,8-CH3-C9-CoA0.022
      2-trans-C10-CoA0.011
      3-OH-C16-CoA0.021
      2-CH3-C18-CoA0.014
      Clofibroyl-CoA0.004
      Choloyl-CoA≈0
      CTE-I was expressed and purified on an affinity column as described under “Experimental Procedures.” Thioesterase activity was measured at various concentrations of straight-chain acyl-CoAs of different chain-lengths (C10–C20:4). Activity was not detectable with acyl-CoAs of 8 or less carbon atoms in length.Km and Vmax were calculated using the Sigma Plot Enzyme Kinetics program. Recombinant CTE-I was also tested for activity with some other CoA-esters at 10 μm; 4,8-CH3-C9-CoA, 4,8-dimethylnonanoyl-CoA; 2-trans-C10-CoA, 2-transdecenoyl-CoA; 3-OH-C16-CoA, 3-hydroxypalmitoyl-CoA; 2-CH3-C18-CoA, 2-methylstearoyl-CoA.
      In view of our observation that the activity of a recently characterized peroxisomal acyl-CoA thioesterase (PTE-2) is highly regulated by CoASH,
      M. Hunt, K. Solaas, B. F. Kase, and S. Alexson, submitted for publication.
      we tested the effect of CoASH on CTE-I activity. In contrast to PTE-2, CTE-I activity was not affected at any of the concentrations tested (up to 500 μm, data not shown).

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