Structural Model of a Malonyl-CoA-binding Site of Carnitine Octanoyltransferase and Carnitine Palmitoyltransferase I
2002; Elsevier BV; Volume: 277; Issue: 13 Linguagem: Inglês
10.1074/jbc.m111628200
ISSN1083-351X
AutoresMontserrat Morillas, Paulino Gómez‐Puertas, Blanca Rubı́, Josep Clotet, Joaquı́n Ariño, Alfonso Valencia, Fausto G. Hegardt, Dolors Serra, Guillermina Asins,
Tópico(s)Mitochondrial Function and Pathology
ResumoCarnitine 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 His340, implicated together with His131 in COT malonyl-CoA sensitivity (Morillas, M., Clotet, J., Rubı́, B., Serra, D., Asins, G., Ariño, J., and Hegardt F. G. (2000) FEBS Lett. 466, 183–186). When we mutated COT His131 the IC50increased, and malonyl-CoA competed with the substrate decanoyl-CoA. Mutation of COT Ala332, present in the domain 8 amino acids away from His340, decreased the malonyl-CoA sensitivity of COT. The homologous histidine and alanine residues of L-CPT I, His277, His483, and Ala478 were also mutated, which decreased malonyl-CoA sensitivity. Natural mutation of Pro479, 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. 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 His340, implicated together with His131 in COT malonyl-CoA sensitivity (Morillas, M., Clotet, J., Rubı́, B., Serra, D., Asins, G., Ariño, J., and Hegardt F. G. (2000) FEBS Lett. 466, 183–186). When we mutated COT His131 the IC50increased, and malonyl-CoA competed with the substrate decanoyl-CoA. Mutation of COT Ala332, present in the domain 8 amino acids away from His340, decreased the malonyl-CoA sensitivity of COT. The homologous histidine and alanine residues of L-CPT I, His277, His483, and Ala478 were also mutated, which decreased malonyl-CoA sensitivity. Natural mutation of Pro479, 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. Interest in malonyl-CoA as a metabolic regulator has increased in recent years, as its function is not restricted to liver regulation of fatty acid oxidation and synthesis. Its contribution to the β-oxidation of long-chain fatty acids in other tissues, such as heart, skeletal muscle, and the β-cell has been widely studied (1.Zammit V.A. Biochem. J. 1999; 343: 505-515Crossref PubMed Scopus (100) Google Scholar). In liver, a low insulin/glucagon ratio decreases malonyl-CoA and shifts the fatty acid traffic throughout the oxidative process. The opposite is also true: when the insulin/glucagon ratio increases, malonyl-CoA concentration also rises, followed by a decrease in β-oxidation (2.McGarry J.D. Foster D.W. Annu. Rev. Biochem. 1980; 49: 395-420Crossref PubMed Scopus (1103) Google Scholar,3.McGarry J.D. Woeltje K.F. Kuwajima M. Foster D.W. Diabetes Metab. Rev. 1989; 5: 271-284Crossref PubMed Scopus (292) Google Scholar). The situation is similar in rat heart: incubation of perfused rat heart with glucose and insulin raises malonyl-CoA concentrations with concomitant suppression of palmitate oxidation (4.Saddik M. Gamble J. Witters L.A. Lopaschuk G.D. J. Biol. Chem. 1993; 268: 25836-25845Abstract Full Text PDF PubMed Google Scholar). Furthermore, when the glucose concentration in β-cell is raised, the malonyl-CoA concentration also increases and the resulting elevation of cytosolic long-chain fatty acyl-CoAs concentration stimulates exocytosis of insulin granules (5.Corkey B.E. Glennon M.C. Chen K.S. Deeney J.T. Matchinski F.M. Prentki M. J. Biol. Chem. 1989; 264: 21608-21612Abstract Full Text PDF PubMed Google Scholar, 6.Prentki M. Vischer S. Glennon M.C. Regazzi R. Deeney J.T. Corkey B.E. J. Biol. Chem. 1992; 267: 5802-5810Abstract Full Text PDF PubMed Google Scholar). Malonyl-CoA acquires its significant regulatory role by virtue of its inhibition of a class of carnitine acyltransferases, carnitine octanoyltransferase (COT) 1The abbreviations used are: COTcarnitine octanoyltransferaseCPT Icarnitine palmitoyltransferase IL-CPT Iliver isoform of CPT IM-CPT Imuscle isoform of CPT ICPT IIcarnitine palmitoyltransferase IICATcarnitine acetyltransferaseCoAcoenzyme A and carnitine palmitoyltransferase I (CPT) I. There are two isoforms of CPT I, produced by different genes: the L (liver) type and the M (muscle) type. In mammals, muscle CPT I has a much lower IC50 for malonyl-CoA than the liver form, whereas the affinity for long chain acyl-CoA is similar (7.Saggerson E.D. Biochem. J. 1982; 202: 397-405Crossref PubMed Scopus (64) Google Scholar, 8.McGarry J.D. Mills S.E. Long C.S. Foster D.W. Biochem. J. 1983; 214: 21-28Crossref PubMed Scopus (467) Google Scholar). This differential response to malonyl-CoA has been associated with the distinct interaction between the NH2- and COOH-terminal domains of the protein, which are exposed to the cytosolic side of the mitochondrial membrane (9.Jackson V.N. Cameron J.M. Fraser F. Zammit V.A. Price N.T. J. Biol. Chem. 2000; 275: 19560-19566Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). In contrast, other members of the family, such as carnitine palmitoyltransferase II (CPT II) and carnitine acetyltransferase (CAT) are not regulated by malonyl-CoA (10.McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1354) Google Scholar). carnitine octanoyltransferase carnitine palmitoyltransferase I liver isoform of CPT I muscle isoform of CPT I carnitine palmitoyltransferase II carnitine acetyltransferase coenzyme A Pioneer studies showed that malonyl-CoA does not bind to the CPT I active site, although there is a competition behavior between malonyl-CoA and palmitoyl-CoA (11.McGarry J.D. Leatherman G.F. Foster D.W. J. Biol. Chem. 1978; 253: 4128-4136Abstract Full Text PDF PubMed Google Scholar, 12.Saggerson E.D. Carpenter C.A. FEBS Lett. 1981; 132: 166-168Crossref PubMed Scopus (40) Google Scholar, 13.Mills S.E. Foster D.W. McGarry J.D. Biochem. J. 1984; 219: 601-608Crossref PubMed Scopus (50) Google Scholar, 14.Cook G.A. J. Biol. 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One site is the low affinity site, near the catalytic acyl-CoA-binding domain, in which the inhibitory effect could be exerted by malonyl-CoA and, a lesser extent, other compounds with the CoA moiety, such as acetyl-CoA, glutaryl-CoA, hydroxymethylglutaryl-CoA, methylmalonyl-CoA or free CoA itself (19.a′Bhaird N. Ramsay R.R. Biochem. J. 1992; 286: 637-640Crossref PubMed Scopus (24) Google Scholar,20.Zierz S. Engel A.G. Biochem. J. 1987; 245: 205-209Crossref PubMed Scopus (31) Google Scholar). The second, high affinity site is separated from the active site and does not compete with acyl-CoA (21.Bird M.I. Saggerson E.D. Biochem. J. 1984; 222: 639-647Crossref PubMed Scopus (42) Google Scholar, 22.Cook G.A. Mynatt R.L. Kashfi K. J. Biol. Chem. 1994; 269: 8803-8807Abstract Full Text PDF PubMed Google Scholar, 23.Kashfi K. Mynatt R.L. Cook G.A. Biochim. Biophys. Acta. 1994; 1212: 245-252Crossref PubMed Scopus (33) Google Scholar, 24.Zammit V.A. Corstorphine C.G. Gray S.R. Biochem. J. 1984; 222: 335-342Crossref PubMed Scopus (28) Google Scholar, 25.Bird M.I. Munday L.A. Saggerson E.D. Clark J.B. Biochem. J. 1985; 226: 323-330Crossref PubMed Scopus (44) Google Scholar). Compounds analog to malonyl-CoA, like 4-hydroxyphenylglyoxylate or its derivative Ro 25–0187, could inhibit CPT I without the presence of the CoA moiety (23.Kashfi K. Mynatt R.L. Cook G.A. Biochim. Biophys. Acta. 1994; 1212: 245-252Crossref PubMed Scopus (33) Google Scholar). The probable locations of the malonyl-CoA-binding sites in L-CPT I were predicted to be at the cytosolic COOH-terminal domain, after the preparation of several L-CPT I chimeras (26.Swanson S.T. Foster D.W. McGarry J.D. Brown N.F. Biochem. J. 1998; 335: 513-519Crossref PubMed Scopus (57) Google Scholar, 27.Jackson V.N. Zammit V.A. Price N.T. J. Biol. Chem. 2000; 275: 38410-38416Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). However, the NH2 terminus of L-CPT I also influences the enzyme-inhibitor interaction, since mutation of either His5or Glu3 reduced malonyl-CoA sensitivity (17.Shi J. Zhu H. Arvidson D.N. Woldegiorgis G. J. Biol. Chem. 1999; 274: 9421-9426Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 26.Swanson S.T. Foster D.W. McGarry J.D. Brown N.F. Biochem. J. 1998; 335: 513-519Crossref PubMed Scopus (57) Google Scholar). Moreover, modification of the interaction between the cytosolic NH2-terminal domain and the cytosolic catalytic COOH-terminal domain of CPT I alters the IC50 values for malonyl-CoA (9.Jackson V.N. Cameron J.M. Fraser F. Zammit V.A. Price N.T. J. Biol. Chem. 2000; 275: 19560-19566Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). In addition, the removal of the segment comprised between amino acids 19 and 40 increases malonyl-CoA sensitivity in L-CPT I, which emphasizes the importance of the NH2terminus before the first transmembrane region as a modulator of malonyl-CoA inhibition (27.Jackson V.N. Zammit V.A. Price N.T. J. Biol. Chem. 2000; 275: 38410-38416Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 28.Jackon V.N. Price N.T. Zammit V.A. Biochemistry. 2001; 40: 14629-14634Crossref PubMed Scopus (24) Google Scholar). The possible influence on malonyl-CoA inhibition of amino acids in the NH2 terminus does not occur in COT protein because the first amino acid of COT corresponds to position 152 of the L-CPT I, and therefore COT does not have the same amino terminus nor the transmembrane domains. Histidine residues are implicated in the malonyl-CoA interaction by the finding that a decrease in pH (associated with the protonation of the imidazol group of histidine) increases the affinity for malonyl-CoA (12.Saggerson E.D. Carpenter C.A. FEBS Lett. 1981; 132: 166-168Crossref PubMed Scopus (40) Google Scholar, 29.Stephens T.W. Cook G.A. Harris R.A. Biochem. J. 1983; 212: 521-524Crossref PubMed Scopus (32) Google Scholar). Our previous work showed that COT-malonyl-CoA interactions implicated two histidine residues, His131 and His340, which could involve two domains of interaction and binding of malonyl-CoA (30.Morillas M. Clotet J. Rubı́ B. Serra D. Asins G. Ariño J. Hegardt F.G. FEBS Lett. 2000; 466: 183-186Crossref PubMed Scopus (19) Google Scholar). The proposal of a structural model of the catalytic core of the COT protein (31.Morillas M. Gómez-Puertas P. Roca R. Serra D. Asins G. Valencia A. Hegardt F.G. J. Biol. Chem. 2001; 276: 45001-45008Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), in which one of these histidines, His340, is located has facilitated the study of one of these malonyl-CoA affinity domains. In this study we identified a putative binding domain of malonyl-CoA located in the model of COT and L-CPT I. From bioinformatic and mutagenic analysis, we define a domain containing amino acids from Ala332 to His340 in COT, and from Ala478 to His483 in L-CPT I (its homologous domain) as the site at which malonyl-CoA could bind to with either protein and induce inhibition. The structural models of COT and L-CPT I locate malonyl-CoA near the catalytic channel. The presence of malonyl-CoA in the groove of ligand-protein interaction would interfere with the positioning of the substrate decanoyl-CoA (in COT) or palmitoyl-CoA (in L-CPT I). To confirm this model, we expressed the cDNA for the wild type and different single and double mutants of COT and L-CPT I in Saccharomyces cerevisiae. A similar picture to that proposed for COT and L-CPT I is impossible with the non-inhibitable malonyl-CoA enzymes CPT II or CAT, although they have the same three-dimensional structure (which is analogous to the 2dubE domain described for COT and L-CPT I), since when we use a docking program, malonyl-CoA cannot be located in the model. Structural models of rat CPT I and COT were obtained as previously described (31.Morillas M. Gómez-Puertas P. Roca R. Serra D. Asins G. Valencia A. Hegardt F.G. J. Biol. Chem. 2001; 276: 45001-45008Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Models of the homologous proteins CAT and CPT II were built using a similar procedure. Briefly, sequences homologous to rat CPT II and mouse CAT were obtained using BLAST (32.Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71456) Google Scholar) and aligned using ClustalW (33.Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56002) Google Scholar). Secondary structure analysis of the sequences was performed using the public servers PHD (34.Rost B. Methods Enzymol. 1996; 266: 525-539Crossref PubMed Google Scholar), Jpred (35.Cuff J.A. Clamp M.E. Siddiqui A.S. Finlay M. Barton G.J. Bioinformatics. 1998; 14: 892-893Crossref PubMed Scopus (921) Google Scholar), and Psi-Pred (36.Jones D.T. Tress M. Bryson K. Hadley C. Proteins Struct. Funct. Genet. 1999; 37: 104-111Crossref Scopus (96) Google Scholar). Protein folds were recognized using the threading programs Threader2 (37.Jones D.T. Miller R.T. Thornton J.M. Proteins. 1995; 23: 387-397Crossref PubMed Scopus (77) Google Scholar), based on solvatation and pair potentials, and FUGUE (38.Shi J. Blundell T.L. Mizuguchi K. J. Mol. Biol. 2001; 310: 243-257Crossref PubMed Scopus (1085) Google Scholar), which uses environment-specific substitution tables and a structure-dependent gap penalty to detect remote structural homologues. The structural templates obtained were analyzed using information from the structure classification data bases CATH (39.Orengo C.A. Michie A.D. Jones S. Jones D.T. Swindells M.B. Thornton J.M. Structure. 1997; 5: 1093-1108Abstract Full Text Full Text PDF PubMed Google Scholar), FSSP (40.Holm L. Sander C. Nucleic Acids Res. 1996; 24: 206-210Crossref PubMed Scopus (232) Google Scholar), and SCOP (41.Murzin A. Brenner S.E. Hubbard T. Chothia C. J. Mol. Biol. 1995; 247: 536-540Crossref PubMed Scopus (5610) Google Scholar). Using the three-dimensioinal structure of rat enoyl-CoA hydratase (PDB entry 2dub chain E), as selected previously for the homologous proteins CPT I and COT (31.Morillas M. Gómez-Puertas P. Roca R. Serra D. Asins G. Valencia A. Hegardt F.G. J. Biol. Chem. 2001; 276: 45001-45008Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), the program Swiss-Pdb Viewer and the SWISS-MODEL server facilities (42.Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9641) Google Scholar, 43.Guex N. Diemand A. Peitsch M.C. Trends Biochem. Sci. 1999; 24: 364-367Abstract Full Text Full Text PDF PubMed Google Scholar, 44.Peitsch M.C. Bio/Technology. 1995; 13: 658-660Crossref Scopus (116) Google Scholar, 45.Peitsch M.C. Biochem. Soc. Trans. 1996; 24: 274-279Crossref PubMed Scopus (900) Google Scholar) were used to build the coordinates for the three-dimensional model of the active site-surrounding regions of CAT and CPT II. Models were validated using ProsaII (46.Sippl M.J. Proteins. 1993; 17: 355-362Crossref PubMed Scopus (1792) Google Scholar), WHAT-CHECK (47.Hooft R.W.W. Vriend G. Sander C. Abola E.E. Nature. 1996; 381: 272Crossref PubMed Scopus (1818) Google Scholar), from WHAT-IF (48.Vriend G. J. Mol. Graph. 1990; 8: 52-56Crossref PubMed Scopus (3377) Google Scholar), and PROCHECK (49.Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Cryst. 1993; 26: 283-291Crossref Google Scholar). Calculation and representation of electrostatic potentials of the obtained models were performed using GRASP (50.Nicholls A. Bharadwaj R. Honig B. Biophys. J. 1993; 64: A166Google Scholar). Docking calculations to obtain a molecular model of the interaction between the inhibitor (ligand) malonyl-CoA and the putative receptor proteins CPT I, COT, CPT II, and CAT were performed using the spherical polar Fourier correlations based program "Hex" (51.Ritchie D.W. Kemp G.J.L. Proteins Struct. Funct. Genet. 2000; 39: 178-194Crossref PubMed Scopus (492) Google Scholar). The coordinates of the malonyl-CoA molecule were obtained from the PDB entry 1hnj(three-dimensional structure of the β-ketoacyl-acyl carrier protein synthase III). Program parameters were adjusted to evaluate the docking score at more than 40,000 distinct rotational orientations at each of 10 different intermolecular distances (at increments of ± 1 Å) between the inhibitor molecule and the molecular models of the four acyltransferases indicated above. The 128 highest scoring (lowest energy) orientations were retained for viewing and evaluation. Conserved differences (tree-determinants) between malonyl-inhibitable (L-CPT I, M-CPT I, COT) and non-inhibitable (CPT II, CAT) carnitine acyltransferases were analyzed with the SequenceSpace algorithm (52.Casari G. Sander C. Valencia A. Nat. Struct. Biol. 1995; 2: 171-178Crossref PubMed Scopus (353) Google Scholar, 53.Pazos F. Sanchez-Pulido L. Garcı́a-Ranea J.A. Andrade M.A. Atrian S. Valencia A. Lundh D. Olsson B. Narayanan A. Biocomputing and Emergent Computation. World Scientific, Singapore1997: 132-145Google Scholar), using the multiple alignment of the carnitine-choline acyltransferase family of proteins as input. Plasmids pYESCOTwt, pYESCOTH131A, pYESCOTH340A, and pYESCOTH131A/H340A were obtained as previously described (54.Morillas M. Clotet J. Rubı́ B. Serra D. Ariño J. Hegardt F.G. Asins G. Biochem. J. 2000; 351: 495-502Crossref PubMed Google Scholar). Plasmid pYESCOTA332Gwas constructed using the QuikChange polymerase chain reaction-based mutagenesis procedure (Stratagene) with the pYESCOTwtplasmid as template and the primer 5′-GCTGTGATCATGCTCCTTATGATGGAATGCTTATGGTGAAC-3′ (the mutated nucleotide is underlined). Plasmid pYESLCPTIwt, which contained nucleotides 103–2701 including the coding region of L-CPT was constructed as described (31.Morillas M. Gómez-Puertas P. Roca R. Serra D. Asins G. Valencia A. Hegardt F.G. J. Biol. Chem. 2001; 276: 45001-45008Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Plasmid pYESLCPTIwt was used for site-directed mutagenesis of His277 to Ala277 by the asymmetric PCR method (55.Datta A.K. Nucleic Acids Res. 1995; 23: 4530-4531Crossref PubMed Scopus (100) Google Scholar). The following primers were used: the mutated megaprimer fragment, obtained with the forward primer CPT583.for: 5′-AGCCCATGTTGTACAGCTTCC-3′ and the reverse primer H277A.rev: 5′-AGTATGGCGGCGATGGTGTTGCCAGC-3′ (the mutated nucleotides are underlined), was used with the reverse primer CPT1607.rev: 5′-CCATCCTCTGAGTAACCCAGC-3′. The fragment obtained was digested with AatII (nucleotides 610–1580) and subcloned into the plasmid pYESLCPTIwt, obtaining plasmid pYESLCPTIH277A. Plasmid pBSLCPTIwt, which contained nucleotides 54–2701 of rat L-CPT I subcloned in the ClaI and EcoRI sites of pBluescript SK+ vector (Stratagene), was used for construction of plasmid pYESLCPTIH483A. The mutated megaprimer fragment was obtained with the forward primer H483A.for: 5′-GTGGGCGCTTTGTGGGAGTATGTCATGGC-3′ (the mutated nucleotides are underlined) and the reverse primer CPT1878.rev: 5′-GGCCTCATATGTGAGGC-3′, which was used with the primer CPT1182.for: 5′-GCAGCAGATGCAGCAGATCC-3′ to obtain a fragment which, after digestion with PstI, was subcloned into the pBSLCPTIwt, obtaining pBSLCPTIH483A. To obtain the plasmid pYESCPTIH483A, the fragment KpnI-EcoRI was digested and subcloned into the pYESCPTIwt. Plasmid pYESLCPTIH277A/H483A was generated by mutagenesis of His277 to Ala using the plasmid pYESLCPTIH483A as template, and the primers described above. Mutant L-CPT I A478G was constructed using the "QuikChange" polymerase chain reaction-based mutagenesis procedure (Stratagene) with pYESLCPTIwt plasmid as template and the primer 5′-CACTCCTGCGCGGACGGGCCCATCGTGGGCCATTTG-3′(mutated nucleotide is underlined). The appropriate substitutions, as well as the absence of unwanted mutations were confirmed by sequencing the inserts in both directions with an Applied Biosystems 373 automated DNA sequencer. The expression of the constructs containing COT and L-CPT I wild type and mutants (see above) in yeast cells and the obtaining of the cell extracts were performed as described in Refs. 30.Morillas M. Clotet J. Rubı́ B. Serra D. Asins G. Ariño J. Hegardt F.G. FEBS Lett. 2000; 466: 183-186Crossref PubMed Scopus (19) Google Scholar and 31.Morillas M. Gómez-Puertas P. Roca R. Serra D. Asins G. Valencia A. Hegardt F.G. J. Biol. Chem. 2001; 276: 45001-45008Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar. Depending on the experiment the time to induce expression of L-CPT I by galactose was varied between 1 and 20 h. Carnitine acyltransferase activity was determined by the radiometric method as described in Ref. 26.Swanson S.T. Foster D.W. McGarry J.D. Brown N.F. Biochem. J. 1998; 335: 513-519Crossref PubMed Scopus (57) Google Scholar with minor modifications. The substrates werel-[methyl-3H]carnitine and, decanoyl-CoA for COT and palmitoyl-CoA for L-CPT I. Enzyme activity was assayed for 4 min at 30 °C in a total volume of 200 μl. For determination of the Km for carnitine, decanoyl-CoA was fixed at 50 μm (for COT) and palmitoyl-CoA was fixed at 135 μm (for L-CPT I). For determination of the Km for acyl-CoA, carnitine concentration was fixed at 400 μm. When malonyl-CoA inhibition was assayed, increasing concentrations of malonyl-CoA were included. The IC50, defined as the malonyl-CoA concentration that produces 50% inhibition of enzyme activity, was determined using 50 μm acyl-CoA and 400 μmcarnitine. Km was estimated by analyzing the data from three experiments using the program Enzifit (Biosoft) and IC50 was calculated by Excel software using linear regression analysis. Values reported in the text are the means and standard deviations of three to five determinations. Curve fitting was carried out using Sigma plot software. All protein concentrations were determined using the Bio-Rad protein assay with bovine albumin as standard. Western blot analysis for COT and L-CPT I were performed as described (31.Morillas M. Gómez-Puertas P. Roca R. Serra D. Asins G. Valencia A. Hegardt F.G. J. Biol. Chem. 2001; 276: 45001-45008Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The specific antibody used for rat COT was directed against peptide 344–360. The antibody for rat L-CPT I was kindly given by Dr. V. A. Zammit (Hannah Research Institute, Ayr, Scotland, United Kingdom) and was directed against peptide 428–441, in the cytosolic catalytic COOH-terminal domain. Rat liver mitochondria were isolated as described elsewhere (56.Derrick P. Ramsay R.R. Biochem. J. 1989; 262: 801-806Crossref PubMed Scopus (61) Google Scholar). Mitochondria were dispersed in 250 mm sucrose, 10 mm Tris-HCl, pH 7.4, and 1 mm EDTA to a final concentration of 40 mg/ml and stored at −70 °C. Once the structural models of COT and L-CPT I catalytic core, including the putative substrate-binding site, active site, were established (31.Morillas M. Gómez-Puertas P. Roca R. Serra D. Asins G. Valencia A. Hegardt F.G. J. Biol. Chem. 2001; 276: 45001-45008Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), we performed exhaustive in silico molecular docking analysis to find clues to the putative binding site of malonyl-CoA. We used the computational system Hex (51.Ritchie D.W. Kemp G.J.L. Proteins Struct. Funct. Genet. 2000; 39: 178-194Crossref PubMed Scopus (492) Google Scholar) that uses spherical polar Fourier correlations. Each molecule was modeled using three-dimensional parametric functions which encode both surface shape and electrostatic charge and electrostatic potential distributions. By using an expression for the overlap of pairs of parametric functions, an expression for a docking score can be derived as a function of the six degrees of freedom in a rigid body docking search. With the appropriate scaling factors, the docking score can be interpreted as an interaction energy that ranks possible docking solutions according to the minimum value. After analysis of the best (minimum energy) solutions given by the program for the interaction between the malonyl-CoA molecule and the structural model for the catalytic core of rat COT, a common position was observed for the five best predicted solutions in terms of low energy of the complex and lowest macromolecular distance. The lowest distance solution is shown in Fig. 1A. The inhibitor molecule fits into a second cavity in the three-dimensional model, which is distinct from the cavity occupied by the substrate. The simultaneous representation of both molecules (decanoyl-CoA (substrate) and malonyl-CoA (inhibitor), (Fig. 1B) offers an explanation for the inhibition process: both molecules compete for a portion of the same spatial groove, despite the different binding site proposed in the model. To analyze the fitting of malonyl-CoA on the surface of the protein, and to improve our definition of this domain, we have performed a site-directed mutagenesis in some of the amino acids involved in this domain. If site-directed mutants show decreased sensitivity to malonyl-CoA, the precise location of this molecule in the COT model would be supported. We expressed COT wild type in S. cerevisiae, an organism devoid of endogenous COT activity, and determined the influence of the concentration of the acyl-CoA substrate, decanoyl-CoA, on the inhibitory pattern produced by malonyl-CoA. As shown in Fig. 2A, there was a competition in COT wild type between the acyl-CoA and the inhibitor, since when the concentration of decanoyl-CoA increased from 5 to 50 or 200 μm, the curves are similar, but the IC50values rose from 3.3 to 76 or 581 μm. We then expressed the COT H131A mutant and compared the kinetics of malonyl-CoA inhibition of this mutant with that of the double mutant H131A/H340A (Fig. 2B). The result of these comparisons shows the particular responsibility of COT His340 in the inhibition. Whereas the double mutant is not inhibited at any malonyl-CoA concentration (1–200 μm), the inhibition of mutant COT H131A was dependent on the concentration of the substrate. A low decanoyl-CoA concentration (5 μm) gives low IC50 values for malonyl-CoA (36 μm). When the substrate concentration is increased to 50 μm or 200 μm the IC50 values increase in a sigmoid kinetic, so that the IC50 values were not calculated. These results implicate His340 in malonyl-CoA inhibition, which was competitive between substrate and inhibitor. They are also consistent with the COT structural model, in which decanoyl-CoA and malonyl-CoA exclude each other. To confirm the model presented in Fig. 1, we mutated another amino acid, in the COT malonyl-CoA domain, to see whether the sensitivity to the inhibitor was modified. COT Ala332 is 8 amino acids away from His340 and therefore could participate in the inhibitory process. Two considerations supported this choice. On the one hand, Ala332 is the first amino acid after the acidic residue (aspartic or glutamic) common to carnitine octanoyl- and palmitoyltransferases (Fig. 3, right). On the other hand, as shown by the tree-determinant analysis of the multiple alignment of the acyltransferase family, alanines at this position are present in all malonyl-CoA inhibitable carnitine acyltransferases (COT and L-CPT I) but absent in non-malonyl-CoA inhibitable carnitine acyltransferases (CPT II and CAT), where they are substituted by glycine. Therefore, we prepared the mutant COT A332G and expressed it in S. cerevisiae. The mutated protein had the same molecular mass as the wild type and was expressed at the same levels (Fig. 4). Moreover, the kinetics toward both carnitine and decanoyl-CoA, were similar to wild type, with normal saturation behavior (Table I). The mutant was measured for malony
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