Structure of Human Carnitine Acetyltransferase
2003; Elsevier BV; Volume: 278; Issue: 15 Linguagem: Inglês
10.1074/jbc.m212356200
ISSN1083-351X
AutoresDonghai Wu, L. Govindasamy, Wei Lian, Yunrong Gu, Thomas Kukar, Mavis Agbandje‐McKenna, Robert McKenna,
Tópico(s)Mitochondrial Function and Pathology
ResumoCarnitine acyltransferases are a family of ubiquitous enzymes that play a pivotal role in cellular energy metabolism. We report here the x-ray structure of human carnitine acetyltransferase to a 1.6-Å resolution. This structure reveals a monomeric protein of two equally sized α/β domains. Each domain is shown to have a partially similar fold to other known but oligomeric enzymes that are also involved in group-transfer reactions. The unique monomeric arrangement of the two domains constitutes a central narrow active site tunnel, indicating a likely universal feature for all members of the carnitine acyltransferase family. Superimposition of the substrate complex of a related protein, dihydrolipoyl trans-acetylase, reveals that both substrates localize to the active site tunnel of human carnitine acetyltransferase, suggesting the location of the ligand binding sites for carnitine and coenzyme A. Most significantly, this structure provides critical insights into the molecular basis for fatty acyl chain transfer and a possible common mechanism among a wide range of acyltransferases utilizing a catalytic dyad. Carnitine acyltransferases are a family of ubiquitous enzymes that play a pivotal role in cellular energy metabolism. We report here the x-ray structure of human carnitine acetyltransferase to a 1.6-Å resolution. This structure reveals a monomeric protein of two equally sized α/β domains. Each domain is shown to have a partially similar fold to other known but oligomeric enzymes that are also involved in group-transfer reactions. The unique monomeric arrangement of the two domains constitutes a central narrow active site tunnel, indicating a likely universal feature for all members of the carnitine acyltransferase family. Superimposition of the substrate complex of a related protein, dihydrolipoyl trans-acetylase, reveals that both substrates localize to the active site tunnel of human carnitine acetyltransferase, suggesting the location of the ligand binding sites for carnitine and coenzyme A. Most significantly, this structure provides critical insights into the molecular basis for fatty acyl chain transfer and a possible common mechanism among a wide range of acyltransferases utilizing a catalytic dyad. acyl coenzyme A human peroxisomal carnitineO-acetyltransferase carnitine palmitoyltransferase mitochondrial liver isoform of CPT I mitochondrial muscle isoform of CPT I root mean square peroxisomal carnitine O-octanoyltransferase helix strand multi-wavelength anomalous dispersion ornithine decarboxylase In eukaryotic cells, the carnitine system plays a vital role in fatty acid β-oxidation and maintenance of acyl coenzyme A (acyl-CoA)1 pools (1Ramsay R.R. Gandour R.D. Van der Leij F.R. Biochim. Biophys. Acta. 2001; 1546: 21-43Crossref PubMed Scopus (299) Google Scholar, 2Ramsay R.R. Arduini A. Arch. Biochem. Biophys. 1993; 302: 307-314Crossref PubMed Scopus (99) Google Scholar). The important physiological functions of carnitine are made possible by the presence of carnitine/acylcarnitine transporters on cellular membranes and a group of enzymes, the carnitine acyltransferases, which catalyze the reversible transfer of acyl groups between CoA and carnitine (1Ramsay R.R. Gandour R.D. Van der Leij F.R. Biochim. Biophys. Acta. 2001; 1546: 21-43Crossref PubMed Scopus (299) Google Scholar, 2Ramsay R.R. Arduini A. Arch. Biochem. Biophys. 1993; 302: 307-314Crossref PubMed Scopus (99) Google Scholar) as shown in Equation 1.AcylCoA+carnitine=CoA+AcylcarnitineEquation 1 The three known classes of carnitine acyltransferases differ in their acyl group specificity, subcellular localization, tissue distribution, and physiological function (3Bieber L.L. Annu. Rev. Biochem. 1988; 57: 261-283Crossref PubMed Scopus (667) Google Scholar, 4Ramsay R.R. Biochem. Soc. Trans. 2000; 28: 182-186Crossref PubMed Scopus (75) Google Scholar, 5Corti O. Finocchiaro G. Rossi E. Zuffardi O. DiDonato S. Genomics. 1994; 23: 94-99Crossref PubMed Scopus (22) Google Scholar). Carnitine acetyltransferase has a substrate preference for short chain acyl-CoAs and is found in the mitochondrial matrix, the endoplasmic reticulum, and the peroxisome. Carnitine octanoyltransferase (COT) is predominantly localized in peroxisomes with a substrate preference for medium length acyl-CoAs. Carnitine palmitoyltransferases (CPT) are found both in the outer mitochondrial membrane (CPT I) and the mitochondrial matrix (CPT II) with a substrate preference for long chain acyl-CoAs.Arguably, the most critical member is CPT I, which is responsible for facilitating the transfer of long chain fatty acids into the mitochondria. There are two different types of CPT I in mammalian tissues (1Ramsay R.R. Gandour R.D. Van der Leij F.R. Biochim. Biophys. Acta. 2001; 1546: 21-43Crossref PubMed Scopus (299) Google Scholar), a liver (L-CPT I) and a muscle isoform (M-CPT I). Both isoforms of CPT I are inhibited by malonyl-CoA, which is the committed intermediate for the biosynthesis of fatty acids. However, each isoform displays a different sensitivity to this metabolite. Malonyl-CoA inhibition regulates and balances β-oxidation and the biosynthesis of fatty acids, depending on the metabolic needs of the cell. Interestingly, CPT I has an extended N terminus, which contains two trans-membrane domains, that is not found in the other carnitine acyltransferases and appears to be important not only in anchoring the enzyme in the outer mitochondrial membrane but also in modulating sensitivity of the CPT I isoforms to malonyl-CoA (1Ramsay R.R. Gandour R.D. Van der Leij F.R. Biochim. Biophys. Acta. 2001; 1546: 21-43Crossref PubMed Scopus (299) Google Scholar). The important metabolic roles of CPT I make it a potential drug target for diabetes, coronary heart disease, and other disorders involving abnormal fatty acid metabolism (6Finocchiaro G. Taroni F. Rocchi M. Martin A.L. Colombo I. Tarelli G.T. DiDonato S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 661-665Crossref PubMed Scopus (107) Google Scholar, 7Anderson R.C. Curr. Pharm. Des. 1998; 4: 1-16PubMed Google Scholar, 8Wagman A.S. Nuss J.M. Curr. Pharm. Des. 2001; 7: 417-450Crossref PubMed Scopus (178) Google Scholar, 9Zarain-Herzberg A. Rupp H. Expert Opin. Investig. Drugs. 2002; 11: 345-356Crossref PubMed Scopus (40) Google Scholar).However, the lack of a molecular structure for any member of this family has hampered our understanding of the biology of these enzymes and the rational design of selective inhibitors. Here we report the x-ray crystal structure of human peroxisomal carnitine acetyltransferase (hpCAT), which represents the first structure for the carnitine acyltransferase family (TableI). To gain more insight into the function of hpCAT, we have also modeled the binding sites of carnitine and CoA using the superimposition of the ternary substrate complex of a structurally and functionally related protein, dihydrolipoyl trans-acetylase (E2p). The results from this work correlate well with previous experimental data and provide further information regarding the catalytic mechanism of hpCAT and ligand binding sites.Table ICrystallographic data, phasing, and refinement statistics of human peroxisomal carnitine acetyltransferaseData setWavelengthReflections (unique)CompletenessRsym(%)1-aRsym = ΣhldΣi‖Ii(hkl) −i(hkl)‖/ΣhklΣiIt(hkl), where i(hkl) is the average intensity over symmetry equivalent reflections. (outer shell)I/ς(I)1-bCompleteness I/I(ς) > 1.0. (outer shell)Beamline1-cF2-CHESS = Cornell High Energy Synchrotron Source, Wilson laboratory, and native data collected from R-AXIS IV++image plate system.ÅSeMet (edge)0.97941,783,752 (84,270)93.59.3 (49.5)17.5 (3.3)F2-CHESSSeMet (peak)0.97911,465,330 (84,002)93.29.2 (45.1)16.6 (4.4)F2-CHESSSeMet (remote)0.96411,597,584 (83,892)93.08.4 (47.6)17.0 (2.4)F2-CHESSNative1.5487,24,938 (80,622)90.24.3 (40.4)22.8 (2.3)R-axis IV++MAD phasingSeMet (edge)SeMet (peak)SeMet (remote)Rcullis1-dRcullis(iso) = Σ∥FPH −FD‖−‖FPH(c)∥/Σ‖FPH−Fr‖, where FPH and FPH(c) are the obser ved and calculated structure factors of a heavy atom derivative.(iso/ano)−0.830.70/0.690.68/0.70Phasing power1-ePhasing power = Σ <‖Fobs −Fcalc‖/Σ Fobs, where summation is over the data used for refinement.(iso/ano)−1.301.55/1.251.18/0.94FOM(before/after DM)1-fFigure of merit (FOM) = ΣP(α)eia/ΣP(α)‖, where α is the phase and P(α) is the phase probability distribution for MAD phasing (30–1.6 Å).0.49/0.97Refinement statisticsResolutionNumber of reflectionsNumber of non-H atomsRfactor1-gRfactor − Σ(∥FF(obs)‖ − ‖FP(calc)∥/Σ‖FP(obs)‖).Rfree1-hRfree was calculated using 5% data excluded from refinement.Average Br.m.s.d. bondsr.m.s.d. anglesÅ%%ÅÅ%1.677,7315,09520.823.217.2/28.60.0051.255For MAD data, the Bijvoet pairs were not merged.1-a Rsym = ΣhldΣi‖Ii(hkl) −i(hkl)‖/ΣhklΣiIt(hkl), where i(hkl) is the average intensity over symmetry equivalent reflections.1-b Completeness I/I(ς) > 1.0.1-c F2-CHESS = Cornell High Energy Synchrotron Source, Wilson laboratory, and native data collected from R-AXIS IV++image plate system.1-d Rcullis(iso) = Σ∥FPH −FD‖−‖FPH(c)∥/Σ‖FPH−Fr‖, where FPH and FPH(c) are the obser ved and calculated structure factors of a heavy atom derivative.1-e Phasing power = Σ <‖Fobs −Fcalc‖/Σ Fobs, where summation is over the data used for refinement.1-f Figure of merit (FOM) = ΣP(α)eia/ΣP(α)‖, where α is the phase and P(α) is the phase probability distribution for MAD phasing (30–1.6 Å).1-g Rfactor − Σ(∥FF(obs)‖ − ‖FP(calc)∥/Σ‖FP(obs)‖).1-h Rfree was calculated using 5% data excluded from refinement. Open table in a new tab RESULTSThe overall structure of hpCAT reveals a monomeric protein with dimensions of ∼75 × 45 × 45 Å (Fig.1). The tertiary structure consists of 17 helices (H1–H17) and 14 strands (S1–S14), which are arranged into two equally sized α/β domains, I (residues 87–385) and II (residues 9–86 and 386–599) (Fig. 2). The domains are tightly associated with each other by interconnecting loops and helices that form a central tunnel of ∼10-Å diameter that transverses through the molecule. This tunnel defines a solvent-accessible surface in the center of the protein, which constitutes the putative active site for the enzyme. Seventy-six amino acids accounting for 3400 Å2 (15% of the total surface area) are buried at the interface between domains I and II (Fig.1).The hpCAT structure is composed of 39% helix (H1–H17), 13% β-strand (S1–S14), and 48% loops and turns. The most noticeable common feature in domains I and II is an "open-faced" core five-stranded-mixed β-sheet consisting of three centrally located parallel β-strands flanked on either side by an anti-parallel β-strand that adopt characteristic left-handed twists. In domain I, this motif is defined by strands S5, S4, S6, S7, and S8, and in domain II, this motif is defined by strands S14, S13, S11, S9, and S10. The β-sheet of domain I has a sixth strand, S1, that runs anti-parallel to strand S8. Domain II provides an additional seventh strand, S12 (looped out between strands S11 and S13 of its five-stranded β-sheet), to the domain I β-sheet that is adjacent and anti-parallel to strand S1. Therefore, strands S1 and S12 create an interface between the two domains (Fig. 2).The N-terminal region of domain II is comprised of four α-helices (H1–H4). Helices H1 and H2 form an anti-parallel bundle that leads to an extended structure made up of helices H3 and H4 that cross over to domain I and contribute part of the floor and front surface of the active site tunnel. Domain I is defined from residues 87 to 385. Residues 306–320 constitute an anti-parallel pair of strands (S7 and S8) linked by a type II turn, which forms the rostra lining on the left side of the active site. An active site histidine (residue 322) is located on a turn at the end of strand S8 and is positioned in the center of the active site tunnel. This turn (residues 321–326) leads to helix H10 that lines the active site tunnel, which is followed by a loop (located behind the β-sheet in domain I) that forms the upper lip of an extended surface groove. Helix H11 is next lying on the front portion of the β-sheet of domain I. A final extended loop after H11 crosses into domain II. This loop leads into helix H12 that runs parallel with the β-sheet motif of domain II. β-Strands S11–S14 form an intricate hydrogen-bonding network with each other to facilitate the "flipping out" of strand S12 to align it with strand S1 in domain I. Four helices, H13-H16, between strands S10 and S11 create a supporting scaffold that positions the domain II β-sheet open face toward the active site and constitutes the top and right lining of the tunnel. Domain II terminates with helix H17 that also supports the β-sheet (Figs. 1 and 2).The structural arrangement of hpCAT appears to be unique for an acyltransferase, although similar structural elements to portions of domains I and II have been observed in some oligomeric enzymes that catalyze similar group-transfer reactions (Fig.3). The secondary structural elements, helices H5, H6, and H10 and strands S1, S4, S5, S6, S7, and S8 of domain I, share structural similarity to the catalytic domain of dihydrolipoyl trans-acetylase (E2p) (116 Cα atoms structurally align with an r.m.s. deviation of 2.0 Å) (Protein Data Bank code 1EAB) (for review see Ref. 24Mattevi A. Obmolova G. Schulze E. Kalk K.H. Westphal A.H. de Kok A. Hol W.G. Science. 1992; 255: 1544Crossref PubMed Scopus (227) Google Scholar). In domain II, helices H12, H13, and H17 and strands S11, S13, and S14 are structurally similar to portions of ornithine decarboxylase (ORD) (55 Cα atoms structurally align with an r.m.s. deviation of 2.1 Å) (Protein Data Bank code 1ORD) (for review see Ref. 25Momany C. Ernst S. Ghosh R. Chang N.L. Hackert M.L. J. Mol. Biol. 1995; 252: 643-655Crossref PubMed Scopus (111) Google Scholar).In addition to structural features, E2p also displays functional similarity to hpCAT. E2p is a component of the multienzyme pyruvate dehydrogenase complex and is responsible for the transfer of acetyl groups between lipoamide and CoA. The catalytically active form of E2p is a trimer with the active site located in the subunit interface. This interaction forms an active site channel for CoA and acetyl lipoamide to bind and is the location of His610, which serves as a general base for catalysis in E2p (24Mattevi A. Obmolova G. Schulze E. Kalk K.H. Westphal A.H. de Kok A. Hol W.G. Science. 1992; 255: 1544Crossref PubMed Scopus (227) Google Scholar). A number of studies have also suggested that the carnitine acyltransferases utilize a histidine as a general base for catalysis (1Ramsay R.R. Gandour R.D. Van der Leij F.R. Biochim. Biophys. Acta. 2001; 1546: 21-43Crossref PubMed Scopus (299) Google Scholar). Based on their structural and functional similarity, the ternary complex of E2p with both substrates (lipoamide and CoA) was superimposed onto domain I of the hpCAT structure (see "Experimental Procedures"). The overlay of these two structures results in the direct superimposition of the catalytic residue His610 of E2p (23Falquet L. Pagni M. Bucher P. Hulo N. Sigrist C.J. Hofmann K. Bairoch A. Nucleic Acids Res. 2002; 30: 235-238Crossref PubMed Scopus (900) Google Scholar) onto His322 of hpCAT, which is completely conserved (Fig.4) and has been previously identified as essential for enzymatic activity in other carnitine acyltransferases (1Ramsay R.R. Gandour R.D. Van der Leij F.R. Biochim. Biophys. Acta. 2001; 1546: 21-43Crossref PubMed Scopus (299) Google Scholar, 28Brown N.F. Anderson R.C. Caplan S.L. Foster D.W. McGarry J.D. J. Biol. Chem. 1994; 269: 19157-19162Abstract Full Text PDF PubMed Google Scholar, 29Morillas M. Gomez-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 (42) Google Scholar). In hpCAT, this histidine residue is located on a turn at the end of strand S8 and is positioned in the center of the active site tunnel (Fig. 6).Figure 4Sequence alignment of all human carnitine acyltransferases. The amino acid numbering and secondary structural elements are based on the structure of hpCAT. Strands S1–S14 (arrows) and helices H1–H17 (cylinders). The extended N terminus of CPT I is not shown. Domains I and II arecolored blue and orange. Identical and conserved residues are indicated by red and yellow blocks, respectively. Residues known to be associated with carnitine binding and CoA binding are indicated by solid colored circles inblue and orange. Residues essential for the activity of the acyltransferases are shown in green (for review see Ref. 1Ramsay R.R. Gandour R.D. Van der Leij F.R. Biochim. Biophys. Acta. 2001; 1546: 21-43Crossref PubMed Scopus (299) Google Scholar).View Large Image Figure ViewerDownload (PPT)Figure 6Modeled binding sites of carnitine and CoA inhpCAT. Coil diagram highlighting the regions of hpCAT shows structural homology with dihydrolipoyl trans-acetylase (red color) and ornithine decarboxylase (yellow color). Ball and stick models of carnitine and CoA are shown on opposite sides of the catalytic His322 in the active site tunnel of hpCAT.View Large Image Figure ViewerDownload (PPT)The location of His322 in the center of the catalytic tunnel allows access to this essential residue from either side, suggesting that the binding sites for CoA and carnitine lie on opposite sides of this tunnel (Fig. 6). Additionally, the superimposition of the ternary complex of E2p places lipoamide and CoA into the active site tunnel of hpCAT. The putative location of the separate binding sites for carnitine (Fig. 7A) and acetyl-CoA (Fig.7B) were then identified by modeling their positions based on the locations of lipoamide and CoA using the superimposition of the E2p complex onto hpCAT. The orientation of the binding pockets allows each substrate to approach the active site tunnel from opposite sides independently, which is consistent with the rapid equilibrium random order kinetics proposed for this enzyme (30Chase J.F. Tubbs P.K. Biochem. J. 1966; 99: 32-40Crossref PubMed Scopus (59) Google Scholar).Figure 7The active site of human peroxisomal carnitine acetyltransferase and putative binding sites of carnitine (A) and CoA (B). The residues surrounding carnitine are shown (His322, Glu326, Tyr431, Arg443, Thr444, Ser531, and Thr532) (A). The residues surrounding CoA are shown (Ser533, Leu142, and Lys398, Ser407, and Asp487) (B).View Large Image Figure ViewerDownload (PPT)DISCUSSIONThe two domain structural architecture of hpCAT represents a general model for the structures of other carnitine acyltransferases based on their sequence homology (Fig. 4). There is a 21–31% identity at the amino acid level among the different human carnitine acyltransferases (1Ramsay R.R. Gandour R.D. Van der Leij F.R. Biochim. Biophys. Acta. 2001; 1546: 21-43Crossref PubMed Scopus (299) Google Scholar). COT shares the highest homology with hpCAT followed by CPT II and CPT I. Of note are two consensus sequences that have been defined for these acyltransferases (23Falquet L. Pagni M. Bucher P. Hulo N. Sigrist C.J. Hofmann K. Bairoch A. Nucleic Acids Res. 2002; 30: 235-238Crossref PubMed Scopus (900) Google Scholar). These are Prosite PS00439 (residues 14–28 in hpCAT) and PS00440 (residues 299–327 in hpCAT), which form characteristic supersecondary structural motifs of an extended proline rich region in helix H1 of domain II and strands S7 and S8 of domain I, respectively (Fig.5). Previously, PS00439 was believed to be important for carnitine binding (6Finocchiaro G. Taroni F. Rocchi M. Martin A.L. Colombo I. Tarelli G.T. DiDonato S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 661-665Crossref PubMed Scopus (107) Google Scholar); however, our structure shows that this is not the case and instead plays a structural role. On the other hand, PS00440 is located in the center of the catalytic tunnel and contains the active site histidine. The regions of highest homology are clustered in domain II, implying potential locations of common structural and/or functional significance such as the binding sites for shared substrates. On the contrary, domain I is more divergent and may contain structural differences for recognition of variable length acyl-CoAs and the physiological regulator malonyl-CoA.Figure 5Functional residues of carnitine acyltransferases. Coil diagram highlights the locations of the two consensus sequences for the carnitine acyltransferases gene family. Prosites PS00439 (residues 14–28, yellow) and PS00440 (residues 299–327, red) are represented by colored coils. Residues known to be associated with carnitine binding and CoA binding are indicated by solid colored circles in blueand orange. Residues essential for the activity of the acyltransferases are shown in green (as in Fig. 3). His322 is shown in the center of the catalytic tunnel active site.View Large Image Figure ViewerDownload (PPT)Active Site Tunnel and Catalytic HistidineThe side chain of His322 of hpCAT adopts an unusual conformation (χ1 = −140°; χ2 = −39°) to form an intraresidue hydrogen bond (2.7 Å) between the imidazole nitrogen N1 and the backbone carbonyl oxygen. In E2p as well as chloramphenicol acetyltransferase, which also catalyzes an acyl group transfer, a similar conformation of the active site histidine has been observed and may represent a conserved structural feature that facilitates catalysis (23Falquet L. Pagni M. Bucher P. Hulo N. Sigrist C.J. Hofmann K. Bairoch A. Nucleic Acids Res. 2002; 30: 235-238Crossref PubMed Scopus (900) Google Scholar, 26Leslie A.G.W. Moody P.C.E. Shaw W.V. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4133-4137Crossref PubMed Scopus (132) Google Scholar). This unusual angle most probably allows positioning of the imidazole N3 to align with carnitine, enabling the extraction of a proton from its primary alcohol. This is consistent with the finding that the reaction of carnitine acetyltransferase with bromoacetylcarnitine only produces labeled N3 carboxymethyl histidine (27Chase J.F. Tubbs P.K. Biochem. J. 1970; 116: 713-720Crossref PubMed Scopus (30) Google Scholar). Additionally, the histidine at this position is highly conserved among this enzyme family, and the removal of the corresponding homologous residues, His473, His372, and His327 in rat CPT I, CPT II, and COT, respectively (Fig.4), has been shown to inactivate these enzymes completely (28Brown N.F. Anderson R.C. Caplan S.L. Foster D.W. McGarry J.D. J. Biol. Chem. 1994; 269: 19157-19162Abstract Full Text PDF PubMed Google Scholar, 29Morillas M. Gomez-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 (42) Google Scholar). When His322 was changed to an alanine, a serine, or a glutamine, the mutant forms of hpCAT were completely inactive, supporting its catalytic role. 2Y. Gu, T. Kukar, and D. Wu, unpublished observations. Catalytic DyadThe modeling of carnitine into the active site reveals a number of residues that may be involved in binding and catalysis. Most importantly, the N3 nitrogen of the catalytic His322 is positioned such that it can form a hydrogen bond and abstract a proton from the β-hydroxyl of carnitine. In addition, a functionally conserved glutamate, Glu326, interacts with His322 and appears to be critical for catalysis (Fig.7A). Carbonyl oxygen 1 (O1) of Glu326 is positioned 4.03 Å from the N3 nitrogen of His322, whereas O2 forms a salt bridge with Arg443 (2.78 Å). This interaction probably further potentiates the activity of His322 and also stabilizes the positive charge that would develop after substrate deprotonation. In almost all of the other members of the family, an aspartate is found at this position, which could functionally substitute for glutamate. In rat CPT II, substituting Asp376, which is homologous to Glu326 in hpCAT, to an alanine produced a completely inactive protein, illustrating the important role of this negative charge in catalysis (28Brown N.F. Anderson R.C. Caplan S.L. Foster D.W. McGarry J.D. J. Biol. Chem. 1994; 269: 19157-19162Abstract Full Text PDF PubMed Google Scholar). Similar interactions have been observed in other enzyme active sites that utilize a Ser-His-Asp catalytic triad including a variety of proteases and lipases as well as acetylcholine esterase (31Polgar L. FEBS Lett. 1992; 311: 281-284Crossref PubMed Scopus (58) Google Scholar, 32Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2397) Google Scholar). Thus, hpCAT as well as the other carnitine acyltransferases appear to utilize a His-Glu/Asp catalytic dyad to carry out catalysis.Proposed Binding Sites for Carnitine and CoASeveral conserved residues in carnitine acyltransferases, in particular, Tyr431 and Thr444 of hpCAT, lie within hydrogen-bonding distance of the O1 and O2 of carnitine and appear to interact directly with the carnitine carboxylate group (Fig.7A). Previously, it has been speculated that a positively charged amino acid forms a salt bridge with the carboxylate group of carnitine in the active site. In bovine COT, the mutation of a conserved arginine to asparagine decreased the binding of carnitine with a greater than 1650-fold increase in the apparentKm(carnitine) (33Cronin C.N. Eur. J. Biochem. 1997; 247: 1029-1037Crossref PubMed Scopus (33) Google Scholar). In hpCAT, the equivalent residue, Arg497, may fulfill this role by forming an electrostatic interaction with carnitine because it is ∼5 Å away. Other naturally occurring and site-directed mutations have been shown to affect carnitine binding (Figs. 4 and 5) (for review see Ref. 1Ramsay R.R. Gandour R.D. Van der Leij F.R. Biochim. Biophys. Acta. 2001; 1546: 21-43Crossref PubMed Scopus (299) Google Scholar).The modeled binding site of CoA (Figs. 6and 7B) reveals that the adenine ring binds to the outer portion of the catalytic tunnel with the pantothenic arm extending into the active site. The reactive sulfur group is positioned for catalysis close to both His322 and the β-hydroxyl of carnitine. Some highly conserved residues cluster around this site and may be involved in CoA binding. The positive charge of Lys398 probably interacts with a negatively charged phosphate group, whereas Leu142 may bind CoA through hydrophobic interactions with the methyl groups of the pantothenic arm. Ser407 and Asp487 are within hydrogen-bonding distance of the modeled position of CoA and thus may contribute to CoA binding. Additional residues that have been shown to be associated with CoA binding and enzymatic activity in hpCAT and other members of the carnitine acyltransferases cluster in the vicinity of the active site as shown in Figs. 4 and 5 (for review see Ref.1Ramsay R.R. Gandour R.D. Van der Leij F.R. Biochim. Biophys. Acta. 2001; 1546: 21-43Crossref PubMed Scopus (299) Google Scholar).Transition State StabilizationThe structure of hpCAT also allows the identification of another critical class of active site residues, those that contribute to the stabilization of the putative transition state intermediate. In chloramphenicol acetyltransferase, which appears to utilize a similar mechanism as hpCAT for acyl transfer, a serine and a water molecule hydrogen-bonded to a threonine stabilize the transition state (34Shaw W.V. Leslie A.G. Annu. Rev. Biophys. Biomol. Struct. 1991; 20: 363-386Crossref Scopus (109) Google Scholar). Interestingly, a Ser-Thr-Ser motif, which corresponds to Ser531, Thr532, and Ser533 in hpCAT, is completely conserved in this family (Fig. 4). Substitution of the first serine and threonine to an alanine in bovine COT increased the Km toward carnitine by factors of 15 and 80 without significantly changingkcat (35Cronin C.N. Biochem. Biophys. Res. Commun. 1997; 238: 784-789Crossref PubMed Scopus (18) Google Scholar). The replacement of the last serine did not affect the Km for carnitine but reducedkcat by a factor of 10. This finding suggests that the first serine and threonine are involved in carnitine binding, whereas the last serine contributes to transition state stabilization. In the structure of hpCAT, all three of these residues are in the active site close to the modeled position of carnitine. Ser531 approaches the quaternary amine group and Thr532 is near the carboxylate moiety. When a putative transition state analog of carnitine and CoA is modeled into the active site, Ser533 is in the proper orientation to interact with the oxyanion of this intermediate, supporting the role of Ser533 in transition state stabilization, although other residues may also be involved.Comparison to Other Carnitine AcyltransferasesDetermination of the structure of hpCAT also provides a more realistic model for the study of other members of the acyltransferase family. Recently, Morillas et al. (29Morillas M. Gomez-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 (42) Google Scholar, 36Morillas M. Gomez-Puertas P. Rubi B. Clotet J. Arino J. Valencia A. Hegardt F.G. Serra D. Asins G. J. Biol. Chem. 2002; 277: 11473-11480Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) reported a model of the active sites of CPT I and COT based on the structure of enoyl-CoA hydratase, which included ∼200 amino acids surrounding the putative catalytic histidine. This model predicts that Ala381 in CPT I (Ala238 in COT) is positioned in the vicinity of the acti
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