Báo cáo khoa học: Insights into substrate and product traffic in the Drosophila melanogaster acetylcholinesterase active site gorge by enlarging a back channel ppt

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Insights into substrate and product traffic in theDrosophila melanogaster acetylcholinesterase active sitegorge by enlarging a back channelFlorian Nachon1, Jure Stojan2and Didier Fournier31De´partement de Toxicologie, CRSSA, Grenoble, France2 Institute of Biochemistry, Medical Faculty, Ljubljana, Slovenia3 IPBS, Universite´Paul Sabatier ⁄ CNRS, Toulouse, FranceAcetylcholinesterase (EC is a serine hydrolasethat catalyzes the cleavage of acetylcholine. Structuralstudies have revealed that its active site is buried in a20 A˚deep gorge with a bottleneck [1]. According toa recently developed kinetic model, substrate andproduct molecules follow the same path [2]. A sub-strate molecule first binds to the peripheral site (PAS)at the entrance of the gorge [3] and slides down tothe acylation site (CAS), where it is hydrolyzed andthe products escape the gorge via the entrance. Theactive site gorge is too narrow to allow the crossingbetween a substrate molecule en route to the CASand a product molecule en route to the exit. Conse-quently, at very high substrate concentrations, thereis a traffic jam preventing the exit of the reactionproduct through the main entrance, resulting in inhi-bition [4].However, molecular dynamics experiments have pro-vided evidence for a loop movement leading to the for-mation of a back door suitable for product exit [5].Locking the loop with salt or disulfide bridges [6,7]had no significant effect on the kinetics parameters,indicating that exit through the back door is not themain exit route for the product. However, residualactivity upon fasciculin binding suggests that the backdoor route might become the most important routewhen the main entrance is blocked [8,9]. Recent kineticcrystallography studies provide some structural insightsregarding the putative backdoor. Conformationchanges of Trp84, which belongs to the backdoorregion of Torpedo californica acetylcholinesterase, sug-gest that this residue might behave like a revolvingdoor [10]. In addition, Nachon et al. [11] reported thatthe back door region of the Drosophila acetylcholines-Keywordsacetycholinesterase; back door; inhibition;substrate; trafficCorrespondenceF. Nachon, Unite´d’enzymologie,De´partement de Toxicologie, Centre deRecherches du Service de Sante´desArme´es (CRSSA), 24 Avenue des Maquisdu Gre´sivaudan, 38700 La Tronche, FranceFax: +33 476636962Tel: +33 476639765E-mail: fnachon@crssa.net(Received 18 December 2007, revised 14March 2008, accepted 18 March 2008)doi:10.1111/j.1742-4658.2008.06413.xTo test a product exit differing from the substrate entrance in the activesite of acetylcholinesterase (EC, we enlarged a channel located atthe bottom of the active site gorge in the Drosophila enzyme. Mutation ofTrp83 to Ala or Glu widens the channel from 5 A˚to 9 A˚. The kinetics ofsubstrate hydrolysis and the effect of ligands that close the main entrancesuggest that the mutations facilitate both product exit and substrateentrance. Thus, in the wild-type, the channel is so narrow that the ‘backdoor’ is used by at most 5% of the traffic, with the majority of traffic pass-ing through the main entrance. In mutants Trp83Ala and Trp83Glu,ligands that close the main entrance do not inhibit substrate hydrolysisbecause the traffic can pass via an alternative route, presumably theenlarged back channel.AbbreviationsCAS, acylation site; DmAChE, Drosophila acetylcholinesterase; PAS, peripheral site.FEBS Journal 275 (2008) 2659–2664 ª 2008 The Authors Journal compilation ª 2008 FEBS 2659terase (DmAChE) is much less stabilized than that ofother cholinesterases, such as Torpedo californicaacetylcholinesterase. Indeed, two key residues for thestabilization of Trp84 are not conserved in DmAChE:Met83, which stabilizes Trp84 in the Torpedo enzymethrough sulfur-p interactions, is replaced by an isoleu-cine; Tyr442, which hydroxyl bridges Trp84 to Trp432and Gly80 via hydrogen bonding, is replaced by anaspartate that is also much less bulky (Fig. 1). In theabsence of these stabilizing elements, Trp83 ofDmAChE is prone to oscillations between two alter-nate conformations, as shown by the crystal structures(protein databank codes 1DX4 and 1QO9). One ofthese conformations results in the formation of a chan-nel approximately 5 A˚in diameter, connecting thegorge to the bulk solvent (Fig. 2A).The present study aimed to progressively enlarge thischannel by mutating Trp83 to Tyr, Glu or Ala to testFig. 1. View of the back door region from the outside of DmAChE(residues and labels in green) and Torpedo californica acetylcholin-esterase (residues and labels in fushia). Residues are representedby sticks. The hydrogen bonds involving the hydroxyl of Tyr442 areindicated by a yellow dash.ABFig. 2. View of the back channel from the active site gorge of wild-type DmAChE (A) and Trp83Ala mutant (B). The protein databankcode for wild-type DmAChE is 1DX4. Residues delimiting the holeare represented by sticks. The solvent accessible surface is repre-sented by a mesh.E K p SpESpEK L ESEAS p ESS p EAEASS S S K p k 2 b k 2 k 3 E a k 3 K p K L L CholineCholineSpEASK p AcetateAcetateS Scheme 1. Reaction scheme for the hydro-lysis of acetylthiocholine by DmAChE. S,acetylthiocholine; E, free enzyme; EA,acetylated enzyme. All other intermediatesrepresent enzyme–substrate complexes andthe subscript ‘p’ denotes the substratebound to the peripheral anionic site.Back channel of Drosophila acetylcholinesterase F. Nachon et al.2660 FEBS Journal 275 (2008) 2659–2664 ª 2008 The Authors Journal compilation ª 2008 FEBSthe effect of an open back door on the kinetics forsubstrate hydrolysis.Results and DiscussionThe effect of substrate concentration on acetylthiocho-line hydrolysis for the four proteins is shown in Fig. 3.These data were fitted using Scheme 1, which permitsthe description of substrate activation and inhibitionin a manner consistent with the structural data[4,12]. The values of the parameters for hydrolysis ofacetylthiocholine by wild-type DmAChE (Table 1) arestrongly restrained because they were deduced fromanalysis of inhibition of substrate hydrolysis and accel-eration of decarbamoylation by substrate analogue [2],inhibition of substrate hydrolysis by reversible inhibi-tors [13–17], hydrolysis of substrate at different tem-peratures [18] and hydrolysis of different substrates[19]. For the purposes of alternative substrate traffic inand out of the active site of DmAChE, the mutationto Tyr had no significant effect. The pS curves (i.e.curves showing enzyme activity at different substrateconcentrations) for acetylthiocholine hydrolysis by Gluand Ala mutants, however, were shifted to higherconcentrations of substrate and became symmetric.Consequently, the best fits (Fig. 3) were obtained byassuming that mutations did not affect binding ofsubstrate at the peripheral site (Kp), the rate constantfor deacetylation (k3) and the acceleration of deacety-lation (a). Any other assumption resulted in an unsta-ble fitting. Consistently, it appears that substitution ofTrp83 by smaller side chains did not affect deacetyla-tion parameters k3and a because the amino acid atposition 83 is too far away from the activated watermolecule during deacetylation. As expected, the maindifference is the affinity for the catalytic site Kc(= Kp· KL) because Trp83 is the main component ofsubstrate stabilization at the catalytic site via cation-pinteraction with the quaternary ammonium moiety ofacetylthiocholine. This is consistent with the highapparent Kmreported for the same mutation in humanacetylcholinesterase [20,21], although the differenceappears at different magnitudes. In addition, mutationof this Trp to Glu or Ala decreased acylation (k2),as reported for human butyrylcholinesterase [22]. Acet-ylation may be subdivided into three steps: accommo-dation of substrate at the CAS, chemicaltransesterification and choline exit. In regard to theeffect on affinity, we can hypothesize that mutationsmodify accommodation of the substrate.Another striking difference is parameter b, whichrepresents the effect of substrate bound at the periph-eral site on acylation and choline exit (Scheme 1).Parameter b for the wild-type enzyme is estimated at0.050 ± 0.025 (i.e. acylation step is reduced to 5%when a substrate molecule is bound to the PAS). Thetraffic of choline outside the gorge is blocked when thePAS is occupied and choline stays inside the activesite, resulting in inhibition [9]. Parameter b is signifi-cantly different from zero, and no combination ofparameters leading to a satisfactory fit can be obtainedif b is restrained to zero. This suggests that an alterna-tive exit for choline may exist when the PAS is occu-pied by a substrate molecule but would account forapproximately 5% of choline traffic. Factor b for theTrp83Ala mutant is estimated at 1.05 (Table 1). Theacetylation step is not reduced in this mutant, suggest-ing that choline can freely exit despite the entrance ofthe gorge being occupied by a substrate molecule. Thisis expected because mutation Trp83Ala enlarges thechannel by up to 9 A˚, thus facilitating the passage ofcholine (Fig. 2B). In the case of the Glu mutation, theb value is linked to the k2value and thus both cannotbe estimated independently. However, if b is set to 1(i.e. the symmetry of the pS curve supporting it), theTable 1. Kinetics parameters obtained for the various mutants.Wild-type Trp83Glu Trp83Alak2(s)1) 52 000 ± 26 000 1818 ± 130 689 ± 30k3(s)1) 396 ± 77 396a396aKp(mM) 0.175 ± 0.02 0.175a0.175aKL4.08 ± 2.41 38.2 ± 7.8 13.1 ± 2.4KLL177 ± 33 851 ± 1.63 336 ± 63a 3.44 ± 0.18 3.44a3.44ab 0.0498 ± 0.0247 1a1.05 ± 0.53aParameters restrained in the simulation.10100 1000 10000100 00002004006008001000WYEAATCh (µM)v/Et (s–1)Fig. 3. Activity of the wild-type (W) and mutated DmAChEs (Y, E,A) at different acetylthiocholine concentrations (pS curves). Theoret-ical curves were calculated according to the Scheme 1 specific rateequation, using the corresponding kinetic parameters from Table 1.F. Nachon et al. Back channel of Drosophila acetylcholinesteraseFEBS Journal 275 (2008) 2659–2664 ª 2008 The Authors Journal compilation ª 2008 FEBS 2661fit is satisfactory. This suggests that choline can exitfreely through the back channel as in the alaninemutant.According to Scheme 1, inhibition by excess ofsubstrate does not only originate from inhibitionof choline exit (b < 1), but also from inhibition ofdeacetylation following the sliding of a molecule ofsubstrate inside the acetylated active site and occupa-tion of the peripheral site by a second substrate mole-cule (complex SpEAS does not deacetylate inScheme 1). This mechanism was suggested by excesssubstrate inhibition of decarbamoylation and the crys-tal structure of the SpEAS obtained by soaking crystalsin a solution containing a high substrate concentration[2,4]. This second mechanism remains active in the Gluand Ala mutants because inhibition was observed at ahigh substrate concentration (Fig. 3). We observed ashift of the pS curve towards higher substrate concen-trations due to the lower affinity of both the free andacetylated mutated active sites.If choline could leave the active site by the backchannel, we might also hypothesize that acetylcholineenters using the same path. To test this hypothesis, weused two inhibitors specific for the peripheral site thatbind to Trp321 close to the entrance: propidium andaflatoxin B1. In the wild-type DmAChE, the affinity ofpropidium for the peripheral site is estimated to be80 pm, and the affinity for aflatoxin to be 3.5 lm,when considering competition between the substrateand inhibitor only at the PAS (Fig. 4A). However,inhibition is completely abolished by the substitutionof Trp83 by Ala or Glu. It should be strongly empha-sized at this point that, according to the proposedreaction scheme (Scheme 1) enlarged by the binding ofinhibitor to the PAS, inhibition at low substrate con-centrations should always be observed. Therefore, thecomplete absence of inhibition by peripheral ligandsdoes not originate from changes in substrate hydrolysisparameters (Table 1), and the simulation can readilyconfirm this. The loss of inhibition following muta-tions of Trp83 might be interpreted as a strongdecrease in affinity of ligand for the peripheral site,resulting from a hypothetic allosteric interaction[23,24]. However, binding to the peripheral site wasnot affected by mutations, as demonstrated by changesin fluorescence. Furthermore, considering that inhibi-tion arises because inhibitors bound to the PAS hinderthe entrance of acetylcholine to the CAS in the wild-type enzyme, it appears that, in the two mutants(Trp83Ala ⁄ Glu), inhibitors that bind to the PAS didnot prevent the entrance of substrate into the activesite. At this point, a plausible explanation is that thesubstrate may enter by an alternative route (i.e. theback channel at the bottom of active site). Thishypothesis is in accordance with reported resultsobserved with Trp83Ala mutants: the strong decreaseof inhibition of propidium [21] and the increase ofremaining activity upon peripheral site saturation byfasciculin [24].Finally, minor deviations of pS curves upon bindingof inhibitors on the PAS (Fig. 4B,C), may be assigned10 100 1000 10 000 100 00002004006008001000ABCprop10 µMAfl50 µMrefprop1 µMprop10 µMAfla10 µMAfla50 µMprop1 µMprop10 µMAfla10 µMAfla50 µMrefATCh ( M)10 100 1000 10 000 100 000ATCh ( M)10 100 1000 10 000 100 000ATCh ( M)01002003004000200400600refv/Et (s–1)v/Et (s–1)v/Et (s–1)Fig. 4. Effect of closing the entrance of the active site with ligands(propidium or aflatoxin) on activity of wild-type DmAChE (A),Trp83Ala (B) and Trp83Glu (C) mutants.Back channel of Drosophila acetylcholinesterase F. Nachon et al.2662 FEBS Journal 275 (2008) 2659–2664 ª 2008 The Authors Journal compilation ª 2008 FEBSto an allosteric interaction between PAS and CAS[8,21,25], to a lower efficiency of the alternative routecompared to the main entrance, or to a partial overlapof the side chain of propidium and aflatoxin with theactive site as it may span into the gorge.Experimental proceduresProtein production and purificationMutations were introduced by site-directed mutagenesisusing the QuickChange XL kit following the manufac-turer’s instructions (Stratagene, La Jolla, CA, USA). ThecDNA encoding DmAChE and mutants were expressedwith the baculovirus system [26]. We expressed a solubledimeric form deprived of a hydrophobic peptide at theC-terminal and with a 3· histidine tag replacing the loopfrom amino acids 103–136. This external loop is at theopposite side of the molecule with respect to the activesite entrance and its deletion does not affect the activityor the stability of the enzyme. Secreted acetylcholinesteras-es were purified to homogeneity using the following steps:ammonium sulfate precipitation, ultrafiltration with a10 kDa cut-off membrane, affinity chromatography withprocainamide as a ligand, nitrilotriacetic acid-nickel chro-matography and anion exchange chromatography [27].Residue numbering follows that of the mature protein.The concentrations of the enzymes were determined byactive site titration using high affinity irreversible inhibi-tors [28].Enzyme activityData acquisition and kinetics were performed with the sub-strate acetylthiocholine as previously described [18]. Briefly,the enzymatic and non-enzymatic hydrolysis of acetylthi-ocholine by the wild-type DmAChE and its three W83mutants was followed using Ellman’s method [29]. Theinitial rate measurements were performed at acetylthiocho-line concentrations from 2 lm to 500 mm in the absenceand presence of two ligands known to close the entrance ofthe active site. We used 1 and 10 lm propidium and 10 and50 lm aflatoxin. The activity was followed for 1 min afterthe addition of acetylcholinesterase to the mixture, and thespontaneous hydrolysis of the substrate was subtracted, ifpresent. Each measurement was repeated at least fourtimes. The experiments were carried out at 25 °Cin25mmphosphate buffer (pH 7.0) without ionic strength compensa-tion to avoid interference with electrostatic components ofbinding and chemical steps of the reaction. Analysis of thekinetics data were performed using gosa-fit, software thatis based on a simulated annealing algorithm (BioLog,Toulouse, France; http://www.bio-log.biz). For analysis ofinitial rate data in the absence of inhibitors, we used thespecific equation in Scheme 1. The effect of two ligands onthe activity of the wild-type DmAChE was evaluated by theequation in Scheme 1 enlarged by the intermediates, repre-senting the competition between the substrate and inhibitorat the peripheral site [2].References1 Sussman JL, Harel M, Frolow F, Oefner C, GoldmanA, Toker L & Silman I (1991) Atomic structure of ace-tylcholinesterase from Torpedo californica: a prototypicacetylcholine-binding protein. Science 253, 872–879.2 Stojan J, Brochier L, Alies C, Colletier JP & FournierD (2004) Inhibition of Drosophila melanogaster acetyl-cholinesterase by high concentrations of substrate. EurJ Biochem 271, 1364–1371.3 Mallender WD, Szegletes T & Rosenberry TL (2000)Acetylthiocholine binds to asp74 at the peripheral siteof human acetylcholinesterase as the first step in thecatalytic pathway. 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J Biol Chem 270, 19694–19701.26 Chaabihi H, Fournier D, Fedon Y, Bossy JP, RavallecM, Devauchelle G & Cerutti M (1994) Biochemicalcharacterization of Drosophila melanogaster acetylcho-linesterase expressed by recombinant baculoviruses.Biochem Biophys Res Commun 203, 734–742.27 Estrada-Mondaca S & Fournier D (1998) Stabilizationof recombinant Drosophila acetylcholinesterase. ProteinExpr Purif 12, 166–172.28 Charpentier A, Menozzi P, Marcel V, Villatte F &Fournier D (2000) A method to estimate acetylcholines-terase-active sites and turnover in insects. Anal Biochem285, 76–81.29 Ellman GL, Courtney KD, Andres V Jr & Feather-Stone RM (1961) A new and rapid colorimetric deter-mination of acetylcholinesterase activity. BiochemPharmacol 7, 88–95.Back channel of Drosophila acetylcholinesterase F. Nachon et al.2664 FEBS Journal 275 (2008) 2659–2664 ª 2008 The Authors Journal compilation ª 2008 FEBS . Insights into substrate and product traffic in the Drosophila melanogaster acetylcholinesterase active site gorge by enlarging a back channel Florian Nachon1,. hydrolysisbecause the traffic can pass via an alternative route, presumably the enlarged back channel. AbbreviationsCAS, acylation site; DmAChE, Drosophila acetylcholinesterase;
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