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

Insights into substrate and product traffic in the Drosophila melanogaster acetylcholinesterase active site gorge by enlarging a back channel Florian Nachon 1 , Jure Stojan 2 and Didier Fournier 3 1De ´ partement de Toxicologie, CRSSA, Grenoble, France 2 Institute of Biochemistry, Medical Faculty, Ljubljana, Slovenia 3 IPBS, Universite ´ Paul Sabatier ⁄ CNRS, Toulouse, France Acetylcholinesterase (EC 3.1.1.7) is a serine hydrolase that catalyzes the cleavage of acetylcholine. Structural studies have revealed that its active site is buried in a 20 A ˚ deep gorge with a bottleneck [1]. According to a recently developed kinetic model, substrate and product 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 to the acylation site (CAS), where it is hydrolyzed and the products escape the gorge via the entrance. The active site gorge is too narrow to allow the crossing between a substrate molecule en route to the CAS and a product molecule en route to the exit. Conse- quently, at very high substrate concentrations, there is a traffic jam preventing the exit of the reaction product 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 the main exit route for the product. However, residual activity upon fasciculin binding suggests that the back door route might become the most important route when the main entrance is blocked [8,9]. Recent kinetic crystallography studies provide some structural insights regarding the putative backdoor. Conformation changes of Trp84, which belongs to the backdoor region of Torpedo californica acetylcholinesterase, sug- gest that this residue might behave like a revolving door [10]. In addition, Nachon et al. [11] reported that the back door region of the Drosophila acetylcholines- Keywords acetycholinesterase; back door; inhibition; substrate; traffic Correspondence F. Nachon, Unite ´ d’enzymologie, De ´ partement de Toxicologie, Centre de Recherches du Service de Sante ´ des Arme ´ es (CRSSA), 24 Avenue des Maquis du Gre ´ sivaudan, 38700 La Tronche, France Fax: +33 476636962 Tel: +33 476639765 E-mail: fnachon@crssa.net (Received 18 December 2007, revised 14 March 2008, accepted 18 March 2008) doi:10.1111/j.1742-4658.2008.06413.x To test a product exit differing from the substrate entrance in the active site of acetylcholinesterase (EC 3.1.1.7), we enlarged a channel located at the bottom of the active site gorge in the Drosophila enzyme. Mutation of Trp83 to Ala or Glu widens the channel from 5 A ˚ to 9 A ˚ . The kinetics of substrate hydrolysis and the effect of ligands that close the main entrance suggest that the mutations facilitate both product exit and substrate entrance. Thus, in the wild-type, the channel is so narrow that the ‘back door’ 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 hydrolysis because the traffic can pass via an alternative route, presumably the enlarged back channel. Abbreviations CAS, acylation site; DmAChE, Drosophila acetylcholinesterase; PAS, peripheral site. FEBS Journal 275 (2008) 2659–2664 ª 2008 The Authors Journal compilation ª 2008 FEBS 2659 terase (DmAChE) is much less stabilized than that of other cholinesterases, such as Torpedo californica acetylcholinesterase. Indeed, two key residues for the stabilization of Trp84 are not conserved in DmAChE: Met83, which stabilizes Trp84 in the Torpedo enzyme through sulfur-p interactions, is replaced by an isoleu- cine; Tyr442, which hydroxyl bridges Trp84 to Trp432 and Gly80 via hydrogen bonding, is replaced by an aspartate that is also much less bulky (Fig. 1). In the absence of these stabilizing elements, Trp83 of DmAChE is prone to oscillations between two alter- nate conformations, as shown by the crystal structures (protein databank codes 1DX4 and 1QO9). One of these conformations results in the formation of a chan- nel approximately 5 A ˚ in diameter, connecting the gorge to the bulk solvent (Fig. 2A). The present study aimed to progressively enlarge this channel by mutating Trp83 to Tyr, Glu or Ala to test Fig. 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 represented by sticks. The hydrogen bonds involving the hydroxyl of Tyr442 are indicated by a yellow dash. A B Fig. 2. View of the back channel from the active site gorge of wild- type DmAChE (A) and Trp83Ala mutant (B). The protein databank code for wild-type DmAChE is 1DX4. Residues delimiting the hole are represented by sticks. The solvent accessible surface is repre- sented by a mesh. E K p S p E S p E K L ES EA S p ES S p EA EAS S S S K p k 2 b k 2 k 3 E a k 3 K p K L L Choline Choline S p EAS K p Acetate Acetate S Scheme 1. Reaction scheme for the hydro- lysis of acetylthiocholine by DmAChE. S, acetylthiocholine; E, free enzyme; EA, acetylated enzyme. All other intermediates represent enzyme–substrate complexes and the subscript ‘p’ denotes the substrate bound 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 FEBS the effect of an open back door on the kinetics for substrate hydrolysis. Results and Discussion The 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 permits the description of substrate activation and inhibition in a manner consistent with the structural data [4,12]. The values of the parameters for hydrolysis of acetylthiocholine by wild-type DmAChE (Table 1) are strongly restrained because they were deduced from analysis 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 in and out of the active site of DmAChE, the mutation to Tyr had no significant effect. The pS curves (i.e. curves showing enzyme activity at different substrate concentrations) for acetylthiocholine hydrolysis by Glu and Ala mutants, however, were shifted to higher concentrations of substrate and became symmetric. Consequently, the best fits (Fig. 3) were obtained by assuming that mutations did not affect binding of substrate at the peripheral site (K p ), the rate constant for deacetylation (k 3 ) and the acceleration of deacety- lation (a). Any other assumption resulted in an unsta- ble fitting. Consistently, it appears that substitution of Trp83 by smaller side chains did not affect deacetyla- tion parameters k 3 and a because the amino acid at position 83 is too far away from the activated water molecule during deacetylation. As expected, the main difference is the affinity for the catalytic site K c (= K p · K L ) because Trp83 is the main component of substrate stabilization at the catalytic site via cation-p interaction with the quaternary ammonium moiety of acetylthiocholine. This is consistent with the high apparent K m reported for the same mutation in human acetylcholinesterase [20,21], although the difference appears at different magnitudes. In addition, mutation of this Trp to Glu or Ala decreased acylation (k 2 ), as reported for human butyrylcholinesterase [22]. Acet- ylation may be subdivided into three steps: accommo- dation of substrate at the CAS, chemical transesterification and choline exit. In regard to the effect on affinity, we can hypothesize that mutations modify accommodation of the substrate. Another striking difference is parameter b, which represents 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 at 0.050 ± 0.025 (i.e. acylation step is reduced to 5% when a substrate molecule is bound to the PAS). The traffic of choline outside the gorge is blocked when the PAS is occupied and choline stays inside the active site, resulting in inhibition [9]. Parameter b is signifi- cantly different from zero, and no combination of parameters leading to a satisfactory fit can be obtained if 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 for approximately 5% of choline traffic. Factor b for the Trp83Ala mutant is estimated at 1.05 (Table 1). The acetylation step is not reduced in this mutant, suggest- ing that choline can freely exit despite the entrance of the gorge being occupied by a substrate molecule. This is expected because mutation Trp83Ala enlarges the channel by up to 9 A ˚ , thus facilitating the passage of choline (Fig. 2B). In the case of the Glu mutation, the b value is linked to the k 2 value and thus both cannot be estimated independently. However, if b is set to 1 (i.e. the symmetry of the pS curve supporting it), the Table 1. Kinetics parameters obtained for the various mutants. Wild-type Trp83Glu Trp83Ala k 2 (s )1 ) 52 000 ± 26 000 1818 ± 130 689 ± 30 k 3 (s )1 ) 396 ± 77 396 a 396 a K p (mM) 0.175 ± 0.02 0.175 a 0.175 a K L 4.08 ± 2.41 38.2 ± 7.8 13.1 ± 2.4 K LL 177 ± 33 851 ± 1.63 336 ± 63 a 3.44 ± 0.18 3.44 a 3.44 a b 0.0498 ± 0.0247 1 a 1.05 ± 0.53 a Parameters restrained in the simulation. 10 100 1000 10000 100 000 0 200 400 600 800 1000 W Y E A ATCh (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 rate equation, using the corresponding kinetic parameters from Table 1. F. Nachon et al. Back channel of Drosophila acetylcholinesterase FEBS Journal 275 (2008) 2659–2664 ª 2008 The Authors Journal compilation ª 2008 FEBS 2661 fit is satisfactory. This suggests that choline can exit freely through the back channel as in the alanine mutant. According to Scheme 1, inhibition by excess of substrate does not only originate from inhibition of choline exit (b < 1), but also from inhibition of deacetylation following the sliding of a molecule of substrate inside the acetylated active site and occupa- tion of the peripheral site by a second substrate mole- cule (complex S p EAS does not deacetylate in Scheme 1). This mechanism was suggested by excess substrate inhibition of decarbamoylation and the crys- tal structure of the S p EAS obtained by soaking crystals in a solution containing a high substrate concentration [2,4]. This second mechanism remains active in the Glu and Ala mutants because inhibition was observed at a high substrate concentration (Fig. 3). We observed a shift of the pS curve towards higher substrate concen- trations due to the lower affinity of both the free and acetylated mutated active sites. If choline could leave the active site by the back channel, we might also hypothesize that acetylcholine enters using the same path. To test this hypothesis, we used two inhibitors specific for the peripheral site that bind to Trp321 close to the entrance: propidium and aflatoxin B1. In the wild-type DmAChE, the affinity of propidium for the peripheral site is estimated to be 80 pm, and the affinity for aflatoxin to be 3.5 lm, when considering competition between the substrate and inhibitor only at the PAS (Fig. 4A). However, inhibition is completely abolished by the substitution of Trp83 by Ala or Glu. It should be strongly empha- sized at this point that, according to the proposed reaction scheme (Scheme 1) enlarged by the binding of inhibitor to the PAS, inhibition at low substrate con- centrations should always be observed. Therefore, the complete absence of inhibition by peripheral ligands does not originate from changes in substrate hydrolysis parameters (Table 1), and the simulation can readily confirm this. The loss of inhibition following muta- tions of Trp83 might be interpreted as a strong decrease in affinity of ligand for the peripheral site, resulting from a hypothetic allosteric interaction [23,24]. However, binding to the peripheral site was not affected by mutations, as demonstrated by changes in fluorescence. Furthermore, considering that inhibi- tion arises because inhibitors bound to the PAS hinder the 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 did not prevent the entrance of substrate into the active site. At this point, a plausible explanation is that the substrate may enter by an alternative route (i.e. the back channel at the bottom of active site). This hypothesis is in accordance with reported results observed with Trp83Ala mutants: the strong decrease of inhibition of propidium [21] and the increase of remaining activity upon peripheral site saturation by fasciculin [24]. Finally, minor deviations of pS curves upon binding of inhibitors on the PAS (Fig. 4B,C), may be assigned 10 100 1000 10 000 100 000 0 200 400 600 800 1000 A B C prop10 µ M Afl50 µ M ref prop1 µ M prop10 µ M Afla10 µ M Afla50 µ M prop1 µ M prop10 µ M Afla10 µ M Afla50 µ M ref ATCh ( M) 10 100 1000 10 000 100 000 ATCh ( M) 10 100 1000 10 000 100 000 ATCh ( M) 0 100 200 300 400 0 200 400 600 ref v/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 FEBS to an allosteric interaction between PAS and CAS [8,21,25], to a lower efficiency of the alternative route compared to the main entrance, or to a partial overlap of the side chain of propidium and aflatoxin with the active site as it may span into the gorge. Experimental procedures Protein production and purification Mutations were introduced by site-directed mutagenesis using the QuickChange XL kit following the manufac- turer’s instructions (Stratagene, La Jolla, CA, USA). The cDNA encoding DmAChE and mutants were expressed with the baculovirus system [26]. We expressed a soluble dimeric form deprived of a hydrophobic peptide at the C-terminal and with a 3· histidine tag replacing the loop from amino acids 103–136. This external loop is at the opposite side of the molecule with respect to the active site entrance and its deletion does not affect the activity or the stability of the enzyme. Secreted acetylcholinesteras- es were purified to homogeneity using the following steps: ammonium sulfate precipitation, ultrafiltration with a 10 kDa cut-off membrane, affinity chromatography with procainamide 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 by active site titration using high affinity irreversible inhibi- tors [28]. Enzyme activity Data 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 W83 mutants was followed using Ellman’s method [29]. The initial rate measurements were performed at acetylthiocho- line concentrations from 2 lm to 500 mm in the absence and presence of two ligands known to close the entrance of the active site. We used 1 and 10 lm propidium and 10 and 50 lm aflatoxin. The activity was followed for 1 min after the addition of acetylcholinesterase to the mixture, and the spontaneous hydrolysis of the substrate was subtracted, if present. Each measurement was repeated at least four times. The experiments were carried out at 25 °Cin25mm phosphate buffer (pH 7.0) without ionic strength compensa- tion to avoid interference with electrostatic components of binding and chemical steps of the reaction. Analysis of the kinetics data were performed using gosa-fit, software that is based on a simulated annealing algorithm (BioLog, Toulouse, France; http://www.bio-log.biz). For analysis of initial rate data in the absence of inhibitors, we used the specific equation in Scheme 1. 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Insights into substrate and product traffic in the Drosophila melanogaster acetylcholinesterase active site gorge by enlarging a back channel Florian Nachon 1 ,. hydrolysis because the traffic can pass via an alternative route, presumably the enlarged back channel. Abbreviations CAS, acylation site; DmAChE, Drosophila acetylcholinesterase;