ToxSci Advance Access originally published online on January 10, 2006
Toxicological Sciences 2006 90(2):460-469; doi:10.1093/toxsci/kfj094
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Concentration-Dependent Kinetics of Acetylcholinesterase Inhibition by the Organophosphate Paraoxon
,1
* Drug Metabolism and Pharmacokinetics, Schering-Plough Research Institute, Lafayette, New Jersey 07843; and Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103
1 To whom correspondence should be addressed. Fax: (973) 972-4554. E-mail: sultatle{at}umdnj.edu.
Received November 7, 2005; accepted January 3, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For decades the interaction of the anticholinesterase organophosphorus compounds with acetylcholinesterase has been characterized as a straightforward phosphylation of the active site serine (Ser-203) which can be described kinetically by the inhibitory rate constant ki. However, more recently certain kinetic complexities in the inhibition of acetylcholinesterase by organophosphates such as paraoxon (O,O-diethyl O-(p-nitrophenyl) phosphate) and chlorpyrifos oxon (O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphate) have raised questions regarding the adequacy of the kinetic scheme on which ki is based. The present article documents conditions in which the inhibitory capacity of paraoxon towards human recombinant acetylcholinesterase appears to change as a function of oxon concentration (as evidenced by a changing ki), with the inhibitory capacity of individual oxon molecules increasing at lower oxon concentrations. Optimization of a computer model based on an Ordered Uni Bi kinetic mechanism for phosphylation of acetylcholinesterse determined k1 to be 0.5 nM–1h–1, and k–1 to be 169.5 h–1. These values were used in a comparison of the Ordered Uni Bi model versus a ki model in order to assess the capacity of ki to describe accurately the inhibition of acetylcholinesterase by paraoxon. Interestingly, the ki model was accurate only at equilibrium (or near equilibrium), and when the inhibitor concentration was well below its Kd (pseudo first order conditions). Comparisons of the Ordered Uni Bi and ki models demonstrate the changing ki as a function of inhibitor concentrations is not an artifact resulting from inappropriate inhibitor concentrations.
Key Words: acetylcholinesterase; organophosphate; paraoxon; kinetics.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Organophosphorus insecticides exert their acute mammalian toxicity through inhibition of the critical enzyme acetylcholinesterase (EC 3.1.1.7 [EC] ) (Mileson et al., 1998
). The inhibition occurs as a result of the phosphylation of Ser-203 (the numbers used in this report refer to amino acid positions in human acetylcholinesterase). Much is known regarding the molecular events that lead to the phosphylation of Ser-203 by certain organophosphorus compounds. Ordentlich et al. (1996) have reported that the acyl pocket of acetylcholinesterase's active gorge (Phe-295 and Phe-297) participates in the positioning of an organophosphorus molecule for the nucleophilic attack by the catalytic Ser-203 (Fig. 1). This same group (Ordentlich et al., 1998) have suggested that the oxyanion hole subsite (Gly-121, Gly-122, and Ala-204) might polarize the P=O bond during formation of the Michaelis complex, thereby activating the phosphorus and promoting the nucleophilic attack by Ser-203 (Fig. 1). This nucleophilic attack is made possible by a proton transfer from Ser-203 to a nitrogen in the imidazole ring of His-447, which in turn transfers a proton from the second nitrogen of the imidazole ring to the carboxyl group of Glu-334 (Soreq and Seidman, 2001) (Fig. 1). Additionally, the peripheral anionic site of acetylcholinesterase, located at the lip of the active site gorge (Fig. 1), has been shown to play an important role in the stereoselectivity towards enantiomers of the chemical warfare agent VX (O-ethyl S-[2-(dissopropylamino)ethyl] methylphosphonothioate) (Ordentlich et al., 2004). Occupation of the peripheral anionic site by certain ligands has also been shown to alter rates of substrate catalysis (Taylor and Radi
, 1994; Masson et al., 2004).
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The interactions between organophosphorus compounds and acetylcholinesterase have been summarized kinetically by an Ordered Uni Bi reaction scheme (Aldridge and Reiner, 1972
; Kardos and Sultatos, 2000):
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| Scheme (1) |
Main (1964) simplified the kinetic description in Scheme 1 by deriving the inhibitory rate constant ki (Scheme 2). This bimolecular rate constant describes both the formation of a Michaelis complex, as well as the phosphylation of Ser-203 by a specific inhibitor, as shown in the following scheme:
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| Scheme (2) |
 | Equation (1) |
 | Equation (2) |
, as well as Kousba et al. (2004) have documented that the kis for inhibition of mouse and rat brain acetylcholinesterase by certain organophosphates appear to change over a wide range of inhibitor concentrations, suggesting that at low organophosphate concentrations, individual inhibitor molecules have a greater capacity to inhibit acetylcholinesterase than individual organophosphate molecules at higher inhibitor concentrations (Fig. 2). If viewed as an enzymatic reaction where the organophosphate is hydrolyzed by acetylcholinesterase, this phenomenon would be considered substrate inhibition. However, the stability of the phosphylated intermediate precludes the use of this terminology. Kardos and Sultatos (2000) and Kousba et al. (2004) speculated that certain organophosphates might bind reversibly to a site on acetylcholinesterase separate from the catalytic triad, and that this binding decreases, through steric hindrance or allosteric modulation, the capacity of other organophosphate molecules to inhibit enzyme activity (Fig. 2). The current study extends the observations of Kardos and Sultatos (2000) and Kousba et al. (2004) to human recombinant acetylcholinesterase, and investigates the appropriateness of Schemes 1 and 2 for the kinetic description of the inhibition of acetylcholinesterase by paraoxon.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
Paraoxon (O,O-diethyl O-(p-nitrophenyl) phosphate) was purchased from Chem Services (West Chester, PA), and was further purified by dissolution in trichloroethylene, followed by washes with 2% sodium carbonate. HPLC analyses of the washed product revealed a single peak of paraoxon (Kousba and Sultatos, 2002
). Acridine, iodomethane, methanol, ether, and Dowex (1wx2, 100 mesh) were purchased from Sigma Chemcial Company (St. Louis, MO). Human recombinant acetylcholinesterase was also purchased from Sigma Chemical Company and was routinely stored at –70°C. Previous investigators have established that recombinant acetylcholinesterase is very unstable in solution unless in the presence of certain "stabilizers" such as bovine serum albumin (Estrada-Mondaca and Fournier, 1998; Shafferman et al., 1992). In the present study, bovine serum albumin at a concentration of 1 mg/ml was required in order to stabilize the enzyme in solution for up to 24 h (data not shown). Equilibrium dialysis at 37°C as described by Kousba and Sultatos (2002) found no evidence of binding or hydrolysis of paraoxon by bovine serum albumin under the conditions of the incubations (data not shown). Additionally, bovine serum albumin had no effect on human recombinant acetylcholinesterase activity (data not shown).
Synthesis of N-methylacridinium.
The fluorophore N-methylacridinium was synthesized and purified as described by Mooser and Sigman (1974). Briefly, 3 g of acridine were slowly dissolved in 15 ml acetone in a 200 ml round-bottom flask. To that solution 10 ml of iodomethane were added and stirred for 18 h. The solution was poured through filter paper using excess acetone to remove the entire product form the flask. The filtrate (consisting of orange crystals) was re-dissolved in 30 ml methanol and the N-methylacridinium (iodide salt) was re-crystallized (red crystals) by the addition of anhydrous ether (about ten volumes of ether were required). The methanol and ether were poured off and the crystals were dried in the hood. The red crystals were re-dissolved in about 5 ml of water to give a fluorescent green color. This solution was passed through a Dowex chloride exchange column (converting the iodide salt into the more stable chloride salt). The chloride salt was recovered and the water was evaporated under a gentle stream of air. N-methylacridinium was scanned in the UV-Vis range on a Shimadzu MPS-2000 spectrophotometer (Shimadzu MPS 2000 UV-Vis spectrophotometer, Shimadzu Scientific Instruments, Inc., Columbia, MD), and fluorescence scans were performed on a Cary Eclipse Fluorescence Spectrophotometer (Varian, Inc., Palo Alto, CA), with an excitation wavelength of 360 nm and an emission wavelength of 490 nm (Mooser and Sigman, 1974). The UV-Vis and fluorescence scans were identical to previously published spectra (Fig. 3) (Chan et al., 1974; Eastman et al., 1995; Happ et al., 1970; Mooser and Sigman, 1974; Rosenberry et al., 1996).
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Determination of active site concentration.
The active site concentration in several enzyme solutions was determined by titration with N-methylacridinium as described by Mooser and Sigman (1974)
. With this approach the fluorescence of N-methylacridinium is reduced when it binds stoichiometrically to the active site, allowing active site quantification. Binding data were analyzed by fitting to the following equation:
 | Equation (3) |
Determinations of ki.
A bottle of human recombinant acetylcholinesterase (0.1 mg protein) was dissolved in 10 ml of 100 mM sodium phosphate buffer (pH 7.4) containing 1 mg/ml bovine serum albumin (this buffer was used throughout). Aliquots of 100 µl were placed into vials and frozen at –70°C until the day of use. For each ki determination a vial was thawed and 14.9 ml buffer were added to give a final volume of 15 ml. A concentrated stock solution of paraoxon was prepared in ethanol and stored at –70°C. Paraoxon solutions to be used for incubations were made from this stock with dilutions made with buffer such that the ethanol concentration within any incubation never exceeded 1%. Incubations consisted of a volume of paraoxon solution in buffer at a given concentration with an equal volume of stock enzyme solution that when mixed together, gave the desired final paraoxon concentration. Incubations were carried out in an orbital shaker bath at the indicated temperatures and time periods. At the end of an incubation, 30 µl were placed into a well on a 96 well plate (Nunc-Immuno Module, Morris Plains, NJ), and 345 µl of buffer with acetylthiocholine (0.44 mM) were added. This dilution along with the acetylthiocholine terminated the further inhibition of enzyme by paraoxon since, in certain samples, addition of paraoxon after, rather than before, the addition of the buffer did not result in enzyme inhibition (data not shown). Next 25 µl of 5,5-dithio-bis (2-nitrobenzoic acid) were added to give a final concentration of 0.1 mM in a final volume of 400 µl. The changes in absorbance at 405 nm were monitored at 5-min intervals for up to 20 min on a microplate reader (BIO-TEK Instruments, Inc., Winoski, VT) (Rosenfeld et al., 2001). The increasing absorbances were fitted to a straight line by Sigmaplot 3 or Sigmaplot 8, and the slopes were used as a measurement of uninhibited acetylcholinesterase activities. From these activities the enzyme active site concentrations were calculated.
The ki's were determined by optimization of the following equations (Kardos and Sultatos, 2000) to the empirical inhibition data with the software ACSL (Advanced Continuous Simulation Language, Aegis, Huntsville, AL). These equations are based on Scheme 2, with the addition of aging and reactivation pathways.
 | Equation (4) |
 | Equation (5) |
 | Equation (6) |
 | Equation (7) |
ET represents the initial active site concentration; and ABT represents the initial paraoxon concentration (Kardos and Sultatos, 2000). All other parameters used in Equations 4–7 have the same meaning as those in Schemes 1 and 2. The rate constants for reactivation (k3) and aging (k4) were taken from Masson et al. (2000), and were 0.005379 h–1 and 0.001575 h–1, respectively.
Determination of the Kd and k2 for paraoxon.
Co-incubation studies where paraoxon and p-nitrophenyl acetate were incubated concurrently with acetylcholinesterase were performed in a manner similar to those described by Hart and O'Brien (1973) and Barak et al. (1995). Incubation volumes were 1.6 ml, and were performed in a cuvette in a Shimadzu MPS 2000 UV-Vis spectrophotometer. Paraoxon and p-nitrophenyl acetate in buffer were initially added to both sample and reference cuvettes to give final inhibitor concentrations of 31 nM–1000 nM, and a 1 mM substrate concentration. The reaction was initiated by addition of enzyme (for a final active site concentration of 0.4 pM) to the sample cuvette, while buffer only was added to the reference cuvette. Product formation was continuously monitored by measuring the increase in optical density at 402 nm. These data were analyzed by the method developed by Hart and O'Brien (1973), as described by Barak et al. (1995).
Determination of k1 and k–1 for paraoxon.
The association (k1) and dissociation (k–1) rate constants for the reversible binding of paraoxon to acetylcholinesterase were determined by optimizing the following equations (referred to as the Ordered Uni Bi model, and based on Scheme 1) to the inhibition profiles used for ki determinations for paraoxon concentrations of 25 nM, 50 nM, and 100 nM, at 24°.
 | Equation (8) |
 | Equation (9) |
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| Equation (10) |
 | Equation (11) |
 | Equation (12) |
Modeling studies.
All modeling studies were carried out on laptap or desktop computers using ACSL 11.8 (Advanced Continuous Simulation Language, Aegis, Hunstville, AL). The sensitivity of a model to an optimized parameter was routinely assessed following optimization in order to assure the optimized value yielded the best fit.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In order to minimize the number of variables determined by the optimization routine in ACSL, acetylcholinesterase active site concentrations in stock solutions were determined by titration with the fluorescent compound N-methylacridinium (Mooser and Sigman, 1974
). Analyses of binding data yielded the enzyme active site concentration (Mooser and Sigman, 1974), as well as the dissociation constant for N-methylacridinium (Table 1). Assessment of acetylcholinesterase activity in appropriate dilutions of the stock enzyme solutions indicated that 5 pM active sites resulted in an increase of 0.088 optical density units per minute in the Ellman assay, performed under the conditions described in Materials and Methods.
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Incubations of 4–6 pM human recombinant acetylcholinesterase active sites with various concentrations of paraoxon at 37° and 24° produced inhibition profiles that could be fitted with the ki computer model to determine the ki at each paraoxon concentration. Figure 4 presents two examples of these analyses.
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A comparison of kis generated over a wide range of inhibitor concentrations revealed a changing ki as a function of paraoxon concentration (Fig. 5). The fitted parameters in Figure 5 indicated that the largest ki (the y intercept) at 37° was 29.14 h–1, while the smallest ki (when paraoxon was 100 nM) was 0.18 h–1. Similarly at 24°, the largest and smallest ki's were 0.61 h–1 and 0.15 h–1, respectively. These data, therefore, confirm the observations of Kardos and Sultatos (2000)
with dilute mouse brain homogenate where the ki of paraoxon appeared to vary over a wide range of oxon concentrations. Interestingly, at 37°, the difference between the highest estimated ki (29.14 nM–1h–1) and the lowest estimated ki (0.18 nM–1h–1) was considerably greater than that same comparison at 24° (0.61 nM–1h–1 vs 0.15 nM–1h–1) (Fig. 5). Furthermore, the determination of the ki by the method of Main (1964) at 24° and 37° yielded a ki nearly equal to the plateau value generated by the method of Kardos and Sultatos (2000) at the corresponding temperature (Fig. 5).
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Incubation of 0.4 pM acetylcholinesterase with variable concentrations of paraoxon and 1 mM p-nitrophenyl acetate (Barak et al., 1995
; Hart and O'Brien, 1973) yielded a Kd and k2 for paraoxon of 339 nM and 58.1 h–1, respectively (Fig. 6). The value for k2 was directly substituted into the Ordered Uni Bi model (Equations 8–12). The dissociation constant Kd established the relationship between the binding k1 and k–1, but could not provide a single set of values for these binding parameters. However, optimization of the Ordered Uni Bi model (including the restrictions on k1 and k–1 imposed by Kd) to inhibition profiles of acetylcholinesterase in the presence of 25 nM, 50 nM, and 100 nM paraoxon determined k1 and k–1 to be 0.5 nM–1h–1 and 169.5 h–1 respectively. The upper panel of Figure 7 shows the final fit of the Ordered Uni Bi model to the 25 nM paraoxon inhibition profile. These paraoxon concentrations were chosen because they yielded essentially constant estimates for ki (Fig. 5), and because the lowest paraoxon concentration used for determination of the Kd and k2 was 31 nM (Fig. 6).
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equals [S]/(Km + [S]), where [S] is the p-nitrophenyl concentration, and Km is 4.39 mM (Hart and O'Brien, 1973
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The construction of the Ordered Uni Bi model where values for all parameters were known allowed a systematic evaluation of the accuracy of the ki model and Scheme 2, since the ki scheme was originally derived from the Ordered Uni Bi scheme (Scheme 1). If the ki scheme and model accurately describe the rate of inhibition of acetylcholinesterase by paraoxon, this rate must equal that of the Ordered Uni Bi scheme and model. When these two rates are assumed to be equal, the following equations can be written:
 | Equation (13) |
 | Equation (14) |
 | Equation (15) |
 | Equation (16) |
 | Equation (17) |
. Simulations of the Ordered Uni Bi model indicated that equilibrium was nearly achieved with paraoxon and acetylcholinesterase, and that both the ki model and the Ordered Uni Bi models yielded nearly identical outputs at paraoxon concentrations well below the Kd (Fig. 8). Interestingly, however, at concentrations of paraoxon approaching or exceeding its Kd, the ki model output differed substantially from that of the Ordered Uni Bi model, thereby revealing a second limitation to the ki scheme. This necessity for inhibitor concentrations well below the inhibitor Kd is an indication of the requirement for psuedo first order conditions in order for ki to be meaningful. The requirement for psuedo first order conditions was discussed by Main (1964), although he nevertheless used inhibitor concentrations larger than their Kds.
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And finally, the Ordered Uni Bi kinetic model which was based on a Kd and k2 determined at relatively high paraoxon concentrations (31 nM–1000 nM), did not accurately simulate the empirically determined inhibition profiles for low paraoxon concentrations, essentially confirming the results of the ki model (Fig. 7, lower panel). The Ordered Uni Bi model could accurately simulate the inhibition profiles when changes were made in k1, k-1, or k2, but not k3 (Fig. 9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A changing ki as a function of paraoxon concentration is inconsistent with the kinetic mechanism described by both the Ordered Uni Bi and ki schemes (Schemes 1 and 2). Therefore a consideration of the possible limitations in the use of ki is appropriate when interpreting these results (Fig. 5). Two important limitations were identified. The first is the requirement for equilibrium (or near-equilibrium) with regard to reversible binding of inhibitor to acetylcholinesterase (formation of the Michaelis complex). The lack of achievement of equilibrium with paraoxon accounted, at least in part, for the non-identical outputs from the Ordered Uni Bi and ki models at paraoxon concentrations lower than 100 nM. However, these differences were likely not great enough to be detected experimentally, and can therefore be considered negligible (Fig. 8). Paraoxon concentrations of 200 nM and greater revealed the second limitation in the use of ki. These high concentrations resulted in more substantial differences between the two models (Fig. 8), even though equilibrium conditions were more closely approximated as the inhibitor concentration increased (Fig. 8). This is reflective of the need for pseudo-first order conditions, since the rate of reversible binding of paraoxon to acetylcholinesterase slows as unbound enzyme molecules become fewer with increasing paraoxon concentrations. The ki does not account for this binding phenomenon, and will therefore overestimate the degree of inhibition observed with high inhibitor concentrations. The need for equilibrium conditions and pseudo-first order conditions are limitations in the use of ki that have largely been ignored for many decades, perhaps leading to many erroneous estimations of this constant. In the case of paraoxon, the use of ki to quantify its inhibitory capacity seems appropriate below inhibitor concentrations of 100 nM (Fig. 8). Moreover, the lower the paraoxon concentration, the more accurate was the ki model (Fig. 8), dispelling the notion that the inhibitor concentration must be many times higher than that of acetylcholinesterase in order for ki to be meaningful.
Since the ki and Ordered Uni Bi models simulated essentially the same inhibition profiles for paraoxon concentrations of 100 nM and below (Fig. 8), the changing ki as a function of paraoxon concentration observed in Figure 5 did not result from an inappropriate application of ki. This is further supported by the observation that the Ordered Uni Bi model, which utilized rate constants determined at paraoxon concentrations of 31 nM–1000 nM, also underestimated the capacity of much lower paraoxon concentrations to inhibit acetylcholinesterase (Fig. 9).
While the presence of two kinetically distinct forms of acetylcholinesterase could account for a changing ki as a function of paraoxon concentration (Fig. 5), reports suggest that such distinct enzymatic forms do not exist. As described by Stojan et al. (1998), all known posttranslational modifications to acetylcholinesterase do not appear to change the kinetic behavior (Estrada-Mondaca and Fournier, 1998; Taylor and Radi, 1994). One report (Bolger and Taylor, 1979) documented binding of certain bisquaternary ammonium ligands to acetylcholinesterase that suggested the existence of two conformational states of the enzyme. However these two states were distinguished only by bisquaternary ammonium ligands, and not by active and peripheral site ligands. Furthermore, Bolger and Taylor (1979) established that bisquaternary ammonium ligands bound to only one of the two possible conformational states—a phenomenon that cannot account for the data presented in Figure 5.
The results of the current study support the suggestion of Kardos and Sultatos (2000) that paraoxon binds reversibly to a site distinct from the active site, and that occupation of this site by paraoxon reduces the capacity of other paraoxon molecules to inhibit the active site, either by allosteric modification or steric hindrance. Since the Ordered Uni Bi model could accurately simulate inhibition profiles at lower paraoxon concentrations by adjusting either k1, k-1, or k2 (Fig. 9), binding of paraoxon to a secondary site might reduce the rate of formation of the Michaelis complex and/or reduce the rate of phosphorylation of Ser-203. While reactivation of phosphorylated enzyme could also be affected by occupation of a secondary site, such a change could not account for the concentration-dependent inhibition kinetics documented in Figure 5 (Fig. 9).
Ligand-induced alterations in acetylcholinesterase activity have been documented previously, and various mechanisms have been proposed to account for these observations, which include both increased and decreased activities (Fig. 10). It has been known for many years that binding of certain ligands to the peripheral anionic site reduces substrate hydrolysis through steric blockade and/or conformation changes in Trp-86 and Tyr-133 residues within the active site gorge (Barak et al., 1995; Bourne et al., 2003; Taylor and Radi, 1994). Such ligands include propidium, thioflavin t, acetylthiocholine, and acetylcholine (DeFerrari et al., 2001; Taylor and Radi, 1994). The drug D-tubocurarine also usually inhibits acetylcholinesterase through binding to the peripheral anionic site of this enzyme (Golicnik et al., 2001). However, in the case of the Drosphila W359L mutant, D-tubocurarine increased hydrolysis of acetylthiocholine as a result of its binding to the peripheral anionic site (Golicnik et al., 2001, 2002). Since in this case, enzyme inhibition from binding of acetylchothiocholine to the peripheral binding site was greater than enzyme inhibition from binding of D-tubocurarine to the peripheral anionic site, the presence of D-tubocurarine appeared to activate enzyme activity.
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Although the apparent activation of the W359L mutant by D-tubocurarine resulted from a "disinhibition," occupation of the peripheral anionic site of wild type Drosophila acetylcholinesterase by this same compound increased the binding affinity of the active site for methanesulfonylfluoride, thereby increasing the methanesulfonylation of the active site serine (Golicnik et al., 2002
). Similarly, binding of the detergent Triton X-100 to the peripheral anionic site of Drosophila acetylcholinesterase resulted in activation of phosphorylation and carbamylation of the active site by certain organophosphates and carbamates, respectively, while inhibiting these same reactions with other organophosphates and carbamates (Marcel et al., 2000).
It must also be noted that binding to the peripheral anionic site is not requisite for activation of acetylcholinesterase. The chemical N-methylacridinium binds reversibly to the active site, and accelerates the hydrolysis of certain neutral acetic acid esters such as methyl and ethyl acetate, while competitively inhibiting the hydrolysis of acetylcholine (Barnett and Rosenberry, 1977). Similarly, small cationic ligands such as tetramethyl- and tetraethyl-ammonium have been shown to bind in the active site and enhance the rate of carbamylation and sulfonylation by carbamyl and sulfonyl fluorides (Kitz and Wilson, 1963; Metzger and Wilson, 1963).
The reports summarized above have established that ligand binding to the peripheral anionic site, or other sites on acetylcholinesterase, can result, in some cases, in inhibition of enzyme activity. In other cases, such binding can lead to apparent activation (from "disinhibition"), and still in other cases true activation of enzyme. The specific action of the ligand on enzyme activity appears to be dependent on the ligand itself, where it binds on the molecule, and the chemicals interacting at the active site. Consequently, it seems plausible, as first suggested by Kardos and Sultatos (2000), that paraoxon could bind to a secondary site on acetylcholinesterase, thereby reducing the capacity of other paraoxon molecules to phosphylate the Ser-203. Regardless of mechanism, the concentration dependent inhibition presented in Figure 5 demonstrates the inability of Schemes 1 and 2 to fully describe the inhibition of acetylcholinesterase by paraoxon. The documentation of concentration dependent inhibition of rat acetylcholinesterase with chlorpyrifos oxon (Kousba et al., 2004) indicates that this phenomenon is not unique to paraoxon.
While the potential binding of certain organophosphorus anticholinesterases to a secondary binding site is of interest for understanding the fundamental interactions of these toxic compounds with acetylcholinesterase, a more important consideration is how such binding might influence our understanding of the health risks posed to the public by these insecticides. Current risk assessments of organophosphorus insecticides are built on the assumption that the inhibitory capacity (ki) of molecules of a given oxon at low concentrations is the same as that of the same oxon molecules at high concentrations. Evidence presented here (Fig. 5), as well as elsewhere (Kardos and Sultatos, 2000; Kousba et al., 2004), suggests that the inhibitory capacity of certain oxons increases as the concentration decreases, thereby raising concern that current risk assessments might underestimate the risk posed to public health by low level exposure to certain organophosphorus insecticides.
And finally, further evidence in support of the interaction of certain organophosphorus compounds with a secondary site on acetylcholinesterase was provided by Howard et al. (2005), who showed that the organophosphorus insecticide chlorpyrifos disrupted the morphogenic function of acetylcholinesterase in the absence of inhibition of the catalytic site. Acetylcholinesterase has been shown to mediate neuronal morphogenesis independently of its catalytic site (Bigbee et al., 1999; Johnson and More, 2000; Sharma et al., 2001; Soreq and Seidman, 2001; Sternfeld et al., 1998). Consequently, binding of certain organophosphorus compounds to a secondary site(s) distinct from the catalytic site could not only reduce the capacity of additional organophosphorus molecules to inhibit the active site (Fig. 5), but might also have adverse developmental consequences unrelated to phosphylation of Ser-203.
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ACKNOWLEDGMENTS |
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This study was supported in part by Grant ES012648 from NIEHS.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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