ToxSci Advance Access originally published online on August 16, 2007
Toxicological Sciences 2007 100(1):128-135; doi:10.1093/toxsci/kfm197
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Concentration-Dependent Binding of Chlorpyrifos Oxon to Acetylcholinesterase
Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103
1 For correspondence via fax: (973) 972-4554. E-mail: sultatle{at}umdnj.edu.
Received June 7, 2007; accepted July 22, 2007
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
The organophosphorus insecticides have been known for many years to cause cholinergic crisis in humans as a result of the inhibition of the critical enzyme acetylcholinesterase. The interactions of the activated, toxic insecticide metabolites (termed oxons) with acetylcholinesterase have been studied extensively for decades. However, more recent studies have suggested that the interactions of certain anticholinesterase organophosphates with acetylcholinesterase are more complex than previously thought since their inhibitory capacity has been noted to change as a function of inhibitor concentration. In the present report, chlorpyrifos oxon (O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphate) was incubated with human recombinant acetylcholinesterase in the presence of p-nitrophenyl acetate in order to better characterize kinetically the interactions of this oxon with enzyme. Determination of the dissociation constant, Kd, and the phophorylation rate constant, k2, for chlorpyrifos oxon with a range of oxon and p-nitrophenyl acetate concentrations revealed that Kd, but not k2, changed as a function of oxon concentration. Changes in p-nitrophenyl acetate concentrations did not alter these same kinetic parameters. The inhibitory capacity of chlorpyrifos oxon, as measured by ki (k2/Kd), was also affected as a result of the concentration-dependent alterations in binding affinity. These results suggest that the concentration-dependent interactions of chlorpyrifos oxon with acetylcholinesterase resulted from a different mechanism than the concentration-dependent interactions of acetylthiocholine. In the latter case, substrate bound to the peripheral anionic site of acetylcholinesterase has been shown to reduce enzyme activity by blocking the release of the product thiocholine from the active site gorge. With chlorpyrifos oxon, the rate of release of 3,5,6-trichloro-2-pyridinol is irrelevant since the active site is not available to interact with other oxon molecules after phosphorylation of Ser-203 has occurred.
Key Words: chlorpyrifos oxon; acetylcholinesterase; inhibition kinetics; organophosphates; pesticides.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Organophosphorus insecticides such as chlorpyrifos (O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphorothioate) are known to cause cholinergic crisis in humans as a result of the inhibition of the critical enzyme acetylcholinesterase [EC 3.1.1.7 [EC] ] (Mileson et al., 1998
). The inhibition of this important enzyme by the activated metabolites (called oxygen analogs or oxons) of organophosphorus insecticides has been studied extensively and occurs through phophorylation of Ser-203 of the catalytic triad (numbers refer to the amino acid position of human acetylcholinesterase) (Aldridge and Reiner, 1972; Kryger et al., 2000; Ordentlich et al., 1996; Shafferman et al., 1992). However, the interactions of chlorpyrifos oxon (O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphate) (Fig. 1), as well as certain other oxons, with acetylcholinesterase have been shown to be more complex than was originally thought. Kinetic evidence has suggested that chlorpyrifos oxon likely reversibly binds to a secondary site that subsequently influences events at the active site (Kaushik et al., 2007). This conclusion was reached because, as the concentration of chlorpyrifos oxon increased, the capacity of individual chlorpyrifos oxon molecules to inhibit acetylcholinesterase was reduced (Kaushik et al., 2007). Therefore, the original kinetic scheme developed to describe phophorylation of acetylcholinesterase by oxons of organophosphorus insecticides (upper panel in Fig. 2) proved to be inadequate in describing the interactions of acetylcholinesterase with a wide range of concentrations of chlorpyrifos oxon. A more accurate description of these interactions was supplied by the kinetic scheme in the lower panel of Figure 2, which includes a secondary, regulatory binding site (Kaushik et al., 2007).
|
|
Concentration-dependent inhibition of acetylcholinesterase by chlorpyrifos oxon could result from changes in binding affinity (Kd) or changes in the rate of the phosphorylation reaction (k2). Kaushik et al. (2007)
attempted to address this issue by utilizing a technique that involves coincubation of the surrogate substrate acetylthiocholine with chlorpyrifos oxon and acetylcholinesterase in order to determine these kinetic constants at different chlorpyrifos oxon concentrations. Not surprisingly, however, it was observed that acetylthiocholine also altered the inhibitory capacity of chlorpyrifos oxon, probably due to its ability to bind to the peripheral anionic site of acetylcholinestersae in addition to the active site (Kaushik et al., 2007). The current report revisits this question by employing the test substrate p-nitrophenyl acetate (Fig. 1), which, to date, appears to follow simple Michaelis Menten kinetics and appears not to bind to any secondary site on acetylcholinesterase. |
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Chemicals.
Chlorpyrifos oxon was purchased from Chem Services (West Chester, PA.). Human recombinant acetylcholinesterase, bovine serum albumin, p-nitrophenyl acetate, p-nitrophenol, and all other chemicals were purchased from Sigma Chemical Company (St Louis, MO).
Determination of Kd, k2, and ki for chlorpyrifos oxon.
The Kd (k-1/k1) and k2 were determined by the zero-time method (Gray and Duggleby, 1989; Hart and O'Brien, 1973), as described by Kaushik et al. (2007). This approach allows the determination of these two important kinetic constants by monitoring the hydrolysis of a test substrate (p-nitrophenyl acetate in the current report) in the absence and presence of a single concentration of inhibitor (chlorpyrifos oxon). Hydrolysis of p-nitrophenyl acetate hydrolysis was monitored at 37° in a Shimadzu UV-2550 spectrophotometer (Shimadzu Scientific Instruments, Inc., Columbia, MD) set to a wavelength of 405 nm to quantify p-nitrophenol production. p-Nitrophenyl acetate has been shown to decompose spontaneously in water and is known to be further hydrolyzed slowly by bovine serum albumin (Means and Bender, 1975; Sakurai et al., 2004), which was present in the buffer in order to stabilize human recombinant acetylcholinesterase. These activities had to be monitored and subtracted from total activity in order to determine acetylcholinesterase-catalyzed hydrolysis. Additionally, p-nitrophenol acetate stock solutions were made up in ethanol for stability and were the last ingredient added (in a volume of 10 µl) to the preheated cuvette to initiate the reaction. The total volume of the incubation within the cuvette was 1.5 ml. The active site concentrations of acetylcholinesterase within the cuvettes were determined as previously described (Rosenfeld and Sultatos, 2006) and ranged from 65 to 70pM.
Briefly, the p-nitrophenol produced from p-nitrophenyl acetate with time was fitted to the following equation:
 | (1) |
represent the product concentrations (p-nitrophenol) at any time t and at t = , respectively, and the constant A represents the apparent rate constant for the formation of inhibited enzyme (Gray and Duggleby, 1989; Liu and Tsou, 1986). The initial velocities of incubations containing inhibitor were determined after cubic spline analyses of the fitted curves from Equation 1, as previously described (Barak et al., 1995; Kaushik et al., 2007). The Kd was calculated from:
 | (2) |
 | (3) |
. In Equation 3, 
(lnv)/t represents the slope obtained from the semilogarithmic plot of the changing slopes of the product formation curves in the presence of inhibitor (obtained through cubic spline analyses) versus t, and where
 | (4) |
The Km for p-nitrophenyl acetate hydrolysis by acetylcholinesterase was determined by monitoring metabolism over a range of p-nitrophenyl acetate concentrations. The Km and Vmax were calculated by fitting the data to the Michaelis Menten equation.
The ki for chlorpyrifos oxon was calculated from the following equation:
 | (5) |
Modeling.
In order to investigate the possible limitations of the zero-time method, hydrolysis of a wide range of p-nitrophenyl acetate concentrations by acetylcholinesterase in the presence of various chlorpyrifos oxon concentrations was modeled according to the kinetic scheme shown in the upper panel of Figure 2. This model is referred to as the zero-time model, and the equations that formed it were as follows:
 | (6) |
 | (7) |
 | (8) |
 | (9) |
 | (10) |
 | (11) |
 | (12) |
 | (13) |
 | (14) |
The k2 for p-nitrophenyl acetate (Fig. 2) was calculated from the following relationship (Boyd et al., 2000):
 | (15) |
 | (16) |
).
Statistical analyses and modeling.
All statistical analyses and curve-fitting procedures were conducted with Sigmastat and Sigmaplot (SPSS Science Inc., Chicago, IL). The model simulations represented by Equations 6–14 were solved on a laptop or desktop computer using ACSL 11.8 (Advanced Continuous Simulation Language, Aegis, Huntsville, AL).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Previous investigators have documented that recombinant acetylcholinesterase is 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 current report, bovine serum albumin was utilized to stabilize human recombinant acetylcholinesterase, thereby necessitating the evaluation of p-nitrophenyl acetate hydrolysis by this protein. Incubation of various concentrations of bovine serum albumin in buffer in the absence of acetylcholinesterase led to the hydrolysis of p-nitrophenyl acetate (Fig. 3), as previously noted (Means and Bender, 1975; Sakurai et al., 2004). Activity has been reported to result from the acetylation of certain tyrosine residues, and the deviation from linearity seen at the higher bovine serum albumin concentrations (Fig. 3) likely resulted from the acetylation of different tyrosines at different rates (Means and Bender, 1975). In the current study, all subsequent incubations contained bovine serum albumin at a concentration of 0.1 mg/ml, which resulted in limited nonspecific breakdown of p-nitrophenyl acetate (Fig. 3). Nevertheless, activity was great enough to warrant careful quantification and subtraction from the total hydrolytic activity measured in all subsequent incubations.
|
With the zero-time method, calculation of Kd and k2 for an organophosphate inhibitor requires knowledge of the Km for the substrate included within the incubation (Gray and Duggleby, 1989
; Hart and O'Brien, 1973). Incubation of concentrations of p-nitrophenyl acetate from 0.5 to 8.75mM with acetylcholinesterase allowed the determination of the Km and Vmax for the hydrolysis of this substrate by this enzyme (Fig. 4). Substrate inhibition, which would be expected if p-nitrophenyl acetate were bound to the peripheral anionic site on acetylcholinesterase, was not seen (Fig. 4).
|
Incubation of increasing concentrations of chlorpyrifos oxon with p-nitrophenyl acetate and human recombinant acetylcholinesterase resulted in progressive inhibition of p-nitrophenol production (Fig. 5). Analyses of the metabolic profiles (upper panel in Fig. 5) and the secondary plots (lower panel in Fig. 5) by the zero-time method yielded estimates of Kd and k2 for chloryrifos oxon at a variety of inhibitor concentrations (Fig. 6). Those Kds determined with chlorpyrifos oxon concentrations from 6.25 to 50nM were not significantly different from one another, whereas the Kd determined with 3.125nM chlorpyrifos oxon was reduced by about one-half (Fig. 6). Unfortunately, chlorpyrifos oxon concentrations lower than 3.125nM did not result in enough inhibition to consistently yield initial velocities less than the velocity of control incubations (without chlorpyrifos oxon; data not shown). Additionally, no evidence of activation of p-nitrophenyl acetate hydrolysis was seen, in contrast to that observed with acetylthiocholine hydrolysis (Kaushik et al., 2007
). No significant differences were observed with chlorpyrifos oxon k2s (data not shown) which had an overall mean and standard deviation of 229.27/h + 56.03. Calculation of chlorpyrifos oxon kis (by Equation 5) at the various oxon concentrations revealed that the ki remained unchanged until 3.125nM chlorpyrifos oxon, at which point it increased, reflecting the decrease in Kd at this same concentration (Fig. 6).
|
|
The values for the parameters contained within the zero-time model are shown in Table 1. Certain parameters were taken from the literature (Table 1), while values for others (k1A, k10A, k1C, k10C, and k2c)(Fig. 2), were selected to give a simulated metabolic profile similar in nature, but not identical, to that of the empirical data (Figs. 5 and 7). It must be noted, however, that the zero-time model was not intended to accurately simulate the empirical data but instead was developed as a tool to rigorously investigate the possible limitations of this methodology. This evaluation was accomplished by constructing a model based on selected values for the Kd and k2 of chlorpyrifos oxon (Table 1). Simulations were performed over a range of p-nitrophenyl acetate and chlorpyrifos oxon concentrations, with Kd and k2 calculated from the output for each simulation. The validity of the zero-time method was tested by comparing the calculated values for Kd and k2 with the selected values used in the zero-time model.
|
|
The zero-time model simulated hydrolysis of p-nitrophenyl acetate in the absence and presence of chlorpyrifos oxon and yielded metabolic profiles characteristic of those observed with empirical data (Figs. 5 and 7). Simulated substrate hydrolysis was reduced as chlorpyrifos oxon concentrations were increased (Fig. 7). Calculation of the Kd and k2 of chlorpyrifos oxon from the simulations over a range of p-nitrophenyl acetate and chloryprifos oxon concentrations yielded accurate values for these kinetic constants, until the p-nitrophenyl acetate concentration exceeded the Km for this substrate, at which point the Kd and k2 were underestimated (Fig. 8). Surprisingly, chlorpyrifos oxon concentrations three times greater than the model's selected inhibitor Kd (50nM) gave accurate Kd's and k2's, provided the p-nitrophenyl acetate levels were equal to or lower than this substrate's Km (Fig. 8).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Previous studies have reported that the inhibitory capacity of chlorpyrifos oxon changes as a function of the chlorpyrifos oxon concentration (Kaushik et al., 2007
; Kousba et al., 2004). Kinetic evidence has suggested the existence of a secondary binding site for this organophosphate, although such a binding site has not yet been directly demonstrated. The concentration-dependent changes in inhibitory capacity of chlorpyrifos oxon were evidenced by changes in the inhibitory rate constant ki, which is a function of binding affinity (Kd) as well as the rate of phosphorylation of Ser-203 (k2) (Equation 5). Kaushik et al. (2007) attempted to determine whether Kd and/or k2 changed as a function of chlorpyrifos oxon concentration using the zero-time method but found that the use of acetylthiocholine as the test substrate introduced confounding factors since this substrate itself altered chlorpyrifos oxon inhibitory kinetics. Conversely, no evidence has been reported that would suggest that the substrate p-nitrophenyl acetate binds to any site on acetylcholinesterase other than the active site, and the kinetic analyses performed in the current study supports a simple Michaelis Menten kinetic model for this substrate (Fig. 4). However, use of p-nitrophenyl acetate was complicated by its spontaneous decomposition as well as breakdown mediated by bovine serum albumin (Fig. 3). The smallest concentration of bovine serum albumin that stabilized the human recombinant acetylcholinesterase (0.1 mg/ml), was used, and bovine serum albumin mediated breakdown as well as spontaneous decomposition was carefully quantified and accounted for in each experiment.
The original zero-time method developed by Hart and O'Brien (1973) was proposed for use when the test substrate (p-nitrophenyl acetate) and organophosphate inhibitor concentrations were well below their Kd's. The computer modeling studies in the current report established that this assumption could be violated without rendering the zero-time method inacurrate since the highest inhibitor concentration was three times greater than its Kd (Fig. 8). Interestingly, the p-nitrophenyl acetate concentration could equal its Kd, but not be higher (Fig. 8). These simulations indicate that the zero-time method is far more robust than originally thought by Hart and O'Brien (1973) for p-nitrophenyl acetate.
The zero-time method established that the binding affinity of chlorpyrifos oxon appeared to increase as the concentration of this inhibitor was reduced to 3.125nM, thereby accounting, at least in part, for the concentration-dependent alteration in its ki previously reported (Kaushik et al., 2007; Kousba et al., 2004). Stated differently, the binding affinity of chlorpyrifos oxon, as measured by its Kd, was decreased as the oxon concentration exceeded 3.125nM (Fig. 6). The lack of an effect of oxon concentration on k2 is not surprising since Ordentlich et al. (1996) have shown, by site-directed mutagenesis, that the nucleophilic attack of Ser-203 on the phosphorus atom of certain organophosphate inhibitors is not very sensitive to changes in the architecture of the active site. Instead, the phosphorylation reaction is dependent primarily on the nucleophilicity of Ser-203 and the electronic properties of the phosphoryl moiety (Kemp and Wallace, 1990; Ordentlich et al., 1996).
While it is tempting to make analogies between concentration-dependent kinetics of chlorpyrifos oxon and the concentration-dependent kinetics of acetylcholine or acetylthiocholine, the mechanisms for these two phenomena are likely quite different. Kinetic studies (Johnson et al., 2003; Mallender et al., 2000; Szegletes et al., 1999), and crystallography (Bourne et al., 2006; Colletier et al., 2006) have documented that the substrate inhibition observed with acetylthiocholine likely results from steric blockade of the release of thiocholine. A molecule of acetylthiocholine bound to the peripheral anionic site seems to prevent the release of thiocholine produced from a second acetylthiocholine molecule at the active site. This mechanism cannot account for the concentration-dependent kinetics observed with chlorpyrifos oxon since phosphorylation of Ser-203 by the oxon yields an intermediate that does not reactivate during the time course of the experiments in the current report. Consequently, whether or not 3,5,6-trichloro-2-pyridinol release was blocked is irrelevant since the phosphorylated Ser-203 was not available to interact with another chlorpyrifos oxon molecule. Instead, the results of the current report are better explained by hypothesizing the existence of a binding site on acetylcholinesterase for chlorpyrifos oxon that allosterically affects the active site gorge in a manner that decreases the binding affinity of other chlorpyrifos oxon molecules. The active site gorge of acetylcholinesterase has been shown to contain regions that help position substrate as well as organophosphate inhibitors to form Michaelis complexes (Ordentlich et al., 1996, 1998, 2004; Soreq and Seidman, 2001). A region identified as the acyl pocket (Phe-295 and Phe-297) has been implicated in the positioning of certain organophosphates for nucleophilic attack by Ser-203, while the region known as the oxyanion hole subsite (Gly-121, Gly-122, and Ala-204) has been proposed to polarize the P=O bond, thereby activating the phosphorus and promoting the nucleophilic attack (Ordentlich et al., 1996, 1998). Allosteric modification that would change position of an important residue in either of these regions could result in a decreased binding affinity for inhibitor. One possible secondary binding site is the peripheral anionic site, and De Ferrari et al. (2001) and Shi et al. (2001) have documented conformational interactions between the peripheral anionic site and different regions of the active site gorge. Additionally, Bourne et al. (2006) have identified possible substrate binding sites on the surface of acetylcholinesterase located distal to the active site gorge. These authors speculated that occupation of these putative sites could allosterically affect substrate binding and catalytic parameters (Bourne et al., 2006).
A change in binding affinity, and therefore inhibitory capacity, of an organophosphate as a function of the concentration of that inhibitor, indicates that a linear relationship between organophosphate concentration and acetylcholinesterase activity does not exist when a broad range of organophosphate concentrations are considered. Since the inhibitory capacity of chlorpyrifos oxon toward human acetylcholinesterase has been shown to decrease as a function of concentration with inhibitor levels up to about 10nM (Kaushik et al., 2007), dose-response curves generated from chlorpyrifos oxon concentrations higher than about 10nM likely underestimate the risk represented by lower oxon levels.
|
FUNDING |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
National Institute of Environmental Health Sciences (ES012648 [GenBank] ) to L.G.S.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Aldridge WN, Reiner E. Enzyme inhibitors as substrates: Interactions of esterases with esters of organophosphorus and carbamic acids. In: "Frontiers of Biology"—Neuberger A, Tatum EL, eds. (1972) vol 26. New York: North-Holland Publishing Co. 9.
Barak D, Ordentlich A, Bromberg A, Kronman C, Marcus D, Lazar A, Ariel N, Velam B, Shafferman A. Allosteric modulation of acetylcholinesterase activity by peripheral ligands involves a conformational transition of the anionic subsite. Biochemistry (1995) 34:15444–15452.[CrossRef][Medline]
Bourne Y, Radi
Z, Sulzenbacher G, Kim E, Taylor P, Marchot P. Substrate and product trafficking through the active center gorge of acetylcholinesterase analyzed by crystallography and equilibrium binding. J. Biol. Chem. (2006) 281:29256–29267.
Boyd AE, Marnett AB, Wong L, Taylor P. Probing the active center gorge of acetylcholinesterase by fluorophores linked to substituted cyteines. J. Biol. Chem. (2000) 275:22401–22408.
Colletier J-P, Fournier D, Greenblatt HM, Stojan J, Sussman JL, Zaccai G, Silman I, Weik M. Structural insights into substrate traffic and inhibition in acetylcholinesterase. EMBO (2006) 25:2746–2756.[CrossRef][Web of Science][Medline]
De Ferrari GV, Mallender WD, Inestrosa NC, Rosenberry TL. Thioflavin T is a fluorescent probe of the acetylcholinesterase peripheral site that reveals conformational interactions between the peripheral and acylation sites. J. Biol. Chem. (2001) 276:23282–23287.
Estrada-Mondaca S, Fournier D. Stabilization of recombinant Drosophila acetylcholinesterase. Protein Expr. Purif. (1998) 12:166–172.[CrossRef][Web of Science][Medline]
Gray PJ, Duggleby RG. Analysis of kinetic data for irreversible enzyme inhibition. Biochem. J. (1989) 257:419–424.[Web of Science][Medline]
Hart GJ, O'Brien RD. Recording spectrophotometric method for determination of dissociation and phosphorylation constants for the inhibition of acetylcholinesterase by organophosphates in the presence of substrate. Biochemistry (1973) 12:2940–2945.[CrossRef][Medline]
Johnson JL, Cusack B, Davies MP, Fauq A, Rosenberry TL. Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate. Biochemistry (2003) 42:5438–5452.[CrossRef][Medline]
Kaushik R, Rosenfeld CA, Sultatos LG. Concentration-dependent interactions of the organophosphates chlorpyrifos oxon and methyl paraoxon with human recombinant acetylcholinesterase. Toxiol. Appl. Pharmacol. (2007) 221:243–250.[CrossRef]
Kemp JR, Wallace KB. Molecular determinants of the species-selective inhibition of brain acetylcholinesterase. Toxicol. Appl. Pharmacol. (1990) 104:246–258.[CrossRef][Web of Science][Medline]
Kousba SA, Sultatos LG, Poet TS, Timchalk C. Comparison of chlorpyrifos-oxon and paraoxon acetylcholinesterase inhibition dynamics: Potential role of a peripheral binding site. Toxicol. Sci. (2004) 80:239–248.
Kryger G, Harel M, Giles K, Toker L, Velan B, Lazar A, Kronman C, Barak D, Ariel N, Shafferman A, et al. Structures of recombinant native and E202Q mutant human acetylcholinestersae complexed with the snake-venom toxin fasciculin-II. Acta Cryst. (2000) D56:1385–1394.[Web of Science]
Liu W, Tsou CL. Determination of rate constants for the irreversible inhibition of acetylcholine esterase by continuously monitoring the substrate reaction in the presence of the inhibitor. Biochim. Biophys. Acta. (1986) 870:185–190.[CrossRef][Medline]
Mallender WD, Szegletes T, Rosenberry TL. Acetylthiocholine binds to asp74 at the peripheral site of human acetylcholinesterase as the first step in the catalytic pathway. Biochemistry (2000) 39:7753–7763.[CrossRef][Medline]
Masson HJ, Sains C, Stevenson AJ, Rawbone R. Rates of spontaneous reactivation and aging of acetylcholinesterase in human erythrocytes after inhibition by organophosphorus pesticides. Hum. Exp. Toxicol. (2000) 19:511–516.
Means GE, Bender ML. Acetylation of human serum albumin by p-nitrophenyl acetate. Biochemistry (1975) 14:4989–4994.[CrossRef][Medline]
Mileson BE, Chambers JE, Chen WL, Dettburn W, Ehrich M, Eldefrawi AT, Gaylor DW, Hamernik K, Hodgson E, Karczmar AG, et al. Common mechanism of toxicity: A case study of organophosphorus pesticides. Toxicol. Sci. (1998) 41:8–20.
Ordentlich A, Barak D, Kronman C, Ariel N, Segall Y, Velan B, Shafferman A. The architecture of human acetylcholinesterase active center probed by interactions with selected organophosphate inhibitors. J. Biol. Chem. (1996) 271:11953–11962.
Ordentlich A, Barak D, Kronman C, Ariel N, Segall Y, Velan B, Shafferman A. Functional characteristics of the oxyanion hole in human acetylcholinesterase. J. Biol. Chem. (1998) 278:19509–195127.
Ordentlich A, Barak D, Sod-Moriah G, Kaplan D, Mizrahi D, Segall Y, Kronman C, Karton Y, Lazar A, Marcus D, et al. Stereoselectivity toward VX is determined by interactions with residues of the acyl pocket as well as the peripheral anionic site of AChE. Biochemistry (2004) 43:11255–11265.[CrossRef][Medline]
Rosenfeld CA, Sultatos LG. Concentration-dependent kinetics of acetylcholinesterase inhibition by the organophosphate paraoxon. Toxicol. Sci. (2006) 90:460–469.
Sakurai Y, Ma S-F, Watanabe H, Yamaotsu N, Hirono S, Kurono Y, Kragh-Hansen U, Otagiri M. Esterase-like activity of serum albumin: Characterization of its structural chemistry using p-nitrophenyl esters as substrates. Pharm. Res. (2004) 21:285–292.[CrossRef][Web of Science][Medline]
Shafferman A, Kronman C, Flashner Y, Leitner M, Grosfeld H, Ordentlich A, Gozes Y, Cohen S, Ariel N, Barak D, et al. Mutagenesis of human acetylcholinesterase. Identification of residues involved in catalytic activity and in polypeptide folding. J. Biol. Chem. (1992) 267:17640–17648.
Shi J, Boyd AE, Radi Z, Taylor P. Reversibly bound and covalently attached ligands induce conformational changes in the omega loop cyc69-cys96, of mouse acetylcholinesterase. J. Biol. Chem. (2001) 276:42196–42204.
Soreq H, Seidman S. Acetylcholinesterase-new roles for an old actor. Nat. Rev. Neurosci. (2001) 2:294–302.[CrossRef][Web of Science][Medline]
Szegletes T, Mallender WD, Rosenberry TL. Nonequilibrium analysis alters the mechanistic interpretation of inhibition of acetylcholinesterase by peripheral site ligands. Biochemistry (1998) 37:4206–4216.[CrossRef][Medline]
Szegletes T, Mallender WD, Thomas PJ, Rosenberry TL. Substrate binding to the peripheral site of acetylcholinesterase initiates enzymatic catalysis. Substrate inhibition arises as a secondary effect. Biochemistry (1999) 38:122–133.[CrossRef][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||