Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark, New Jersey 07103
Received May 23, 2000; accepted August 2, 2000
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ABSTRACT |
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Key Words: paraoxon; methyl paraoxon; organophosphate; acetylcholinesterase; peripheral binding site; computer modeling.
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INTRODUCTION |
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Subsequent to the derivation of ki (Main, 1964), a variety of approaches were undertaken to determine experimentally ki, as well as the affinity constant (Ka in Figure 1B
) and the phosphorylation constant (k2 in Figure 1A
) (Hart and O'Brien, 1973
, 1976
; Main and Iverson, 1966
). However, all have been based upon the original procedure developed by Main (1964), and all have been limited by the assumption that the reaction in vitro approximates first-order conditions with respect to uninhibited enzyme (i.e., the oxon concentration is much higher than the enzyme concentration). In contrast, using a new approach capable of utilizing any organophosphate concentration relative to the enzyme concentration for determination of ki, preliminary studies from this laboratory suggested that the appki of paraoxon for mouse brain acetylcholinesterase changed as a function of the paraoxon concentration. This report was undertaken to characterize this relationship and to explore possible mechanisms that might account for such a phenomenon.
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MATERIALS AND METHODS |
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Animals and tissue preparations.
Male TAC:(SW)FBR Swiss mice (2030 g) were purchased from Taconic Farms (Germantown, NY). They were housed under standard laboratory conditions and given free access to water and food (Purina Rodent Chow 5001). Recombinant monomeric mouse brain acetylcholinesterase was kindly provided by Dr. Palmer Taylor (University of California, San Diego, La Jolla, CA).
Mice were decapitated, and their brains were immediately removed and placed on ice. They were weighed, pooled, and homogenized in 9 volumes of 100 mM sodium phosphate buffer (pH 7.4) (phosphate buffer), using a Polytron® homogenizer (Brinkmann Instruments, Westbury, NY). The homogenates were stored in individual 1-ml aliquots at 70°C.
Incubations and assay.
After thawing, a 1-ml aliquot of homogenate was added to 13 ml of phosphate buffer. All homogenate incubations included 200 µl of this diluted mouse brain homogenate in a total volume of 400 µl, with the indicated oxon concentrations, as described by Wang and Murphy (1982a). Incubations were done at either 23°C or 37°C in a shaking water bath. Controls did not include organophosphate, or received the organophosphate at the end of the incubation, just before addition of buffer for the determination of activity (see below). No differences in results were observed with or without the addition of 3 mM EDTA to the buffer to inhibit A-esterase (Sultatos, 1994; data not shown), so EDTA was not routinely included. Similarly, inclusion of 100 µM iso-OMPA to inhibit butyrylcholinesterase (EC 3.1.1.8) (Mortensen et al., 1998) had a negligible effect on the results (data not shown), and was therefore not routinely included in the incubations.
Due to the instability of recombinant monomeric mouse brain acetylcholinesterase, incubations containing this enzyme preparation and oxon were performed at 4°C in phosphate buffer containing 50% glycerol in a total volume of 3050 µl. The final enzyme concentration was 0.4318 nM. Previous studies have documented that glycosylation, which is lacking in recombinant monomeric mouse brain acetylcholinesterase, is required for thermal stability but not catalytic activity (Velan et al., 1993).
Uninhibited mouse brain acetylcholinesterase was determined by the Ellman method (Ellman et al., 1961). In the case of mouse brain homogenate assays, incubations were terminated by dilution with 4.6 ml phosphate buffer. ATC was added to give a final concentration of 0.4 mM, and DTNB was added to give a final concentration of 0.1 mM, with a final volume of 5 ml. These incubations were carried out for 30 min in a shaking water bath at 23°C. The change in absorbance at 412 nm was used as an indication of uninhibited acetylcholinesterase. Measurement of uninhibited recombinant monomeric mouse brain acetylcholinesterase was performed in a similar manner, except that 500 µl of the recombinant acetylcholinesterase incubation was removed and diluted with 10 ml phosphate buffer with 0.4 mM ATC and 0.1 mM DTNB. This incubation was carried out at 23°C for 7 min, and the change in absorbance at 412 nm was recorded.
Model structures.
Several computer models were used. The first, referred to as the ki model, was based on the equation derived by Main (1964) (Fig. 1B). The equations descriptive of this model were as follows:
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Determination of parameters through model optimization.
Appkis were determined by optimization of the ki model to experimental data. The optimization procedure consisted of determination of that appki that gave the best-fit (as judged by the smallest sum of squares) to the experimental data. Optimization for acetylcholinesterase active site concentration in mouse brain homogenate was accomplished with experimental data transformed as percent of control to account for heterogeneous variance. In certain instances, Monte Carlo simulations were incorporated into the modeling in order to estimate standard deviations for optimized appkis. In these cases, Monte Carlo simulations were performed with a computer program modified from that of Thomas et al. (1996), written in ACSL. Simulations were truncated at zero, as negative values (which otherwise would occasionally appear) for the parameters simulated cannot occur biologically. Means and standard deviations of experimental data for organophosphate-induced inhibition of acetylcholinesterase activity over time were incorporated into Monte Carlo simulations to generate 1000 optimizations, from which the appki standard deviations were calculated. All experimental data used in the Monte Carlo simulations were judged normally distributed by the Kolmogorov-Smirnov test for normality.
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RESULTS |
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DISCUSSION |
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Anomalous kinetic results for the interaction of diisopropylfluorophosphate with acetylcholinesterase have been attributed previously to multiple forms of the enzyme with differential sensitivities to this organophosphate (Main, 1969). The existence of multiple forms of mouse brain acetylcholinesterase with differential sensitivities towards paraoxon and methyl paraoxon could provide an explanation for the change in appki as a function of inhibitor concentration. However, this explanation seems unlikely, as their differential sensitivities would have to be striking (the appki for paraoxon changed from 2.94 nM1h1 to 0.19 nM1h1; Fig. 6
). Although multiple forms of acetylcholinesterase exist in the mouse, they are all the product of a single gene and have a constant catalytic core. Their differences arise through alternative mRNA processing and association of the catalytic subunits with structural subunits (Taylor and Radic, 1994
). In addition, Friboulet et al. (1990) have demonstrated that all the molecular forms of muscle acetylcholinesterase have similar susceptibilities to inhibition by O-ethyl S-(2-(diisopropylamino)ethyl) methyphosphonothioate (MPT). Furthermore, Mortensen et al. (1998) have shown that no age-dependent or tissue-specific differences in acetylcholinesterase sensitivity towards chlorpyrifos oxon exist in the rat after immunoprecipitation of the enzyme.
Although the existence of multiple acetylcholinesterases with differential sensitivities toward paraoxon and methyl paraoxon cannot be entirely discounted, a more credible explanation for the unusual kinetic behavior presented in the current report involves a second binding site on acetylcholinesterase, distinct from the active site, to which paraoxon and methyl paraoxon could bind reversibly, and through allosteric modification or steric hindrance impede the subsequent phosphorylation of the active site by additional oxon molecules (hence decreasing the appki) (Fig. 1C). This hypothesis is supported by the observation that recombinant monomeric mouse brain acetylcholinesterase also displayed an appki that changed as a function of the paraoxon concentration (Fig. 9
). A second or peripheral binding site on acetylcholinesterase, first suggested by Changeux (1966), was implied by an early study of Aldridge and Reiner (1969), which proposed that haloxon (di-(2-chloroethyl) 3-chloro-4-methylcoumarin-7-yl phosphate), coroxon (diethyl 3-chloro-4-methylcoumarin-7-yl phosphate), coumarin, acetylcholine, and acetylthiocholine can bind to this second site, thereby decreasing the reactivity of the active site. Subsequent studies have established the existence of a peripheral binding site that has been characterized by site-directed mutagenesis (Barak et al., 1994
, 1995
; Radic et al., 1993
; Shafferman et al., 1992
; Velan et al., 1996
). Binding of ligands such as propidium and fasciculin, specific for this peripheral site, appears to inhibit enzymatic activity by allosteric modification of the active site through affecting the orientation of the Trp-86 indole moiety located at the base of the gorge that contains the active site (Barak et al., 1995
; Berman et al., 1980
; Bourne et al., 1995
, 1999
; Radic et al., 1984
, 1991
, 1995
; Taylor and Lappi, 1975
; Taylor and Radic, 1994
; Velan et al., 1996
).
Whereas the report of Aldridge and Reiner (1969) implied that the organophosphates haloxon and coroxon bound reversibly to a peripheral binding site, Friboulet et al. (1990) established directly that the organophosphorus compound MPT bound reversibly to the peripheral binding site, thereby reducing its own turnover at the active site. Other organophosphates have been thought not to bind to this site, as they have been reported to follow simple second-order kinetics (Aldridge and Reiner, 1972; Friboulet et al., 1990
; Hart and O'Brien, 1973
). However, the current study indicates that, at least with paraoxon and methyl paraoxon, inhibitor concentrations similar to that of enzyme (i.e., not many times higher than the acetylcholinesterase concentration) must be used in order to observe their anomalous kinetic behavior (Fig. 6
), conditions that have not been satisfied by previous kinetic studies. In that regard, the appki determined at a paraoxon concentration (1000 nM) far greater than the enzyme concentration (0.4398 nM) (Fig. 5
) was similar to that determined by the method developed by Main (1964) (Figs. 7 and 8
). Moreover, the appkis determined at high oxon levels in the current study (0.19 nM1h1) (Figs. 68
) are in close agreement to those reported previously by Wang and Murphy (1982b) (0.11 nM1h1) for paraoxon and acetylcholinesterase in rat brain homogenate.
In order to determine if the binding of paraoxon (or methyl paraoxon) to such a peripheral binding site could account for the changes in appki as a function of paraoxon concentration (Fig. 6), a kinetic model incorporating appki and a peripheral binding site (Fig. 1C
, referred to as the p-site model) was constructed. In this model, appki had two possible values depending on whether or not the peripheral binding site was occupied by paraoxon. It should be noted that this exercise was not intended to yield a best-fit simulation for in vitro inhibition of acetylcholinesterase by paraoxon (such as that shown in Figs. 2 and 5
), but was instead meant to determine if the interaction of paraoxon with a peripheral binding site could, in theory, account for the relationship observed in the current study between paraoxon concentration and appki (Fig. 6
). Nonlinear regression analyses revealed the relationship between appki and paraoxon concentration was best-fit by a rectangular hyperbola, with a theoretical appki of 2.94 nM1h1 at 0 nM paraoxon, and an appki of 0.19 nM1h1 when paraoxon was infinitely high (Fig. 6
). Therefore, with the peripheral binding site unoccupied (at 0 nM paraoxon) the appki was 2.94 nM1h1. Similarly, paraoxon's binding to the peripheral binding site, which became saturated at about 3 nM paraoxon (Fig. 6
), reduced the appki to 0.19 nM1h1. Consequently 2.94 nM1h1 and 0.19 nM1h1 represented appki and appki`, respectively, in the p-site model, based on the scheme outlined in Figure 1C
. Although optimization studies could not reveal the actual values for appk+1 and appk1 in Figure 1C
, it could be shown that
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In summary, the data presented in the present study suggest that the reversible binding of paraoxon or methyl paraoxon to a site on acetylcholinesterase distinct from the active site reduces their subsequent capacity to phosphorylate the active site. However it cannot be ascertained from these data whether this putative second site is identical to or overlaps with the well-established propidium-specific peripheral binding site of acetylcholinesterase. In that regard, Radic and Taylor (1999) have reported that the peripheral site ligands propidium, gallamine, D-tubocurarine, and atropine enhanced paraoxon's rate of inhibition of wild-type mouse acetylcholinesterase, whereas coumarin derivatives inhibited this same reaction. However, the absence of details regarding the paraoxon and enzyme concentrations make the interpretation of these results difficult within the context of the current report.
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NOTES |
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REFERENCES |
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Aldridge, W. N., and Reiner, E. (1969). Acetylcholinesterase. Two types of inhibition by an organophosphorus compound: one the formation of phosphorylated enzyme and the other analogous to inhibition by substrate. Biochem. J. 115, 147162.[ISI][Medline]
Aldridge, W. N., and Reiner, E. (1972). Enzyme Inhibitors as Substrates. Interactions of Esterases with Esters of Organophosphorus and Carbamic Acids. North Holland, Amsterdam.
Barak, D., Kronman, C., Ordentlich, A., Ariel, N., Bromberg, A., Marcus, D., Lazar, A., Velan, B., and Shafferman, A. (1994). Acetylcholinesterase peripheral anionic site degeneracy conferred by amino acid arrays sharing a common core. J. Biol. Chem. 269, 62966305.
Barak, D., Ordentlich, A., Bromberg, A., Kronman, C., Marcus, D., Lazar, A., Ariel, N., Velan, B., and Shafferman, A. (1995). Allosteric modulation of acetylcholinesterase activity by peripheral ligands involves a conformational transition of the anionic subsite. Biochemistry 34, 1544415452.[ISI][Medline]
Berman, H. A., Yguerabide, J., and Taylor, P. (1980). Fluorescence energy transfer on acetylcholinesterase: Spatial relationship between peripheral site and active center. Biochemistry 19, 22262235.[ISI][Medline]
Bourne, Y., Taylor, P., and Marchot, P. (1995). Acetylcholinesterase inhibition by fasciculin: crystal structure of the complex. Cell 83, 503512.[ISI][Medline]
Bourne, Y., Taylor, P., Bougis, P. E., and Marchot, P. (1999). Crystal structure of mouse acetylcholinesterase. J. Biol. Chem. 274, 29632970.
Changeux J-P. (1966). Responses of acetylcholinesterase from Torpedo marmorata to salts and curarizing drugs. Mol. Pharmacol. 2, 369392.[Abstract]
Ellman, G. L., Courtney, K. D., Andres, V., and Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 8895.[ISI][Medline]
Friboulet, A., Rieger, F., Goudou, D., Amitai, G., and Taylor, P. (1990). Interaction of an organophosphate with a peripheral site on acetylcholinesterase. Biochemistry 29, 914920.[ISI][Medline]
Fukuto, T. R. (1990). Mechanism of action of organophosphorus and carbamate insecticides. Environ. Health Perspect. 87, 245254.[ISI][Medline]
Hart, G. J., and O'Brien, R. D. (1973). Recording spectrophotometric method for determination of dissociation and phosphorylation constants for the inhibition of acetylcholinesterase by organophosphates in the presence of substrate. Biochem. 12, 29402945.[ISI][Medline]
Hart, G. J., and O'Brien, R. D. (1976). Dissociation and phosphorylation constants for the inhibition of acetylcholinesterase by a series of novel O-ethyl O-phenyl S-n-propyl phosphorothioates. Pestic. Biochemistry. Physiol. 6, 8590.
Levine, B. S., and Murphy, S. D. (1977). Esterase inhibition and reactivation in relation to piperonyl butoxide-phosphorothionate interactions. Toxicol. Appl, Pharmacol. 40, 379391.[ISI][Medline]
Main, A. R. (1964). Affinity and phosphorylation constants for the inhibition of esterases by organophosphates. Science 144, 992993.[ISI][Medline]
Main, A. R. (1969). Kinetic evidence of multiple reversible cholinesterases based on inhibition by organophosphates. J.Biol. Chem. 244, 829840.[ISI][Medline]
Main, A. R., and Iverson, F. (1966). Measurement of the affinity and phosphorylation constants governing irreversible inhibition of cholinesterases by di-isopropyl phosphorofluoridate. Biochem. J. 100, 525531.[ISI][Medline]
Mileson, B. E., Chambers, J. E., Chen, W. L., Dettburn, W., Ehrich, M., Eldefrawi, A. T., Gaylor, D. W., Hamernik, K., Hodgson, E., Karczmar, A.G., Padilla, S., Pope, C. N., Richardson, R. J., Saunders, D. R., Sheets, L. P., Sultatos, L. G., and Wallace, K. B. (1998). Common mechanism of toxicity: a case study of organophosphorus pesticides. Toxicol. Sci. 41, 820.[Abstract]
Mortensen, S. R., Brimijoin, S., Hooper, M. J., and Padilla, S. (1998). Comparison of the in vitro sensitivity of rat acetylcholinesterase to chlorpyrifos-oxon: what do tissue IC50 values represent? Toxicol. Appl. Pharmacol. 148, 4649.[ISI][Medline]
Rachinsky, T. L., Camp, S., Li, Y., Ekstrom, T. J., Newton, M., and Taylor, P. (1990). Molecular cloning of mouse acetylcholinesterase: tissue distribution of alternatively spliced mRNA species. Neuron 5, 317327.[ISI][Medline]
Radic, A., Reiner, E., and Simeon, V. (1984). Binding sites on acetylcholinesterase for reversible ligands and phosphorylating agents. Biochem. Pharmacol. 33, 671677.[ISI][Medline]
Radic, Z., Reiner, E., and Taylor, P. (1991). Role of the peripheral anionic site on acetylcholinesterase: inhibition by substrates and coumarin derivatives. Mol. Pharmacol. 39, 98104.[Abstract]
Radic, Z., Pickering, N., Vellom, B., Camp, S., and Taylor, P. (1993). Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors. Biochemistry 32, 1207412084.[ISI][Medline]
Radic, Z., and Taylor, P. (1999). The influence of peripheral site ligands on the reaction of symmetric and chiral organophosphates with wildtype and mutant acetylcholinesterase. Chem. Biol. Interact. 119-120, 111117.
Radic, Z., Quinn, D. M., Vellom, D. C., Camp, S., and Taylor, P. (1995). Allosteric control of acetylcholinesterase catalysis by fasciculin. J. Biol. Chem. 270, 2039120399.
Segel, I. H. (1975). Enzyme Kinetics. John Wiley & Sons, New York.
Sultatos, L. G. (1994). Mammalian toxicology of organophosphorus pesticides. J. Toxicol. Environ. Health 43, 271289.[ISI][Medline]
Shafferman, A., Velan, B., Ordentlich, A., Kronman, C., Grossfeld, H., Leitner, M., Flashner, Y., Cohen, S., Barak, D., and Ariel, N. (1992). Substrate inhibition of acetylcholinesterase: residues affecting signal transduction from the surface to the catalytic center. EMBO J. 11, 35613568.[Abstract]
Sussman, J. L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L., and Silman, I. (1991). Atomic structure of acetylcholinesterase from Torpedo californica: a prototype acetylcholine-binding protein. Science 253, 872879.[ISI][Medline]
Taylor, P., and Lappi, S. (1975). Interaction of fluorescence probes with acetylcholinesterase. The site and specificity of propidium binding. Biochemistry 14, 19891997.[ISI][Medline]
Taylor, P., and Radic, Z. (1994). The cholinesterases: from genes to proteins. Annu. Rev. Pharmacol. Toxicol. 34, 281320.[ISI][Medline]
Thomas, R. S., Lytle, W. E., Keefe, T. J., Constan, A. A., and Yang, R. S. (1996). Incorporating Monte Carlo simulation into physiologically based pharmacokinetic models using advanced continuous simulation language (ACSL): a computational method. Fundam. Appl. Toxicol. 31, 1928.[ISI][Medline]
Velan, B., Kronman, C., Ordentlich, A., Flashner, Y., Leitner, M., Cohen, S., and Shafferman, A. (1993). N-glycosylation of human acetylcholinesterase: effects on activity, stability, and biosynthesis. Biochem. J. 296, 649656.[ISI][Medline]
Velan, B., Barak, D., Ariel, N., Leitner, M., Bino, T., Ordentlich, A., and Shafferman, A. (1996). Structural modifications of the loop in human acetylcholinesterase. FEBS Lett. 395, 2228.[ISI][Medline]
Wang, C., and Murphy, S. D. (1982a). The role of non-critical binding proteins in the sensitivity of acetylcholinesterase from different species to diisopropyl fluorophosphate (DFP), in vitro. Life Sci. 31, 139149.[ISI][Medline]
Wang, C., and Murphy, S. D. (1982b). Kinetic analysis of species difference in acetylcholinesterase sensitivity to organophosphate insecticides. Toxicol. Appl. Pharmacol. 66, 409419.[ISI][Medline]