Inhibition of Acetylcholinesterase by (1S,3S)-Isomalathion Proceeds

An aliquot of the enzyme solution was then diluted 1/300 (v/v) in 0.1 M ... (1S)-isomers; 0−4 min, (1R)-isomers, oxime-mediated reactivation; and 0â...
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Chem. Res. Toxicol. 2000, 13, 1313-1320

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Inhibition of Acetylcholinesterase by (1S,3S)-Isomalathion Proceeds with Loss of Thiomethyl: Kinetic and Mass Spectral Evidence for an Unexpected Primary Leaving Group Jonathan A. Doorn,† Douglas A. Gage,‡ Michael Schall,‡ Todd T. Talley,§ Charles M. Thompson,§,| and Rudy J. Richardson*,† Toxicology Program, Department of Environmental Health Sciences, The University of Michigan, Ann Arbor, Michigan 48109, Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, and Department of Chemistry and Department of Pharmaceutical Sciences, The University of Montana, Missoula, Montana 59812 Received August 22, 2000

Previous work demonstrated kinetically that inhibition of mammalian acetylcholinesterase (AChE) by (1S)-isomalathions may proceed by loss of thiomethyl instead of the expected diethyl thiosuccinate as the primary leaving group followed by one of four possible modes of rapid aging. This study sought to identify the adduct that renders AChE refractory toward reactivation after inhibition with the (1S,3S)-stereoisomer. Electric eel acetylcholinesterase (EEAChE) was inhibited with the four stereoisomers of isomalathion, and rate constants for spontaneous and oxime-mediated reactivation (k3) were measured. Oxime-mediated k3 values were >25-fold higher for enzyme inhibited by (1R)- versus (1S)-stereoisomers with the greatest contrast between the (1R,3R)- and (1S,3S)-enantiomers. EEAChE inactivated by (1R,3R)isomalathion reactivated spontaneously and in the presence of pyridine-2-aldoxime methiodide (2-PAM) with k3 values of 1.88 × 105 and 4.18 × 105 min-1, respectively. In contrast, enzyme treated with the (1S,3S)-enantiomer had spontaneous and 2-PAM-mediated k3 values of 0 and 6.05 × 103 min-1, respectively. The kinetic data that were measured were consistent with those obtained for mammalian AChE used in previous studies. Identification of the adduct that renders EEAChE stable toward reactivation after inhibition with (1S,3S)-isomalathion was accomplished using a peptide mass mapping approach with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). A peak with a mass corresponding to the active site peptide containing the catalytic Ser with a covalently bound O-methyl phosphate adduct was found in the mass spectra of (1S,3S)-treated EEAChE but not control samples. Identities of the modified active site peptide and adduct were confirmed by fragmentation in MALDI-TOF-MS post-source decay (PSD) analysis, and peaks corresponding to the loss of an adduct as phosphorous/phosphoric acid methyl ester were observed. The results demonstrate that inhibition of EEAChE by (1S,3S)-isomalathion proceeds with loss of thiomethyl as the primary leaving group followed by rapid expulsion of diethyl thiosuccinate as the secondary leaving group to yield an aged enzyme.

Introduction Malathion, S-[1,2-bis(ethoxycarbonyl)ethyl]-O,O-dimethyl phosphorothioate, is an organophosphorus (OP)1 pesticide that has activity against a broad range of insects and low mammalian toxicity. Metabolic activation of malathion to malaoxon results in an effective inhibitor * To whom correspondence should be addressed: Toxicology Program, The University of Michigan, 1420 Washington Heights, Ann Arbor, MI 48109-2029. Telephone: (734) 936-0769. Fax: (734) 6479770. E-mail: [email protected]. † The University of Michigan. ‡ Michigan State University. § Department of Chemistry, The University of Montana. | Department of Pharmaceutical Sciences, The University of Montana. 1 Abbreviations: AChE, acetylcholinesterase; ATCh, acetylthiocholine; BSA, bovine serum albumin; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); EEAChE, electric eel acetylcholinesterase; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MH+, protonated molecule; OP, organophosphorus; PSD, post-source decay; 2-PAM, pyridine-2-aldoxime methiodide.

of acetylcholinesterase (AChE, EC 3.1.1.7) (1, 2). Thermal or photochemical isomerization of malathion can occur to yield racemic isomalathion, S-[1,2-bis(ethoxycarbonyl)ethyl]-O,S-dimethyl phosphorodithiolate, as an impurity (3, 4). Isomalathion contains two asymmetric centers with one at the phosphorus and the other at a carbon, yielding four stereoisomers (Figure 1). Previous work has demonstrated that AChE is stereoselectively inhibited by isomalathion with potencies differing by 29-fold for rat brain AChE (5, 6). In all the cases that have been reported, the (1R,3R)-isomer is the most potent, while its enantiomer, (1S,3S)-isomalathion, is the least potent. It has also been shown that a difference in postinhibitory kinetics exists for AChE treated with the four stereoisomers (7, 8). Although AChE inhibited with (1R)-isomers reactivates readily both spontaneously and in the presence of an oxime, the enzyme inhibited with (1S)-stereoisomers is intractable

10.1021/tx000184v CCC: $19.00 © 2000 American Chemical Society Published on Web 11/10/2000

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cially available purified electric eel acetylcholinesterase (EEAChE) was selected for this study due to its availability and solubility in the absence of salts and detergents, which can interfere with mass spectrometric analysis (9). Rates of reactivation were measured for enzyme inhibited by the four stereoisomers of isomalathion to verify that EEAChE postinhibitory kinetic data were consistent with those determined for mammalian AChE used in previous studies. Figure 1. The four stereoisomers of isomalathion with asymmetric phosphorus and carbon atoms designated as positions 1 and 3, respectively.

toward reactivation. A recent study compared the rates of reactivation and aging for the stereoisomers of isomalathion with those of isoparathion methyl and found similarity between (1R)-isomalathion isomers and the configurationally equivalent (S)-isoparathion methyl, but dissimilarity between (1S)-isomalathion isomers and the configurationally equivalent (R)-isoparathion methyl (8). Both enantiomers of isoparathion methyl are thought to modify covalently the active site Ser of AChE with an O,S-dimethylphosphoryl group differing only in stereochemistry. The dissimilarity in postinhibitory kinetics between enzyme treated with (R)-isoparathion methyl and (1S)-isomalathion implies that inhibition of AChE with (1S)-isomalathion does not yield the expected O,Sdimethylphosphoryl adduct, and that a difference in the mechanism of inactivation between the (1R)- and (1S)stereoisomers of isomalathion exists. To account for this dissimilarity, it has been postulated that the primary leaving group may be the thiomethyl instead of the diethyl thiosuccinate moiety in the reaction of (1S)isomers with the enzyme (7). Subsequent postinhibitory reactions could yield an aged enzyme that is intractable toward reactivation (Scheme 1). The AChE adduct depicted in each of the four mechanisms shown in Scheme 1 has a mass that differs from the others by at least 16 Da. Therefore, the mechanistic outcomes could be distinguished from each other using mass spectrometry. In addition to the difference in postinhibitory kinetics between AChE inhibited by (1R)- versus (1S)-isomers of isomalathion, there is also an apparent difference in the postinhibitory kinetics for AChE inhibited with (1S,3R)versus (1S,3S)-isomalathion (8). Respective kinetic constants for reactivation and aging were dissimilar for the enzyme inhibited with each stereoisomer. Moreover, it was discovered that AChE inactivated with (1S,3S)isomalathion aged at an extremely rapid rate with an estimated t1/2 of 99% as assessed by 1H and 31P NMR, GC, HPLC, and combustion analysis. Isomalathion stereoisomer configurations were assigned on the basis of single-crystal X-ray analysis of a strychnine salt precursor. Optical rotations and stereoisomeric purities (all >90%) were determined by polarimetry and chiral HPLC, respectively. EEAChE Type V-S, acetylthiocholine iodide (ATCh), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), bovine serum albumin (BSA), human angiotensin I, melittin, diethyl-p-nitrophenyl phosphate (paraoxon), and pyridine-2-aldoxime methiodide (2-PAM) were purchased from Sigma Chemical Co. (St. Louis, MO). Sequencing grade modified porcine trypsin used for protein digestion was purchased from Promega (Madison, WI). All other chemicals were reagent grade. Aqueous solutions were prepared in doubly deionized water. AChE Reactivation. AChE was incubated with each of the four stereoisomers of isomalathion with concentrations to yield 80% [(R)-isomers] and 90% inhibition [(S)-isomers] in 15 min. Reactions were performed in 0.1 M sodium phosphate buffer (pH 7.6) at 25 °C. Concentrations used for inhibition of EEAChE were as follows: (1R,3R), 4.3 µM; (1R,3S), 13 µM; (1S,3R), 1.2 µM; and (1S,3S), 21 µM. An aliquot of the enzyme solution was then diluted 1/300 (v/v) in 0.1 M sodium phosphate buffer (pH 7.6, 25 °C) and 1 mg/mL BSA to stabilize the enzyme. Aliquots were withdrawn at timed intervals and assayed for activity by the method of Ellman et al. (11) with 1.0 mM ATCh and 0.32 mM DTNB using a SPECTRAmax 340 microplate reader (Molecular Devices Corp., Sunnydale, CA). All assays were performed in 0.1 M sodium phosphate buffer (pH 7.6) at 25 °C. Oxime-mediated reactivation was accomplished using the same procedure except that the initial reactivation mixture contained 50 µM 2-PAM. A separate control was run to determine the effect of 2-PAM on ATCh hydrolysis, and the rate of nonenzymatic ATCh hydrolysis was subtracted from control and treated samples. Determination of k3. The apparent first-order rate constant of reactivation (k3) was determined according to the method of Clothier et al. (12). The following equation was used to calculate k3:

ln(100/% inhibition) ) k3t; % inhibition ) [(A - At)/(A - A0)] × 100 where A is the activity of the control, At is the activity of the inhibited enzyme at time t, and A0 is the activity of the inhibited enzyme at 0 min. The slope of the least-squares best-fit line was determined using linear regression, and only the linear portion of the graph was used in the calculation [0-12 min, (1S)isomers; 0-4 min, (1R)-isomers, oxime-mediated reactivation; and 0-8 min, (1R)-isomers, spontaneous reactivation].

Mechanism of AChE Inhibition by (1S,3S)-Isomalathion

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Scheme 1. Proposed Postinhibitory Reactions of AChE Inhibited with (1S)-Isomalathion To Account for Apparent Rapid Aging Involving (A) Intramolecular SN2 Reaction, (B) SN2 Reaction of a Nucleophile Such as Water with the OP Adduct, (C) Intramolecular E2 Reaction, or (D) Hydrolysis of One of the Two Ester Groups on the Diethyl Thiosuccinyl Moiety

Statistical Analysis. All values are reported as means ( SE from at least three separate experiments. All statistics were calculated using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA). The significance of differences between pairs of means was determined by twotailed unpaired t tests (p < 0.05). An F test was performed to determine whether slopes of regression lines of reactivation plots were significantly non-zero (p < 0.05). Preparation of AChE for MALDI-TOF-MS Analysis. EEAChE (250 units) in 500 µL of 25 mM Tris/HCl buffer (pH 7.6) at 37 °C, containing 1 mM CaCl2, was incubated with 20 µM (1S,3S)-isomalathion for 2 h at 25 °C. The protein content of each sample was ∼0.5 mg/mL. The concentration of acetone was 0.05). A significant difference in k3 for 2-PAM-mediated reactivation was found between the enzyme inhibited with (1S,3R)- and (1S,3S)-isomalathion. EEAChE inhibited with (S)-isoparathion methyl spontaneously reactivated with a k3 (mean ( SE) of 175.5 ( 5.3 min-1 × 103 (n ) 2). This value was not significantly different from spontaneous k3 values measured for the enzyme inhibited with (1R,3R)- or (1R,3S)-isomalathion (p < 0.05). The (R)-enantiomer of isoparathion methyl was an ineffective inhibitor of EEAChE. Treatment of the enzyme with 230 µM (R)-isoparathion methyl for 15 min resulted in a 0.05).

difference in k3 was observed for AChE treated with (1R,3R)- versus (1R,3S)-isomalathion. On the contrary, EEAChE inhibited with (1S)-isomalathions was refractory toward reactivation in the presence and absence of 50 µM 2-PAM (Figure 3). The presence of 50 µM 2-PAM increased the rate of reactivation for enzyme inhibited by (1R)- and (1S,3R)-isomers by about 2-fold. Plots used to calculate the spontaneous k3 for AChE treated with (1S,3S)-isomalathion had slopes that were not signifi-

MALDI-TOF-MS Analysis. A positive control experiment was performed using EEAChE inhibited with diethyl-p-nitrophenyl phosphate (paraoxon) to test the suitability of using peptide mass mapping and MALDITOF-MS to approach this problem. Paraoxon is thought to yield a diethyl phosphate adduct covalently bound to the active site Ser when reacted with AChE (14). After tryptic digestion, a peak in the MALDI spectrum corresponding to the putative active site peptide with the expected P(O)(OCH2CH3)2 (137 Da) adduct was observed in the treated sample but not in the control sample (results not shown). Following this positive result, 250 units of EEAChE was inhibited to 10-fold) than (1S)-isomalathion-inhibited AChE (7, 8). EEAChE treated with the four stereoisomers falls into this pattern as well, with a remarkable difference in the rates of reactivation for the enzyme inhibited with (1R)- versus (1S)-isomers. The k3 values measured for this enzyme inactivated by (1R)isomalathions in the presence and absence of 2-PAM are the highest yet reported for any species of AChE despite the fact that a lower temperature of 25 °C was used for the reactivation experiments described in this paper.

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Table 1. Masses of Theoretical and Observed MH+ Ions for Unmodified and Modified Active Site Peptides of EEAChEa MH+ (average mass) preparation unseparated HPLC separated

peptideb unmodified modified unmodified modified

theoretical 2657.0 2751.0 2657.0 2751.0

∆m (theoretical)c

observed

∆m (observed)c

((1.7)d

94.0 94.0

2657.6 2752.3 ((1.6)d 2658.0 ((0.9)e 2751.9 ((1.0)e

94.7 ((2.3) 93.9 ((1.3)

a

Conditions used for the preparation and analysis of the active site peptide containing the catalytic Ser were as described in Materials and Methods. b Active site peptide containing the catalytic Ser in the control (unmodified) and (1S,3S)-isomalathion-treated (modified) samples. c Difference in mass between the modified and unmodified active site peptide that takes into account the Ser proton lost in the phosphorylation reaction. d Mean ( SE (n ) 2 experiments). e Mean ( SE (n ) 4 or 5 experiments for unmodified or modified samples, respectively).

Figure 6. Representative spectrum from MALDI-TOF-MS analysis in reflectron mode of the peak with a mass (monoisotopic) corresponding to that of the active site peptide with an O-methyl phosphate adduct (2750.4 Da). The three peaks to the left of the precursor ion peak (2750.4 Da) demonstrate its fragmentation. The masses of the observed peaks represent loss of mass corresponding to that of phosphoric/phosphorous acid methyl ester from the modified active site peptide. The m/z 2621.5 fragment ion may correspond to loss of ammonium or water from the m/z 2638.6 fragment ion (16). Experimental conditions used to generate the spectrum are described in Materials and Methods.

Diethyl thiosuccinate has long been thought to be the primary leaving group in the inhibition of AChE by isomalathion (12). Since Clothier et al. first postulated the identity of the primary leaving moiety, evidence has been generated to support this hypothesis. Mammalian AChE inhibited with (1R)-isomalathions had k3 values that were not significantly different from each other or from k3 values for the enzyme inhibited with the configurationally equivalent (S)-isoparathion methyl (7, 8), which suggests that both (1R)-isomalathions and (S)isoparathion methyl generate a stereochemically identical O,S-dimethyl phosphate adduct upon reaction with AChE. In the study presented here, a similar kinetic result was found. The k3 values for EEAChE inhibited with (1R,3R)versus (1R,3S)-isomers were not significantly different from each other for either spontaneous or 2-PAMmediated reactivation (p < 0.05). Moreover, the enzyme inactivated with the configurationally equivalent (S)isoparathion methyl spontaneously reactivated with a k3 that was not significantly different from spontaneous k3 values for EEAChE inhibited with either (1R,3R)- or (1R,3S)-isomalathion (p < 0.05). Recent kinetic data suggest that inhibition of mammalian AChE with (1S)-isomalathion proceeds with loss of thiomethyl instead of diethyl thiosuccinate. It was found that mammalian AChE inactivated by (1S,3R)versus (1S,3S)-isomalathion had k3 values significantly

different from each other and from that for the enzyme inhibited with the configurationally equivalent (R)-isoparathion methyl (7, 8). In the study presented here, (R)isoparathion methyl proved to be an ineffective inhibitor of EEAChE; therefore, it was not possible to obtain k3 values for this compound to compare with spontaneous or oxime-mediated values for the enzyme inhibited with the configurationally equivalent (1S)-isomalathions. Nevertheless, just as observed with mammalian AChE, not only were the k3 values for EEAChE inhibited by (1S)isomers different from each other for a given reaction condition, but the enzyme inhibited by the (1S,3S)-isomer was completely intractable to spontaneous reactivation. Thus, the kinetic results obtained in the study presented here for EEAChE inactivated by (1S)-isomers of isomalathion are consistent with those from previous studies on mammalian AChE and support the idea that thiomethyl may be the primary leaving group. Confirmation of these and prior kinetic studies predicting an unexpected change in the primary leaving group in the case of AChE inhibition by (1S)-isomers of isomalathion requires direct chemical identification of the OP adduct formed with the enzyme. In the study presented here, the identity of the adduct resulting from inhibition of EEAChE with (1S,3S)-isomalathion was determined using peptide mass mapping and MALDI-TOF-MS analysis and is strikingly different from that predicted by the conventional mechanism of inhibition and aging (Scheme 2). Our analysis of the inhibited electric eel enzyme did not reveal any tryptic peptides in the unseparated or HPLC-separated fractions with a mass representing the active site peptide containing an O,S-dimethyl phosphate or O-methyl thiophosphate adduct. Although the latter species could theoretically form the O-methyl phosphate adduct by loss of HS- or H2S, this possibility is unlikely at the pH used in our experiments. Loss of HS- or H2S requires protonation of the phosphorothiolate anion, which is improbable at pH 7.6 given the highly acidic nature of O,O-dialkyl phosphorothioic acids (17). Hydrolysis of the O-methyl thiophosphate adduct with loss of methoxide would yield a phosphorothiolate adduct with a mass of 96 Da, similar to the mass of the O-methyl phosphate adduct (94 Da). This possibility is also unlikely, because of the mild alkaline conditions used in our experiments; furthermore, base-catalyzed hydrolysis would require a nucleophile (HO-) to approach the methoxy group apically, placing it in close and unfavorable proximity to the phosphorothiolate anion. An O-methyl phosphate covalently bound to the active site peptide containing the catalytic Ser corresponds to the adduct formed from one of the predicted postinhibitory mechanisms proposed by Berkman et al. to account for the nonreactivation experienced by AChE inhibited

Mechanism of AChE Inhibition by (1S,3S)-Isomalathion

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Scheme 2. Inhibition and Aging of AChE with (1S,3S)-Isomalathion According to (A) Conventional Mechanism and (B) Proposed Mechanism Based on Kinetic and MALDI-TOF-MS Dataa

a

Resulting adducts in each case differ by a mass of 16 Da.

with (1S)-isomalathion (Scheme 1B) (7). The mechanism by which (1S,3S)-isomalathion inhibits AChE thus appears to proceed with loss of the thiomethyl as the primary leaving group to yield an enzyme with an initial O-methyl-S-diethylsuccinyl phosphate adduct. Subsequent reaction of water or hydroxyl ion with the phosphorus atom that displaced diethyl thiosuccinate instead of the enzyme as the secondary leaving group would generate a negative charge in the active site (Scheme 2B). Such a modification yields an aged enzyme intractable toward reactivation (18). Methoxy was discounted as the primary leaving group in the inhibition of AChE by (1S,3S)-isomalathion for the following reasons. First, thiomethyl is predicted to be a better leaving group than the methoxy ligand. It is known that methanethiol (pKa ) 10.0) is a stronger acid than methanol (pKa ) 15.5), and its conjugate base is therefore better able to accommodate a negative charge (19). Second, Thompson et al. demonstrated that inhibition of AChE with methamidophos proceeds primarily with loss of thiomethyl instead of the methoxy group (20). The aging reaction for organophosphorylated AChE is thought to proceed with dealkylation of the OP adduct involving C-O bond cleavage and the formation of a carbocation on the leaving alkyl group in an SN1 manner (21, 22). This reaction yields a negative charge in the active site and an enzyme intractable toward reactivation (18). Evidence presented in this paper suggests otherwise for aging of AChE inhibited with (1S,3S)-isomalathion, which appears to proceed via an SN2 reaction with P-S bond scission. This contention could be proved through the use of H218O and mass spectrometry. If the mechanism postulated in Scheme 2B is correct, then the 18O would be covalently bound to the OP adduct. Rapid aging via an SN2 mechanism has been previously observed for AChE inhibited by phosphonodichloridates, which involves cleavage of a P-Cl bond (23, 24). Recently, it has been shown that aging of AChE inhibited by racemic tabun, a phosphoramidate, may involve an SN2 reaction leading to scission of a P-N bond (25). It has been proposed that residues in or near the active site may participate in the aging process (26, 27). The rapid aging of AChE treated with (1S,3S)-isomalathion may be facilitated by amino acid side chains in the catalytic pocket. Appropriately positioned residues such as His or Lys could serve as proton acceptors in the base catalysis of hydrolysis of the OP adduct, resulting in the expulsion of diethyl thiosuccinate. Replacement of such residues via site-directed mutagenesis would address this question. Fragmentation involving net loss of HPO3 (80 Da) and H3PO4 (98 Da) has previously been observed for phos-

phopeptides analyzed with reflectron MALDI-TOF-MS (15, 28). This has been shown to be a useful approach for the identification of phosphorylated peptides and the location of phosphorylation sites on a protein (29). In the study presented here, metastable fragmentation of a peptide containing an organophosphorylated Ser with subsequent loss of the OP adduct has been demonstrated for the first time using PSD analysis in MALDI-TOFMS (Figure 6). The fragment ion of highest intensity observed had a mass corresponding to the loss of CH3OP(O)(OH)2 (112 Da) from the active site peptide (Figure 6). Note that the loss of CH3OP(O)(OH)2 does not yield the unmodified peptide, because the Oγ of the catalytic Ser is lost in the fragmentation and the fragment ion contains dehydroalanine instead of the organophosphorylated Ser. A second peak of lower intensity with a mass corresponding to the modified active site peptide after loss of CH3OP(O)(OH) (95 Da) was observed in the PSD spectrum. This unique approach may prove to be valuable in studies involving Ser proteases and esterases by providing a means of identifying proteins or peptides of proteins that react with OP inhibitors and locate the catalytic Ser. In summary, EEAChE inactivated by isomalathion stereoisomers reactivates in a pattern similar to that seen previously for mammalian AChE (7, 8), but with a much larger difference in k3 values for enzyme inhibited with (1R)- versus (1S)-isomers. For the first time, direct chemical characterization of the adduct resulting from treatment of AChE with (1S,3S)-isomalathion has been performed using a mass spectrometric-based approach, thus elucidating the mechanism of inhibition by this stereoisomer (Scheme 2B). Work is in progress to identify the inhibitory adduct formed for each of the four stereoisomers using a species of cholinesterase that does not rapidly reactivate when inactivated by (1R)-isomalathions. In addition to its mechanistic interest, a practical application anticipated for this work is the development of biomarkers of exposure to OP compounds. Because the crystal structure of AChE has been determined in recent years (30), molecular modeling studies could be a viable option for gaining insight into the mechanisms proposed in this paper (31). AChE is a member of a large family of proteins termed the R/βhydrolase fold (32), and it would be of biochemical and toxicological interest to determine whether related enzymes are stereoselectively inhibited by isomalathion. Future work might also address whether the shift in the mechanism of inhibition that appears to occur between the (1R)- and (1S)-isomers is conserved for serine hydrolases related to AChE.

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Acknowledgment. We thank Mr. William Trzos at the NIH Mass Spectrometry Facility, Michigan State University, for technical assistance. This work was supported by a donation in support of research from Dow AgroSciences, NIH Grants ES07062 and RR00480, and NSF Grant MCB9808372.

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