©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Amino Acid Residues Controlling Reactivation of Organophosphonyl Conjugates of Acetylcholinesterase by Mono- and Bisquaternary Oximes (*)

(Received for publication, August 22, 1994; and in revised form, November 28, 1994)

Yacov Ashani (1)(§) Zoran Radic (2)(¶) Igor Tsigelny (2) Daniel C. Vellom (2) Natilie A. Pickering (2) Daniel M. Quinn (2)(**) Bhupendra P. Doctor (1)(§§) Palmer Taylor (2)

From the  (1)Division of Biochemistry, Walter Reed Army Institute of Research, Washington, D. C. 20307-5100 and the (2)Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0636

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS (¶¶)
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Single and multiple site mutants of recombinant mouse acetylcholinesterase (rMoAChE) were inhibited with racemic 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide (MEPQ) and the resulting mixture of two enantiomers, CH(3)P(O)(OC(2)H(5))-AChE(EMP-AChE), were subjected to reactivation with 2-(hydroxyiminomethyl)-1-methylpyridinium methanesulfonate (P2S) and 1-(2`-hydroxyiminomethyl-1`-pyridinium)-3-(4"-carbamoyl-1"-pyridinium)-2-oxapropane dichloride (HI-6). Kinetic analysis of the reactivation profiles revealed biphasic behavior with an approximate 1:1 ratio of two presumed reactivatable enantiomeric components. Equilibrium dissociation and kinetic rate constants for reactivation of site-specific mutant enzymes were compared with those obtained for wild-type rMoAChE, tissue-derived Torpedo AChE and human plasma butyrylcholinesterase. Substitution of key amino acid residues at the entrance to the active-site gorge (Trp-286, Tyr-124, Tyr-72, and Asp-74) had a greater influence on the reactivation kinetics of the bisquaternary reactivator HI-6 compared with the monoquaternary reactivator P2S. Replacement of Phe-295 by Leu enhanced reactivation by HI-6 but not by P2S. Of residues forming the choline-binding subsite, the E202Q mutation had a dominant influence where reactivation by both oximes was decreased 16- to 33-fold. Residues Trp-86 and Tyr-337 in this subsite showed little involvement. These kinetic findings, together with energy minimization of the oxime complex with the phosphonylated enzyme, provide a model for differences in the reactivation potencies of P2S and HI-6. The two kinetic components of oxime reactivation of MEPQ-inhibited AChEs arise from the chirality of O-ethyl methylphosphonyl moieties conjugated with Ser-203 and may be attributable to the relative stability of the phosphonyl oxygen of the two enantiomers in the oxyanion hole.


INTRODUCTION

Inhibition of acetylcholinesterase (AChE; EC 3.1.1.7) (^1)and butyrylcholinesterase (BChE; EC 3.1.1.8) by organophosphorus (OP) esters is attributed to the formation of a covalent conjugate between the OP moiety and the active-site serine of the enzyme(1) . OP-inhibited cholinesterases (ChEs) (^2)can be reactivated by certain oxime nucleophiles, if the enzyme does not undergo a prior ``aging'' reaction(1, 2) . Since the discovery of powerful reactivators of OP-inhibited ChEs, 2-(hydroxyiminomethyl)-1-methylpyridinium iodide (2-PAM; Fig. 1)(3) , and the bispyridinium dioxime 1,1`-trimethylene bis(4-hydroxyiminomethylpyridinium)-dibromide(4) , several reports have described the preparation, structure, and biochemical properties of mono- and bisquaternary oximes. The limited scope of antidotal activity of commonly used reactivators such as the methanesulfonate salt of 2-PAM (P2S; Fig. 1) or 1,1`-trimethylene bis(4-hydroxyiminomethylpyridinium)-dibromide against certain types of OP anti-ChE, prompted the evaluation of a new series of oxime reactivators(5) . One such compound, 1-(2`-hydroxyiminomethyl-1`-pyridinium)-3-(4"-carbamoyl-1"-pyridinium)-2-oxapropane dichloride (HI-6; Fig. 1) is among the most potent reactivating agents that serve as antidotes against organophosphate toxicity(6, 7) .


Figure 1: Structures of 2-PAM, P2S, and HI-6. The syn configurations shown are in accordance with the crystal structure of 2-PAM (19) and HI-6(20) .



The effectiveness of oxime reactivators as antidotes is primarily attributed to the nucleophilic displacement rate of the OP moiety from the inhibited enzyme (Fig. 2) and varies with the structure of the bound OP, the source of the enzyme, and the oxime. In spite of three decades of progress in improving the reactivation properties of the lead compounds, structure-function relationships are not clearly understood.


Figure 2: Chemical pathways and kinetic schemes of the reaction of oximes with MEPQ (a), and the inhibition and oxime-induced reactivation of ChEs (b). The structure in parentheses depicts the assumed pentacoordinate transition state having a trigonal bipyramidal geometry. Both the nucleophile and the leaving group occupy apical positions when assuming an in-line S(N)2 displacement reaction.



The elucidation of the three-dimensional structure of Torpedo AChE (TcAChE)(8) , together with kinetic and mechanistic studies of site-directed mutants(9) , have added a new dimension to the study of organophosphate inhibition and reactivation. In this report we describe studies on P2S- and HI-6-induced reactivation of wild-type recombinant mouse AChE (rMoAChE) and its mutants inhibited with 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide (MEPQ; Fig. 2) (10) . Delineation of amino acid residues that are important for reactivation highlight several aspects of the mechanism by which oximes enhance displacement of an OP from EMP-ChEs and provide an explanation for differences between the reactivation potency of HI-6 and P2S. In addition, owing to chirality of the phosphorus in MEPQ and the potent anti-ChE activity of both of its enantiomers(10, 11) , the inhibited enzyme consists of two enantiomeric components, EMP-ChE, and EMP-ChE, that are amenable to analysis of the stereospecificity of the reactivation process.


EXPERIMENTAL PROCEDURES

Materials

MEPQ was prepared as described elsewhere(10) . O,O`-Diethyl p-nitrophenyl phosphate (paraoxon) was purchased from Sigma. P2S and HI-6 were obtained from the Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Washington, D. C.

Wild-type and mutant rMoAChEs were prepared as described previously (12, 13) . The cDNA insert encompassing exons 2, 3, 4, and 6 was placed behind the human cytomegalovirus promoter. Most of the expression plasmids exist as stable transfectants in human embryonic kidney (HEK-293) and Chinese hamster ovary (CHO-K1) cells. They secrete into the medium hydrophilic enzyme which was concentrated by ultrafiltration for the kinetic studies. Torpedo californica AChE, wild-type mouse AChE, and some of the mutant enzymes were purified by affinity chromatography as described previously(14) . HuBChE was purified by procainamide-Sepharose 4B gel affinity chromatography. One mg of pure enzyme contained approximately 11 and 14 nmol of active sites of BChE and AChE, respectively. Inhibition and reactivation experiments were carried out in enzyme solutions prepared in microfiltered 0.05% bovine serum albumin containing 25 mM phosphate buffer, pH 7.8, at 25 °C.

pK(a) Determinations

pH-optical density profiles were recorded in 25 mM phosphate buffer (pH 6.4-8.3; 25 °C) at 336 and 354 nm for P2S and HI-6, respectively. Extinction maxima were measured in 0.1 M NaOH and 1% carbonate buffer at pH 10.2 for P2S and HI-6, respectively; pK(a) values were calculated as described by Albert and Serjeant(15) .

Hydrolysis of MEPQ

Rate constants of the hydrolysis of MEPQ were determined in 25 mM phosphate, pH 7.8 and 7.0, at 25 °C. Release of the leaving group 7-hydroxy-1-methylquinolinium ion (7-HQ, Fig. 2) was monitored at 406 nm(10) .

Enzyme Assays

AChE and BChE activities were determined by the method of Ellman et al.(16) , using 1.5 mM acetylthiocholine (ATC) and butyrylthiocholine (BTC) as substrates, respectively. Assays were carried out in 0.05% bovine serum albumin, 50 mM phosphate buffer, pH 8.0, at 25 °C. Due to the low catalytic efficiency of the W86A (^3)mutant and the need to minimize nonspecific hydrolysis of ATC, its assay was carried out with 60 mM ATC in the above buffer adjusted to pH 6.3.

Determination of the Dissociation Constants of AChEbulletOxime Complexes

The dissociation constants, K and alphaK, were determined by examining the dependence on the concentration of P2S or HI-6 for the K(m) and V(max) of the enzyme-catalyzed hydrolysis of ATC. ATC between 0.01 and 0.4 mM was added to the enzyme that had been incubated for 5 min with specified concentration of oxime and 5,5`-dithiobis(2-nitrobenzoic acid) at 25 °C. Activities were corrected for oxime catalyzed hydrolysis of ATC. Secondary plots of the slopes and the intercepts derived from Lineweaver-Burk plots against the concentration of the oxime were used to obtain K, and alphaK, respectively(17) .

Titration of the AChE Active Center by MEPQ and Paraoxon

Increasing amounts of MEPQ, in 5-20-µl aliquots, were added to 0.3-1 unit of wild-type or mutant enzyme in 0.6-2 ml of 25 mM phosphate buffer, pH 7.8. Final concentrations of enzyme and MEPQ ranged between 1 and 10 nM with MEPQ in slight substoichiometric amounts to minimize the presence of appreciable ChE inhibitor in the reactivation media. Active-site titrations of W86A were carried out with 10-20 nM enzyme. Inhibition was allowed to proceed until no further changes in enzyme activity were observed. Titration curves were constructed by plotting residual enzyme activity against the number of equivalents of MEPQ. Wild-type rMoAChE and HuBChE were incubated for 20 h at room temperature with substoichiometric amounts of paraoxon. Approximately 90% of enzyme activity was inhibited.

Reactivation of MEPQ- and Paraoxon-inhibited ChEs

Reactivation was started by mixing 2-5 µl of 2-20 mM oxime stock solution in water with 0.1-0.2 ml of OP-ChE conjugate equilibrated for 5 min at 25 °C. Final concentration of oximes in the reactivation media ranged between 0.01 and 3 mM. At specified time intervals, 5-10 µl of reactivation mixture were diluted into 0.6-1 ml of assay mixture and enzyme activity (E(t)) was monitored as described above. Control activity (E(c)) was measured in the same volume ratio of oxime to nonphosphonylated enzyme. Both E(t) and E(c) were corrected for oxime-induced hydrolysis of ATC and BTC. Inhibited enzyme without oxime was monitored for spontaneous reactivation and/or the presence of residual anti-ChE activity. Fluoride-induced reactivation was conducted as described above except the final concentration of NaF was 0.005-0.01 M.

Calculations of Enzyme Activity

Prior to reactivation OP-ChEs contained 4-14% residual activity (E(0)). The actual percentage of reactivatable enzyme at time t was calculated according to :

where E(max) = 100 E(t)/E(c).

Since reactivation profiles of EMP-ChEs displayed marked deviations from a first-order approach to reactivation of a single reactivatable species, was used to determine the best-fit values of the following parameters:

where E(1) and E(2) are the percent-amplitudes of two reactivatable forms of MEPQ-inhibited ChE and the parameters k(1) and k(2) are the corresponding fast and slow pseudo first-order rate constants of the reactivation of E(1) and E(2), respectively. Ratios of E(1)/E(2) ranged between 0.8 and 1.2. In those cases where nonlinear regression did not converge due to insufficient data points, curve fittings were processed assuming an E(1) to E(2) ratio of 1. To fit the data to a single exponential curve, E(2) in was set to zero. A statistical F test was used to compare single and biexponential nonlinear regression fits to the data. E(1), E(2), k(1), and k(2) were determined by computer iterations to give best-fit values for these parameters. Nonlinear regression and statistical F test analyses were performed by Graphpad Inplot Software, version 4.01, 1992 (GraphPad Software, Inc., San Diego, CA).

Molecular Modeling

Molecular modeling was done on Silicon Graphics Indigo Elan using Discover 2.9, a module of InsightII 2.2.0 program (Biosym, San Diego). Coordinates from the crystal structure of TcAChE (8) and coordinates from a model of HuBChE (18) were used in calculation of energy-minimized conformations of oximes in phosphonylated TcAChE and HuBChE. Coordinates of crystal structures of 2-PAM (19) and HI-6 (20) were used in docking the respective oximes in the models of EMP-ChEs.

Modelling was done in vacuo, with the dissociation state of ionizable groups set equivalent to pH 7.8. First, the O-ethyl methylphosphonyl group was covalently attached to the O of the active-site serine and the conformation of the conjugate minimized. By the initial placement of the oxygen of P=O bond in the oxyanion hole, the phosphonyl moiety is susceptible to an ``in-line'' S(N)2 displacement. Oxime groups were ionized and then partial charges of oximes calculated using the MOPAC module of InsightII. Then 2-PAM and HI-6 were minimized in the model of the phosphonylated enzymes leaving specified residues to rotate freely.

The assumed complex of the transition state for the reactivation of EMP(R)(S)-AChE by HI-6 (Fig. 2b) was analyzed by initially constraining the three putative hydrogen bonds between the phosphonyl oxygen and the oxyanion hole to distances <2.7 Å. Water molecules found in the gorge in the crystal structure were included. Amino acid side chain residues surrounding the EMP conjugate and EMP itself were allowed to rotate, whereas the peptide backbone and distal side chains were fixed. The pentacoordinate complex containing the covalently attached HI-6 to the phosphorus was initially minimized by 100 iterations, equilibrated by running dynamics at 300 K for 100 fs and then at 700 K by 50 subsequent runs of 50 fs. The seed number of the random number generator is changed after each 50-fs run. Data on possible structures were collected from the last 0.5 ps of each run. These structures were then slowly cooled using steps of 50° from 700 to 300 K. Then, the system was minimized with 1000 iterations using a conjugate gradient method. A dielectric constant of 4.0 was used. The above algorithm was created to avoid falling into local energy minima. This algorithm raises the temperature of the EMP-AChE conjugates to an equilibrium state where upon cooling they should approach global energy minima.


RESULTS (^4)

Nucleophilicity of 2-PAM, HI-6, and Fluoride

To compare chemical reactivity with the reactivation rate constants, the relative nucleophilicities of reactivators were ranked by determining bimolecular rate constants of MEPQ hydrolysis (Fig. 2a). Semilogarithmic plots conforming to a pseudo first-order reaction resulted in straight lines (not shown), indicating that the two enantiomers of MEPQ react at similar rates. The following bimolecular rate constants (k, M min) were calculated for the nucleophiles: 2-PAM, 34.6 ± 1.4; HI-6, 34.2 ± 3.1; NaF, 27.7 ± 0.9. Lowering the pH from 7.8 to 7.0 decreased k of both oximes by 3.5-fold, consistent with the actual nucleophile being the oximate anion rather than its conjugate acid. The rate constant of spontaneous hydrolysis of MEPQ was 0.005 (± 0.001) min at either pH, and the bimolecular rate constant of the reaction between MEPQ and water was estimated to be 9 times 10M min.

To compare reactivity of the actual nucleophiles, bimolecular rate constants were corrected by dividing k by the fraction of the oximate anion at pH 7.8, [1 + 10]. Using pK(a) values 8.07 ± 0.02 (2-PAM) and 7.47 ± 0.01 (HI-6), k increased to 98.9 and 49.9 M min, respectively. Fluoride anion is approximately 1.8 and 3.6-fold less potent as a nucleophile than the oximate forms of HI-6 and 2-PAM, respectively.

Dissociation Constants of 2-PAM and HI-6 for Nonphosphonylated rMoAChE

A comparison of K and alphaK (Fig. SI) for wild-type and mutant rMoAChE may provide insight into the binding regions of mono- and bisquaternary oximes at the entrance or within the active center gorge of both the free and substrate-bound enzyme. In several cases an increase in oxime concentration appeared to enhance enzyme-catalyzed hydrolysis of either ATC or BTC. To calculate K and alphaK, the concentrations of both oximes were adjusted to a range that enabled the use of the secondary plots(17) .


Scheme I: Scheme I. Equilibria for reversible inhibition of AChE by oximes. The [AChEbulletoximebulletATC] complex is assumed not to lead to hydrolysis.



Plots of 1/Vversus 1/S were linear, and, for most of the enzyme preparations, the intersections from Lineweaver-Burk plots occurred in the upper left quadrant (not shown). These observations suggest that both 2-PAM and HI-6 are likely to exhibit linear mixed-type inhibition in accordance with Fig. SI(17) . For the series of enzymes, dissociation constants of HuBChEbulletoxime and rMoAChE-W286RbulletHI-6 could not be defined due to significant deviations from simple equilibrium schemes for reversible association of inhibitor with an enzyme. The high concentrations of ATC required to saturate mutant W86A enzyme (^5)precluded an accurate determination of K and alphaK for either oxime.

The dissociation constants of 2-PAM (Table 1) for the mutants tested were only moderately perturbed relative to wild-type rMoAChE. The largest destabilization was observed with Y337A and W86F as reflected by an approximately 6-7-fold increase in the corresponding K compared with that of wild-type enzyme. Replacement of Tyr-337 by phenylalanine produced a mutant with the same aromatic amino acid residues within the active-site gorge as TcAChE. K values for Y337F and TcAChE were virtually equivalent. These findings are consistent with previous observations that established a role for aromatic residues at positions 337 (13, 21, 22) and 86 (21, 22, 23) in the stabilizing interactions of quaternary ammonium-containing ligands within the active-site gorge. However, the influence of aromatic side chains at these positions is relatively small. Similarly K of F295Lbullet2-PAM decreased less than 3-fold and single (W286R) and triple (W286A/Y72N/Y124Q) mutations at the entrance to the gorge increased the dissociation constants of 2-PAM 2.7- and 4.7-fold, respectively.



As summarized in Table 2, K of HI-6 for wild-type rMoAChE is 4-fold lower than that of 2-PAM, suggesting that the second pyridinium ring of HI-6 contributes less than 1 kcal/mol to the stabilization of the bisquaternary oxime-enzyme complex relative to the monoquaternary pyridinium oxime. Replacement of Tyr-337 by alanine increased HI-6 dissociation constant 8-fold compared to the wild-type enzyme, whereas K of HI-6 with E202Q, F295L, and wild type rMoAChE were essentially equivalent. The three aromatic amino acid residues at the entrance to the gorge have been shown to constitute part of the peripheral anionic site for bisquaternary ligands(13, 24) . Their replacement with the residues found in BChE increased K of the association of HI-6 with mutant W286A/Y72N/Y124Q only 6.5-fold relative to wild-type rMoAChE.



In Fig. SI, alphaK is the dissociation constant for the AChEbulletoximebulletATC complex. If association of substrates decreases the affinity of the oxime relative to the free enzyme, then alpha should be >1. Whenever plots of V(max)versus [oxime] gave straight lines, alphaK was calculated from the intercept on the x axis. Both 2-PAM and HI-6 displayed alpha values of 1.5 to 6 with the following rank order: for 2-PAM, TcAChE approx wild-type rMoAChE > W286R approx W86F > E202Q approx triple mutant; for HI-6, wild-type rMoAChE > triple mutant approx E202Q. Thus, binding of substrate to AChEs moderately increased the dissociation constant of either oxime. The relative destabilization of the ternary complex of substrate, oxime, and enzyme complex is in agreement with the dissociation constants determined for the corresponding EMP-AChEbulletoxime complexes (see below).

Inhibition and Stability of MEPQ-inhibited Enzymes

MEPQ-ChE conjugates are likely to be composed of a mixture of two enantiomers (10, 11) , designated as EMP(R)-ChE and EMP(S)-ChE. The symbols R and S denote absolute configuration around the phosphorus atom of the inhibited enzyme.

Rates of spontaneous reactivation of EMP(R)(S)-ChE conjugates were slow; all were less than 0.5% h, with the exception of EMP(R)(S)-W86A which was slightly increased to 0.8% h. The extent of reactivation did not differ significantly for preparations that were allowed to incubate for 1-24 h at 25 °C before the addition of reactivator. The almost negligible spontaneous reactivation and aging reactions simplify evaluation of the kinetic profiles. The absence of competing processes, together with the high bimolecular rate constants for the inhibition of AChE (11, 25) and HuBChE (11) by MEPQ, resulted in rapid formation of an inhibited enzyme with defined species that could be transferred immediately to reactivation buffer. It should be pointed out that the concentration of the product 7-HQ in the reactivation medium is equal to the active site concentration of the inhibited enzyme (Fig. 2b). To ascertain whether 7-HQ interferred with reactivation, up to 5 times the initial concentration of MEPQ-inhibited enzyme in reactivation medium of 7-HQ was added to reactivation buffer. Increased 7-HQ did not significantly affect reactivation rate constants.

Is Reactivated Enzyme Subsequently Inhibited by the Phosphonyl-oxime?

A kinetic study of 2-PAM-induced reactivation of EMP-AChE, prepared from bovine erythrocyte AChE using R(p) and S(p) enantiomers of CH(3)P(O)(OC(2)H(5))X, demonstrated inhibition of reactivated AChE by a reaction product under certain conditions(26) . This phenomenon was attributed to formation of a phosphonyl-oxime intermediate (Fig. 2a)(27) . While preparation of EMP-AChE conjugates from MEPQ ensures that the phosphonyl-oxime will not be generated through direct reaction between either 2-PAM or HI-6 and MEPQ, it might be formed upon addition of oxime to EMP(R)(S)-ChE conjugates. To examine this, we measured rates of reactivation as a function of increasing the initial concentration of EMP(R)(S)-ChEs with a fixed concentration of oxime. Representative data are shown in Fig. 3(panels A and B).


Figure 3: Time course of reactivation of MEPQ- and paraoxon-inhibited ChEs, by 1 mM 2-PAM (P2S). Broken and solid lines were fitted to the open squares using mono- and biexponential equations, respectively. A, MEPQ-inhibited wild-type rMoAChE, box, 2 nM; bullet, 5 nM; , 20 nM. B, MEPQ-inhibited HuBChE, box, 3.4 nM; bullet, 10 nM. C, paraoxon-inhibited wild-type rMoAChE, box, 2 nM; bullet, 10 nM. D, paraoxon-inhibited HuBChE, box, 0.5 nM; bullet, 34 nM. The ordinates show percent of maximal reactivation after 48 h.



At concentrations of EMP(R)(S)-ChE(0), ranging between 2 and 20 nM of EMP(R)(S)-AChE and between 3.4 and 10 nM EMP(R)(S)-BChE the kinetics of oxime reactivation were virtually identical. Hence, inhibition by reactivation product in these concentration ranges is not likely to influence reactivation rates.

Kinetics of Reactivation of EMP-ChEs

Analysis of kinetic data by curve fitting clearly showed that oxime-induced reactivation of all EMP-ChEs progressed in a manner that indicates non-homogeneity in reactivatable enzyme (Fig. 3, panels A and B). A curve constructed in accordance with a two-component reactivation model provided a better fit than that obtained for a single exponential equation. The difference in component rate constants was even more pronounced for HuBChE. Semilogarithmic plots of the reactivation of MEPQ-rMoAChE by three different reactivators showed approximately equal distribution of fast and slow components irrespective of the nucleophile used (Fig. 4).


Figure 4: Semilogarithmic plots of % EMP-rMoAChE versus time of incubation with various nucleophiles. Solid lines were fitted to the data assuming two distinguishable reactivatable components. The broken lines were fitted to data (n = 5-6 each) that represent the slow component, in accordance with a single species of reactivatable enzyme, and extrapolated to t = 0 to calculate the ratios of the two reactivatable components. A, 0.9 mM 2-PAM (P2S), [fast]/[slow] = 0.89; B, 0.12 mM HI-6, [fast]/[slow] = 0.96; C, 10 mM NaF, [fast]/[slow] = 1.2. The ratios of k(fast) to k(slow) were 5.5, 7.1, and 8.0 for 2-PAM, HI-6, and NaF, respectively.



These findings are satisfactorily explained by the presence of two kinetically distinguishable EMP-ChE enantiomers. However, since it was observed for bovine erythrocyte AChE that the achiral inhibitor, paraoxon, also produces more than one class of reactivatable species (26) , it was necessary to compare the reactivation profile of paraoxon- and MEPQ-ChE conjugates under the same experimental conditions. Results are depicted in Fig. 3(panels C and D). Indeed, for both achiral paraoxon-inhibited rMoAChE and HuBChE, the biexponential equation improved the fit only slightly, but significantly, better than a single component model.

Although the deviations from kinetics of a homogenous class of inhibited enzyme species were substantially larger for the EMP(R)(S)-ChE conjugates compared to similar preparations using paraoxon, we could not ascertain the basis of an intrinsic, but small, contribution to the deviation from monoexponential decay function for the achiral O,O`-diethylphosphoryl-AChE conjugate. Therefore, we categorize the two phases in as fast and slow components of oxime-induced reactivation, and k(1) and k(2) are the corresponding first-order rate constants of the fast and slow components, respectively, in the presence of large stoichiometric excesses of the reactivator.

The mathematical solution for the kinetic scheme of the reactivation depicted in Fig. 2b is:

where k is either k(1) or k(2), and K` = (k + k`(max))/k. Assuming k k`(max), K` is approximated by k/k which is the corresponding dissociation constant of the complex EMP-ChEbulletoxime. The individual constants k`(max) and K` were determined by nonlinear regression analysis of the data shown in Fig. 5, according to . The bimolecular rate constant of reactivation (k(r)) was obtained by dividing k`(max) by K`.


Figure 5: Representative plots of kversus [oxime] for reactivation of EMP-AChE. Lines were fitted to the data in accordance with except for panel E that was fitted to . The left- and right-hand side panels of each pair show the fast and the slow rate constants, respectively. A and B, P2S with wild-type rMoAChE; C and D, 2-PAM (P2S) with W86A; E and F, HI-6 with W286R; G and H, HI-6 with W286A/Y72N/124Q.



In several cases (Fig. 5, panel E) the plots of kversus [oxime] were linear rather than asymptotically approaching a constant value. In this situation, the individual component constants could not be resolved. Assuming K` [oxime], is approximated by the following expression:

Thus, k(r) = k`(max)/K` was obtained from the slopes of straight lines constructed by plots of kversus [oxime].

Table 1and Table 2summarize the kinetic constants of the reactivation of EMP-ChEs by 2-PAM and HI-6, respectively. The last column also gives the bimolecular rate constant for the reactivation by the oximate ions, RCH=N-O(k). In general, dissociation constants of the reactivators for EMP(R)(S)-AChEs (K`) are increased compared with nonphosphonylated enzyme (i.e.K<K`). The magnitude of destabilization of the inhibited enzyme-oxime complex was in the range obtained for the ratio alphaK/K, an observation that is consistent with the interpretation of alpha. Thus, the presence of bound ATC or its reaction product and conjugated EMP decreased the affinity of the oximes to an equivalent extent.

Mutations at the Choline Binding Subsite

The side chains of Trp-86, Tyr-337, and Glu-202 appear to contribute to the stabilization of binding of both the choline moiety of ATC and the positive poles of quaternary reversible inhibitors of AChE(13, 21, 22, 25) . Replacement of Glu-202 by glutamine (EMP(R)(S)-E202Q) remarkably decreased the bimolecular rate constant of reactivation by 2-PAM or HI-6. Both reactivatable components were affected similarly. In the case of HI-6, the decrease in k (13-16-fold) compared with wild-type enzyme almost exclusively arises from a smaller unimolecular rate constant k`(max). By contrast, the reduction in k of 2-PAM (23-33-fold) contains contributions from both the affinity of the oxime for the phosphonylated enzyme (K`) and k`(max). These findings indicate that the stability of the initial complex EMP(R)(S)-AChEbulletHI-6 is largely controlled by residues located outside the choline-binding region. The 13-33-fold decrease in k of both reactivators, compared with wild-type EMP(R)(S)-rMoAChE, is highlighted by the fact that k values of mutants of other constituents of the choline binding site, namely, W86F, W86A, Y337F, and Y337A, are approximately within 2-fold of the wild-type enzyme, for the fast reactivatable component.

Replacement of the -electron-rich indole side chain of Trp-86 by alanine decreased only slightly (1.2-2.3-fold) and moderately (2.2-5.1-fold) k (slow) of 2-PAM and HI-6, respectively, compared with wild-type phosphonylated rMoAChE. Furthermore, k (fast) of both 2-PAM and HI-6 were slightly enhanced with the EMP-Y337A conjugate relative to the corresponding reactions with wild-type rMoAChE. These findings suggest that Trp-86, and Tyr-337 play only a limited role in binding the oxime in a conformation suitable for reactivation of phosphonylated AChEs.

The reactivatability of the two components of EMP(R)(S)-TcAChE by 2-PAM was comparable to that of mutant Y337F. Replacement of tyrosine by phenylalanine in rMoAChE produces a mutant that contains aromatic side chain residues of the choline subsite identical to TcAChE(8, 13, 28) .

Mutation at the Acyl Pocket

Of the two aromatic side chains that constitute the acyl pocket, Phe-295 and Phe-297, mutation of the former (F295L) produced the greater enhancement of butyrylthiocholine hydrolysis (k/K(m))(12) . Dimensions of the acyl pocket are likely to determine, in part, the stability of the two enantiomeric O-ethyl methylphosphonyl conjugates of AChE in a manner analogous to their influence on carboxyl ester specificity. Since F295L appears to place the essential constraint in limiting butyrylthiocholine hydrolysis by AChE, it is interesting to compare k of R(p) and S(p) enantiomers of EMP-F295L with wild-type EMP(R)(S)-rMoAChE. MEPQ-inhibited F295L reactivated only up to 50-65% of the expected activity at t. By contrast, the extent of reactivation of other MEPQ-inhibited mutants, as well as that of wild-type rMoAChE and tissue-derived TcAChE and HuBChE, ranged from 80 to 98%. The reactivation profiles of EMP(R)(S)-F295L constructed for either 2-PAM or HI-6 were fitted significantly better to a two-component model rather than to a single class of inhibited enzyme (not shown), but the difference in the ratio of k(r) (fast) and k(r) (slow) was markedly reduced from the ratio found for the wild-type enzyme.

One enantiomeric form of EMP(R)(S)-F295L reactivated profoundly faster than the other, which appeared resistant to reactivation. Since the extent of maximal reactivation was independent of the time of prior incubation of F295L with MEPQ, slow dealkylation (i.e. aging) cannot explain the relative resistance of the second component to oxime-induced reactivation. Similar observations were made previously with the O-cycloheptyl methylphosphonyl-TcAChE conjugate(29) .

Interestingly, replacement of phenylalanine in position 295 by leucine had opposing effects on k for 2-PAM (decreased 3.7-fold) and HI-6 (increased 2.2-fold). These findings are consistent with the relative changes observed in the affinity of the oximes (K and K`) for F295L (Table 1). F295L alters the spatial constraints surrounding the O Ser-203-bound phosphonyl moiety and thereby changes the stereochemical requirements of the reactivation process.

Mutations at the Entrance to the Gorge

The potency of 2-PAM in reactivating both the fast and slow components decreased only 1.6 to 3-fold with the triple mutant involving residues at the entrance to the gorge (W286A/Y72N/Y124Q) and with W286R, relative to the wild-type enzyme. By contrast, HI-6-induced reactivation of the triple mutant decreased 70- and 6-fold for the fast and slow components, respectively. Similarly, k of HI-6 with EMP-W286R, a mutation to the residue found in mouse BChE, was 40- and 5-fold lower for the fast and slow components, respectively, compared with those observed for wild-type rMoAChE.

Finally, k values of EMP(R)(S)-HuBChE that contains aliphatic amino acid residues in positions homologous to 286, 124, and 72 of AChE, revealed that 2-PAM is superior to HI-6 in reactivating HuBChE, and the k ratio of rMoAChE to HuBChE is >25 with HI-6, whereas it is <2 for 2-PAM. This further underscores the importance of the aromatic amino acids at the entrance to the gorge of AChEs in enhancing reactivation potency of HI-6 as compared with 2-PAM.

Molecular Modeling

A stereo view of energy minimized conformations of complexes between EMP(R)-AChE, EMP(S)-AChE, EMP(R)-HuBChE, and EMP(S)-HuBChE with 2-PAM is shown in Fig. 6. In addition, the EMP(R)bulletAChE and EMP(R)bulletHuBChE complexes with HI-6 are also shown. The overall geometries of the side chain residues that are lining the gorge of EMP-ChEbullet2-PAM complexes were similar to the starting models of EMP(R)(S)-ChE conjugates with one exception. The indole ring of Trp-86 that is aligned with the gorge axis of TcAChE (22) and HuBChE model (18) is slightly moved to face the gorge entrance, a rotation that appears to increase parallel contacts between the aromatic rings of 2-PAM and Trp-86.


Figure 6: Stereo views of the final conformations of energy minimized 2-PAM and HI-6 in models of EMP-AChE, EMP-AChE, EMP-HuBChE, and EMP-HuBChE. Energy minimizations were carried out using the atomic coordinates obtained from the crystal structure of TcAChE(8) , and HuBChE model based on the TcAChE structure(18) . Amino acid residues are labeled in accordance with the numbering system of rMoAChE (EMP-AChE) and HuBChE (EMP-HuBChE). Hydrogen bonds between the phosphonyl oxygen and the amide hydrogens in the oxyanion hole are shown by dotted lines. The hydrogen bond formed between the hydroxyl of Tyr-124 and the etheral oxygen of HI-6 is not shown. Selected final geometries of energy minimized 2-PAM within the corresponding conjugates were included to illustrate differences in steric hindrance around the P-atom. Dotted spheres are van der Waals surfaces of CH(3)-P, CH(3)CH(2)O-P, and RC=N-O of 2-PAM modeled and energy minimized within the corresponding EMP-AChE conjugates. The final conformation of 2-PAM shown is one of several closely related overlapping structures with similar energy content.



Although the charge on the pyridinium nitrogen (N) is delocalized(30) , it is interesting to measure distances between N and some of the atoms surrounding 2-PAM. Trp-86 C is 5.4 and 4.8 Å from N of the R(p) and S(p) enantiomers, respectively. Both Tyr-337 C, and Phe-338 C of the enantiomeric EMP-AChE conjugates are >6.2 Å away from the pyridinium nitrogen. These distances are in fair agreement with experimental observations showing that k is not significantly affected by single replacement of an aromatic side chain by aliphatic amino acids at positions 86 and 337.

Of the two carboxylate side chains that are projected into the gorge, the carboxylate oxygen of Asp-74 is 4.2 and 5.0 Å from the quaternary nitrogen of 2-PAM modeled in the R(p) and S(p) conjugates, respectively, whereas Glu-202 carboxylate is about 9.5 Å from nitrogen in both enantiomers. Despite the greater distance of Glu-202 carboxylate from the pyridinium nitrogen compared with Asp-74 carboxylate, perturbation of reactivation with 2-PAM was significantly greater with E202Q compared to D74N. We note that the distance of either Glu-202 carboxylate or His-447 N from the oximate oxygen ranged from 5.8 to 8.8 Å in all EMP(R)(S)-AChEbulletoxime complexes.

The oxime-containing pyridinium ring of HI-6 is oriented essentially as described above for the 2-PAM complex. The carbamoyl, C(O)NH(2) moiety of the distal pyridinium ring is projected toward the peripheral binding site and forms close contacts with aromatic side chains of Trp-286, Tyr-72, and Tyr-124. Computer-simulated molecular dynamics of the ground state (not shown) and the pentacoordinate transition state (Fig. 7a) of EMP(R)(S)-AChEbulletHI-6 clearly point to the ability of the hydroxyl of Tyr-124 to hydrogen bond to the oxygen of the bismethylene ether moiety that connects the two pyridinium rings. These interactions appear to restrict movements of HI-6 within the gorge. Apparently, anchoring of the distal pyridinium moiety results in shortening of the distances between N of the proximal pyridinium ring and Trp-86 C (4.6 Å), Tyr-337 C (3.9 Å), and Phe-338 C (5.1 Å), compared to EMP(R)-AChEbullet2-PAM. The model of EMP(R)-AChEbulletHI-6 is consistent with the finding that mutations of residues Trp-286, Tyr-72, and Tyr-124 decreased dramatically k of HI-6 but not of 2-PAM. Molecular dynamics carried out by equilibration of the EMP(R)(S)-AChEbulletHI-6 complexes at high temperature followed by cooling yields a dramatic difference for the EMP(R)-AChE and EMP(S)-AChE enantiomers. In the case of the R enantiomer the phosphonyl oxygen remains within the oxyanion hole (Fig. 7b) while the S enantiomer assumes multiple conformations. Binding in the oxyanion hole should enhance reactivity by lowering the energy of the transition state and this factor could account for the different rates of reaction of the R and S enantiomers.


Figure 7: Molecular dynamics of the pentacoordinate transition state between EMP-AChE and HI-6. A stereoview of EMP-AChEbulletHI6. Geometry around phosphorus was pentacoordinate with the oxime and Ser-203 oxygen assuming apical positions. Shown are the results of five simulations with heating and equilibrating at 700 K with subsequent cooling to 300 K. Simulations were constructed for R and S enantiomers of EMP. B shows an enlarged view in the region of the pentacovalent phosphorus for the EMP-AChEbulletHI-6 transition state. Note the fixed position of the phosphonyl oxygen. C shows an identical simulation for EMP-AChEbulletHI-6 where a large variation of position of the residues around the phosphorus are noted.




DISCUSSION

The overall mechanism of displacement of the phosphonyl-bound moiety of EMP(R)(S)-AChE by oxime reactivators is assumed to parallel analogous reactions with low molecular weight organophosphate model compounds. Thus, reactivation is expected to proceed from a tetrahedral ground state of the phosphonyl moiety to a trigonal bipyramidal transition state (Fig. 2a)(31) . For both oximes the carboxylate side chain of Glu-202 appears to be important for stabilizing intermediates along the chemical pathway by an inductive electronic influence on the phosphorus which facilitates the nucleophilic attack. This was suggested previously to account for the diminished rates of phosphorylation of the E202Q mutant(25) . The acquisition of a negative charge of the transition state is likely to be stabilized by both the amide hydrogens in the oxyanion hole and the positive charge of the pyridinium ring. The contribution of the latter interaction to the overall stabilization of the transition state is evident from the high ratios of k for 2-PAM (>125) and for HI-6 (>500) compared with the estimated bimolecular rate constant of the reactivation by NaF (<10 M min), even though the nucleophilicity of fluoride is only 3.6- and 1.8-fold smaller than that of 2-PAM and HI-6, respectively.

The unimolecular rate constants of the reactivation of wild-type phosphonylated AChE by both oximes (k`(max), 0.25-0.58 min) are 2500-fold higher than the estimated rate constants for spontaneous restoration of enzyme activity. Such an enhancement suggests a decrease of more than 4.5 kcal/mol in energy barrier, in going from initial state of oxime-bound complex to the activated state, compared with a general base or H(2)O-catalyzed reactivation. This consideration together with results shown here reveals a specific molecular recognition of quaternary oximes in the active-site gorge.

Comparative Reactivating Potencies of HI-6 and 2-PAM

The nucleophilicity of the oximate anion of 2-PAM is 2-fold greater than that of HI-6, as would be expected from the stronger basicity of 2-PAM(32) . Nevertheless, variations of k ratio for HI-6 to 2-PAM with wild-type EMP(R)(S)bulletrMoAChE is approximately 4 yielding an overall enhancement of 8-fold for k of HI-6 over 2-PAM. A similar trend was reported for human erythrocyte EMP-AChE that was obtained using O-ethyl p-nitrophenyl methylphosphonate(33) . The superiority of HI-6 over 2-PAM is well established with respect to other O-alkyl methylphosphonyl conjugates of rat, bovine, and human AChEs(34, 35, 36, 37) .

Considerations of energy barriers suggest that reactivation will be favored by an in-line displacement where the nucleophile and the enzyme occupy apical positions in the trigonal bipyramidal transition state (31) . Molecular modelling showed that the oximate oxygen of various EMP(R)(S)-AChEbulletoxime complexes is positioned 4.4 to 4.9 Å from the P atom, suggesting that a change in conformation is required in order for the hydroxamate oxygen atom to form a covalent bond with the phosphonyl moiety. This view is supported by the observation that the ratio of dissociation constants of the HI-6 complexes with nonphosphonylated (K) and EMP conjugate (K`) of the triple mutant is 6 to 9 as opposed to a ratio of 70 for wild-type rMoAChE. Of all the models examined, the bond angle RCH=NO-P-O Ser-203 that approaches an optimal 180° for in-line displacement was found to be 169° for EMP(R)-AChEbulletHI-6. The most likely explanation for the overall 8-fold increase in k of HI-6 over 2-PAM stems from a better orientation of the oximate oxygen of the former oxime toward an apical approach to the phosphorus atom from the face formed by three atoms and perpendicular to P-O Ser-203 bond. Eventually, this might lead to greater stabilization of the transition state.

Using similar arguments, the smaller molecular volume of 2-PAM may confer to this reactivator sufficient flexibility to accommodate itself at various overlapping orientations within the gorge of wild-type phosphonylated AChE as well as within a gorge of diminished aromaticity seen with BChE. This view is supported by energy minimization yielding different final conformations with similar energy depending on the starting positions of the oxime within EMP(R)(S)-ChEs (not shown). This flexibility may diminish the dependence of k on structural changes with the active-site gorge and give rise to unproductive binding conformations.

Sequence alignments of AChE and BChE reveal that aromatic amino acids at positions Trp-286, Tyr-124, and Tyr-72 in mammalian AChE are replaced by aliphatic residues in BChE(13, 28) . For EMP(R)(S)-HuBChE the reduced k for HI-6 to values less than k of 2-PAM further substantiates the contribution of the aromatic cluster at the gorge entrance to the enhanced potency of HI-6. Interestingly, the ratio k(fast)/k(slow) for the reactivation of EMP(R)(S)-HuBChE by HI-6 was similar to wild-type rMoAChE, whereas the ratio approached one for the mutant enzymes W286A/Y72N/Y124Q and W286R. The diminished differences in the susceptibility of the two enantiomeric mutant EMP conjugates to undergo reactivation with HI-6 show that productive binding of the fast reactivatable component by peripheral residues is significantly greater than for the slow enantiomer of wild-type rMoAChE. Initial experiments show that replacement of aspartic acid by asparagine at position 74 produced approximately 24- and 3-fold decreases in k(fast) for HI-6 and 2-PAM, respectively. These observations suggest that the role of the conserved carboxylate side chain of Asp-74 in stabilizing a productive conformation of AChEbulletHI-6 is manifest mainly in combination with the aromatic residues of the peripheral site.

An interesting feature of the energy minimized EMP(R)-AChEbulletHI-6 complex is hydrogen bonding of the hydroxyl of Tyr-124 to the etheral oxygen of HI-6. The proposed stabilization of HI-6 is consistent with a reported decrease in reactivation potency of a congener of HI-6 in which a three carbon methylene chain (CHS-6) was substituted for the bisoxymethylene bridge (HS-6)(34) .

Finally, it is of interest to point out that k of 2-PAM was reported to be significantly larger than k of HI-6 for the reactivation of homologous EMP conjugate of electric eel AChE(33) . The anomalously low potency of HI-6 as reactivator of phosphonylated eel AChE allows one to speculate that one or more amino acids that control the enhanced reactivity of HI-6 toward EMP-AChE from mammals are not conserved in eel AChE.

Stereospecificity of Oxime-induced Reactivation

Molecular modeling of R(p) and S(p) enantiomers of O-isopropyl methylphosphonyl-AChE conjugates predicted distinct enantiomeric selectivity for nucleophilic displacement of the organophosphonyl moiety(38) . However, O-isopropyl- and O-3,3-dimethylbutyl methylphosphonyl-TcAChE conjugates were found to undergo reactivation by 1 mM HI-6 and 1,1`-trimethylene bis(4-hydroxyiminomethylpyridinium)-dibromide at rates that were largely independent of configuration around the P atom of the inhibitor or the structure of the alkyl group(29) . The concentration of the oximes could be well above K` and therefore the observed rate constant might actually reflect k`(max). Our data show that the rank orders of k`(max) do not correlate closely with the bimolecular rate constants of reactivation and therefore comparative analysis should include K`. Below we rationalize the two kinetically distinguished components in terms of stereospecific reactivation of the enantiomeric conjugates.

Energy minimization of the putative covalent enantiomeric conjugates revealed that the bond angles P=O- - -H-N(C=O) of either EMP(R)-AChE (residues 121, 122, and 204) or EMP(R)-HuBChE (corresponding residues 116, 117, and 199), as well as the relevant interatomic distances, should produce three hydrogen bonds between the phosphonyl oxygen and the backbone nitrogen atoms of the oxyanion hole region (Fig. 6, broken lines). By contrast, the bond lengths increase and only a single hydrogen bond appears to stabilize the S(p) enantiomers of O-ethyl methylphosphonyl conjugates of AChE and HuBChE. Furthermore, molecular dynamic simulations (Fig. 7) indicate that stabilization of the putative P-O of the transition state (Fig. 2b), by hydrogen bonding to the oxyanion hole, is likely to be greater for EMP(R)-ChE than for the EMP(S)-ChE conjugates. In the latter conjugate the P-O moiety of several of the lowest energy conformers are shifted out of the oxyanion hole. These considerations predict that the R(p) enantiomer is the fast reactivatable component. The importance of the oxyanion hole in stabilizing the phosphonyl oxygen is underscored in the recently reported crystal structures of phosphonate complexes with lipases that are homologous to the ChE's(39) .

The van der Waals surfaces of the methyl group CH-P of EMP(S)-AChE, as well as of EMP(S)-HuBChE, that are aligned in both cases toward the oxime moiety, reveal that the oximate oxygen should experience greater steric hindrance for its approach toward the P atom, compared with the homologous EMP(R)-ChE conjugates (Fig. 6). In the latter conjugate, CH-P is projected toward the acyl pocket (Phe-295, Phe-297), the methylene of the ethyl moiety that faces the oximate oxygen is removed from the phosphorus by an oxygen ester linkage (P-OCHCH), and thereby a larger space is opened to the oxime from the side envisaged for the nucleophilic attack. These observations are also consistent with EMP(R)-ChE being the enantiomer of MEPQ-inhibited ChEs exhibiting rapid reactivation.


FOOTNOTES

*
This work was supported in part by U. S. Army Research and Material Command Grant DAMD17-91-C-1056 (to P. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Visiting scientist from the Israel Institute for Biological Research, Ness-Ziona, Israel.

Visiting fellow from the Institute for Medical Research and Occupational Health, University of Zagreb, Croatia.

**
Visiting scientist from the Department of Chemistry, University of Iowa, Iowa City, IA.

§§
To whom correspondence should be addressed. Tel.: 202-782-3001; Fax: 202-782-6304.

(^1)
The abbreviations used are: AChE, acetylcholinesterase; BChE, butyrylcholinesterase; OP, organophosphorus; ChE, cholinesterase; 2-PAM, 2-(hydroxyiminomethyl)-1-methylpyridinium iodide; P2S, 2-(hydroxyiminomethyl)-1-methylpyridinium methanesulfonate; HI-6, 1-(2`-hydroxyiminomethyl-1`-pyridinium)-3-(4"-carbamoyl-1"-pyridinium)-2-oxapropane dichloride; rMoAChE, recombinant mouse AChE; MEPQ, 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide; EMP-ChE, O-ethyl methylphosphonyl-ChE; HuBChE, human BChE; TcAChE, Torpedo californica AChE; paraoxon, diethyl p-nitrophenyl phosphate; ATC, S-acetylthiocholine iodide; BTC, S-butyrylthiocholine iodide; 7-HQ, 7-hydroxy-1-methylquinolinium ion.

(^2)
The abbreviation ChE was used whenever AChE was not distinguished from BChE.

(^3)
MoAChE numbering system.

¶¶
Since 2-PAM and P2S generate the same 2-(hydroxyiminomethyl)-1-methylpyridinium cation, the abbreviation 2-PAM was used for both salts.

(^5)
D. M. Quinn and Z. Radic, unpublished data.


ACKNOWLEDGEMENTS

We acknowledge the expert technical assistance of Ann M. Gallaher and Anthony Schmidt in carrying out this investigation.


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