This work was supported by US Army Medical Research and Materiel Command grants DAMD17-97-1-7349, DAMD17-01-2-0036 (to O.L.) and by a Center Grant to UNMC from the National Cancer Institute, P30CA36727-19. The opinions or assertions contained herein belong to the authors and should not be construed as the official views of the US Army or the Department of Defense.

abstract

Human butyrylcholinesterase (BChE, EC 3.1.1.8) is an efficient scavenger of nerve agents and organophosphorus (OP) pesticides; one molecule of BChE inactivates one molecule of OP in a suicide reaction that irreversibly inhibits BChE. By contrast the BChE mutant, G117H, inactivates many molecules of OP. The OP makes a covalent bond with the active site serine and then the serine is dephosphorylated by the action of His117. In an effort to understand the mechanism by which His 117 achieves dephosphorylation, 62 new mutants of human BChE were tested for OP hydrolase activity, using a new screening assay. It was found that not only G117H, but also G117D, G117E, and L286H mutants were OP hydrolases. These results support the hypothesis that a hydrogen-bond acceptor acts as a general base to activate a water molecule which in turn dephosphorylates the active site serine. The screening assay provides a convenient means for identifying cholinesterase mutants with OP hydrolase activity.

introduction

Organophosphorus cholinesterase inhibitors are widely used as pesticides and have been stockpiled and sometimes used as chemical warfare agents. Each year thousands of people are exposed to these compounds by accident or in attempts at suicide. The threat of the use of anticholinesterases in warfare or in terrorist attacks has increased greatly in recent years. Progress in protection against OP toxicity was achieved by using human butyrylcholinesterase (BChE) as a scavenger (Broomfield et al., 1991; Raveh et al., 1997). Laboratory animals including monkeys are protected from up to 4 LD₅₀ doses of nerve agent by pretreatment with BChE (Raveh et al., 1997; Wolfe et al., 1992). A genetically engineered human BChE containing a single amino acid substitution, Gly 117 His, has the unusual property of being resistant to OP inhibition by virtue of its ability to hydrolyze OP (Millard et al., 1995; Lockridge et al., 1997; Millard et al., 1998). The G117H mutant hydrolyzes paraoxon (the active form of the common pesticide parathion) 120,000 times faster than spontaneous hydrolysis of paraoxon in water at pH 7 occurs (Lockridge et al., 1997). The G117H mutant also hydrolyzes acetylcholine. Transgenic mice expressing human G117H BChE are resistant to OP toxicity (Wang et al., 2004).

Our goals were to identify additional mutations in BChE that would provide resistance to OP and to determine the mechanism of OP resistance. A new OP hydrolase screening assay was developed; activity was inhibited with echothiophate [CASRN: 6736-03-4], and, the rate at which activity reappeared was taken as the rate of dephosphorylation. Sixty-two mutants were tested. Twenty-nine were single mutants, 31 were double mutants and two were triple mutants. Of these, 13 were found to be resistant. The OP resistant mutants all had a nucleophile at a distance less than 6—8 Angstroms from the phosphorylated active site serine. The nucleophile was a histidine, aspartic acid, or glutamic acid. This led to the conclusion that an activated water molecule was responsible for the dephosphorylation.

methods and materials

Mutagenesis and Expression of Recombinant BChE. Mutagenesis and expression were performed as previously described (Xie et al., 1999; Josse et al., 1999). Briefly, mutations in human BChE were made by PCR with Pfu polymerase. Fragments containing the mutation were cloned into the plasmid pGS and the DNA sequenced to ensure that no unwanted mutations were present. Both stably transfected and transiently transfected cell lines were used. Stably transfected CHO-K1 cells (American Type Culture Collection, No CCL61) were selected in 50 µM methionine sulfoximine. Expressed BChE was secreted from these cells and collected into serum free medium (Ultraculture 12-725B, BioWhittaker). For transient transfections, the plasmid was transfected into 293T/17 human embryonic kidney cells (American Type Culture Collection, No CRL 11268, used with permission of David Baltimore (Pear et al., 1993), by calcium phosphate-DNA coprecipitation as previously described (Josse et al., 1999). After four days, there was enough transiently expressed BChE to test. Untransfected media had no background hydrolysis of butyrylthiocholine (BTC) or OP.

Purification of BChE. In some instances, mutant forms of BChE were purified from culture medium taken from permanent cell lines, as previously described (Xie et al., 1999). Briefly, 1 to 10 liters of culture medium were passed over a 100 to 300 ml procainamide-Sepharose affinity column, which retained the BChE. Contaminating proteins were eluted with 0.1 M NaCl. BChE was selectively eluted with 0.2 M procainamide hydrochloride. Further purification was obtained by ion exchange chromatography on 400 ml of DE52 (Whatman) using a sodium chloride gradient (0 to 0.5 M NaCl in 20 mM Tris/Cl buffer pH 7.4) for elution. Purified enzyme was dialyzed into 20 mM potassium phosphate buffer pH 7.0, containing 0.3 mM EDTA. Enzyme was either stored at 4 °C, or after addition of glycerol to 30%, was frozen at –80 °C. Wild-type BChE, used in this work, was purified from human serum using ion exchange chromatography at pH 4.0, followed by affinity and DE52 chromatography at neutral pH (Lockridge, 1990).

Organophosphatase screening assay. Reacting small amounts of OP with cholinesterases results in a gradual decrease in cholinesterase activity (Aldridge and Davison, 1952), which is referred to as progressive inhibition (Main, 1979). Main first introduced the idea of including substrate in the inhibition reaction so that the progress of the inhibition could be continuously monitored (Main and Dauterman, 1963). We have adapted Main’s assay for progressive inhibition in the presence of substrate, to screen for OP resistance and OP hydrolysis activity in BChE mutants. After the enzyme, the inhibitor (echothiophate iodide: from Wyeth Ayerst, Rouses Point, NY), and the substrate (BTC and 5,5’-dithiobis-2-nitrobenzoic acid [DTNB]) were mixed in a cuvette, product formation was monitored spectrophotometrically at 412 nm. Progressive decrease in the rate of product formation reflected inhibition of the enzyme. All assays were performed at 25 °C, in 0.1 M potassium phosphate buffer, pH 7.0, containing 1 mM BTC, 0.5 mM DTNB and variable amounts of echothiophate. Variable amounts of echothiophate (1 to 90 µM, depending upon the enzyme under study) were used to improve detection of dephosphorylation. When phosphorylation was too fast, the assay failed to detect OP hydrolysis. Therefore, the rate of phosphorylation was minimized by adjusting the concentration of echothiophate to require about 30 minutes to achieve maximum inhibition. If, after about 30 minutes of reaction with echothiophate, a linear steady rate of BTC hydrolysis was observed, that linear rate was taken to be an indication of OP turnover. As an illustration of this point, the reaction of the double mutant G117H/E197Q and the resulting linear rate of BTC hydrolysis is shown in figure 1.

The lower limit for detection was a dephosphorylation rate of 0.001 min^-1. A dephosphorylation rate as low as 0.001 min^-1 could be observed when the rate of phosphorylation was not faster than 0.1 min-1. The rate of phosphorylation was about 0.1 min^-1 when the echothiophate concentration was low enough so that 30 minutes elapsed before a linear steady state level of BTC hydrolysis was observed. The exact concentration on echothiophate needed to achieve this condition varied from 1 to 90 µM, depending upon the enzyme under study.

No attempt was made to correct the observed rate of product formation from BTC for contribution from hydrolysis of echothiophate. Though both compounds generate thiol products, which would be detected in the Ellman assay, the rate for echothiophate turnover was found to be four orders of magnitude slower than for BTC (Lockridge et al., 1997). In addition, any residual product formation found in the screening assay, regardless of the origin, would qualify the candidate enzyme as a potential OP-hydrolase and make it subject to additional study.

Spontaneous reactivation in the presence of BTC. Mutants that gave an indication of OP hydrolysis in the screening assay were directly assayed for spontaneous reactivation. For this assay, echothiophate-inhibited enzyme was diluted into buffer containing BTC and DTNB, without removing excess inhibitor. Reactivation was monitored by the increase in rate of product formation. This method is similar to the approach described by Skrinjaric-Spoljar et al. (1973), and was chosen because some of the mutants reactivated so quickly that the conventional sampling method was impractical.

The first step in the spontaneous reactivation assay was to prepare inhibited BChE. BChE mutant was mixed with the minimum concentration of echothiophate needed to inhibit activity by 90-95% in 1-2 hours (1 to 90 µM echothiophate depending upon the enzyme). It should be noted that active organophosphatases, such as G117H, could be inhibited only about 80% because the phosphorylation and dephosphorylation rate constants were similar. The amounts of enzyme and inhibitor used depended on the activity of the particular enzyme under investigation. The inhibition mixture was incubated at pH 7.0, 25 °C, in a sealed plastic microfuge tube, and aliquots were withdrawn periodically to measure residual activity. When maximal inhibition had been achieved, a 10-50 µL aliquot was removed and added to a cuvette containing 2 mL of 0.1 M potassium phosphate buffer, pH 7.0, plus 1 mM BTC and 0.5 mM DTNB, at 25 °C. Product formation was monitored continuously at 412 nm. Reactivation was allowed to proceed until a constant rate of product formation was obtained (see figure 2 for an example).

This assay relies on the assumption that the magnitude of the dilution and competition from BTC are sufficient to prevent re-inhibition by residual echothiophate. This assumption was validated by control experiments in which the reactivation time course was shown to be unaffected by the concentration of echothiophate.

The mechanism for spontaneous reactivation in the presence of substrate is illustrated in Scheme 1.

Equation 1 describes the time dependence of product formation for scheme 1. It was derived by the general principles outlined by Tsou (1988).

k_react is the reactivation rate constant. V is the linear, steady state turnover rate (delta in absorbance at 412 nm per min) at the end of the reactivation portion of the time course. V/k_react is the y-axis intercept, extrapolated from the linear portion of the time course. Values for V and k_react were obtained by fitting the observed time course for reactivation to equation 1, using SigmaPlot (Jandel Scientific). Equation 1 is identical to the reactivation expression reported by Tsou if the re-inhibition terms in that equation are set to zero (see equation 13 in Tsou, 1988). The only assumptions involved in deriving equation 1 are a) that the rates of substrate binding and turnover are very fast relative to the dephosphorylation rate, and b) that the substrate concentration is much larger than the enzyme concentration.

Aging in the absence of substrate. Aging kinetics were determined directly for BChE mutants that showed reactivation rates >0.01 min^-1 because, at 0.01 min^-1, the reactivation process is well separated from aging, and the kinetics of the two do not interact significantly. An aging assay consisted of a series of spontaneous reactivation assays from a single inhibition reaction. The inhibition mixture was prepared as described in the reactivation section, and was incubated at 25 °C for up to several days. Reactivation assays were conducted periodically to determine the amount of residual activity remaining after reactivation. The limiting steady state rate of substrate turnover at the end of each reactivation assay (V) is directly proportional to the concentration of active (unaged) enzyme remaining. A parallel incubation of uninhibited enzyme was assayed periodically to control for spontaneous loss of enzymatic activity.

results

Design of BChE mutants. A model (Millard and Broomfield, 1992) of the three-dimensional structure of human BChE, shown later to be consistent with the crystal structure of human BChE (Nicolet et al., 2003; Protein Data Bank 1P0i), was used to design mutants that might have OP hydrolase activity. Histidine was moved to various locations in the active site to test the effect of the position of the positive charge. Acidic residues were added to ion pair with the extra histidines, thereby creating more protonated histidine character at neutral pH. The G117D mutation was introduced to correspond with the naturally occurring mutation in the sheep blowfly, whose carboxylesterase is an OP hydrolase (Newcomb et al., 1997; Claudianos et al., 1999). The mutations in the OP resistant acetylcholinesterase (AChE) of Drosophila melanogaster (Mutero et al., 1994) were introduced into BChE. A variety of amino acid residues at position 117 tested the absolute requirement for a positive charge in that location. Many of these mutants hydrolyzed echothiophate, but none had better echothiophate-hydrolysis activity than G117H.

Figure 3 is a schematic representation of the active site of BChE showing the catalytic triad, S198, H438, and E325, at the bottom of the active site gorge. G117 is part of the oxyanion hole that stabilizes the transition state intermediate. The acyl binding pocket is defined by L286 and V288. Two tryptophan residues, W231 and W82, are prominent components of the wall of the active site gorge. Positively charged choline is oriented toward W82 and A328. The leaving group of echothiophate is oriented toward the mouth of the gorge.

Screening for resistance to OP inhibition. When echothiophate was added to wild-type BChE, product formation came to a complete stop, indicating complete inhibition of the BChE by OP. When the mutant was OP resistant, product formation slowed to a constant rate, reflecting a steady state level of active enzyme. For example in figure 1, the double mutant G117H/E197G was still able to hydrolyze BTC in the presence of 100 µM echothiophate. The rate slowed to a steady state at 55 minutes, which is about 30 minutes after addition of echothiophate.

Screening for reactivation of echothiophate-inhibited mutants. An example of the reactivation assay results is shown in figure 2, where the L286H mutant achieved a constant rate of product formation after 300 minutes. Sixty-two BChE mutants were screened for reactivation after echothiophate inhibition. Thirteen, including G117H, showed significant rates of reactivation in the organophosphatase screening assay. Spontaneous reactivation rate constants were determined for these thirteen mutants by fitting the time course data to equation 1. Results are summarized in table 1.

The G117H mutant exhibited the fastest reactivation rate constant, 1.15±0.15 min^-1. This is identical to the value reported by Lockridge et al., 1.2 min^-1, pH 7.0, 25 °C (Lockridge et al., 1997); and it is slightly faster than that reported by Broomfield et al., 0.48 min 1, pH 7.4, 25 °C (Broomfield et al., 1998). Echothiophate creates a diethylphosphoryl-adduct of BChE. Therefore, these dephosphorylation rate constants can be compared to that for reactivation of wild-type BChE, after inhibition by the diethylphosphoryl-organophosphate paraoxon (0.000016 min^-1, pH 7.8, 37 °C, Davison, 1955). The G117H mutant reactivates about 70,000-fold faster than wild-type BChE.

Addition of other mutations to G117H slowed reactivation. Even attempts at ion-pairing, which were designed to enhance the nucleophilicity of the histidine at position-117, were unproductive, e.g., G117H/Q119E, G117H/A199E, and G117H/F329E/V288A.

With the exception of L286H, moving the histidine to other locations in the active site was also unproductive, e.g., V288H, Q119H, T120H, and A277H. Though the L286H mutant reactivated 400-fold more slowly than G117H, it was still 250-fold faster than wild-type BChE. Adjusting the position of histidine 286 by adding glycine residues to the sequence, e.g., L286GH, L286HG, and L286GHG, did not improve the reactivation rate.

Changing the residue at position-117 revealed that acidic residues G117E and G117D were significantly reactive, but less nucleophilic residues such as G117S, G117C, and G117Y were not. The mutation G117S is homologous to the mutation G119S found in ace-1 of the insecticide resistant mosquitoes Anopheles gambiae and Culex pipiens (Weill et al., 2003). Mosquitoes were resistant to the carbamate propoxur but were not shown to be resistant to OP.

The following 49 mutants gave no convincing evidence for reactivation: G117H/G115H, G117H/G439H, G117H/V288H, G117H/A199H, G117H/L286H, G117H/A328H, G117H/F329H, V288H/F329D, V288H/F329E, V288H/G291D, G117H/F329E/V288A, V288H, V288GH, V288GHG, Q119H, T120H, A277H, G117C, G117K, G117S, G117Y, G117L, G117H/Q119E, G117H/A199E, G117H/L286Y, G117H/F329Y, F329E, F329S, K339M, F329S/E197Q, F398I, G117H/F398I, P285L, P285L/F398I, G117H/P285L, G117H/P285L/F398I, G225A, L286Y, G225A/L286Y, E197Q, G117H/W82A , G117H/Y332A, G117H/E197H, W430A, F329S/E197Q, E325Q, G117H/E325Q, G117H/A328W, and G117H/L286R.

Determination of the dephosphorylation rate constants from the spontaneous reactivation rate constants. Spontaneous reactivation is the combination of two parallel processes: dephosphorylation and aging. Aging is defined as dealkylation of the phosphoryl adduct to give a negatively charged adduct that is stabilized by interaction with the catalytic histidine (Millard et al., 1999). Aging irreversibly prevents dephosphorylation and limits the amount of enzyme that becomes reactivated. The fraction of enzyme that becomes reactivated is described by equation 2.

[E] is the concentration of reactivated enzyme at time t. [Eo] is the total concentration of enzyme. k_dephos is the dephosphorylation rate constant; k_aging is the aging rate constant; and (k_dephos +k_aging) is the observed rate constant for spontaneous reactivation (k_react). This equation comes directly from the theory of parallel first-order reactions (Frost and Pearson, 1961) and was described first in the cholinesterase literature by Skrinjaric-Spoljar et al. (1973). If the reactivation is allowed to run to completion, then the exponential term becomes very small relative to one, and equation 2 can be re-arranged to give equation 3.

The ratio [E]/[Eo] is the fraction of enzyme, which becomes reactivated. This fraction is equal to the measured, linear, steady state velocity at the end of reactivation (V) divided by the velocity for an equal amount of uninhibited enzyme. Multiplying the observed k_react by the fraction of reactivated enzyme gives k_dephos. Calculated values for k_dephos are listed in table 1. For most of the mutants k_dephos was essentially equal to k_react.

A complication for the values reported in table 1 derives from the fact that reactivation of echothiophate-inhibited BChE is slowed by the presence of 1 mM BTC. For example, with G117H reactivation was slowed by about 30%, i.e., 1.4 min^-1 at zero BTC and 1.0 min^-1 at 1 mM BTC (see table 2). Therefore, the values reported in table 1 must be considered as approximate.

Aging. In all cases, the concentration of reactivatable enzyme decreased in a first-order manner with time, yielding the half-life values for aging. Aging for all six mutants studied was substantially slower than aging for G117H (t_1/2 = 5.5 hour, (Lockridge et al., 1997), see table 1.

Characterization of the OP hydrolysis properties of G117H. In order to obtain a better appreciation of the capabilities of G117H, kinetics for phosphorylation, dephosphorylation and aging were determined with: methyl-paraoxon (which forms a dimethylphosphoryl-adduct), echothiophate (which forms a diethylphosphoryl-adduct) and diisopropylfluorophosphate (which forms a diisopropylphosphoryl-adduct).

Dephosphorylation rate constants were calculated from the reactivation rate constants and the fractions of G117H reactivated, using equation 3. Reactivation measurements were made in the presence of BTC because the reactivation rates of G117H were too rapid to allow the measurements to be made in the absence of substrate. In order to determine the reactivation rate constant in the absence of BTC, a series of experiments at different BTC concentrations were made and the value for the apparent reactivation rate constant was extrapolated to zero BTC (see figure 4 for an example and details of the extrapolation). In addition to providing a basis for calculating the dephosphorylation rate constant at zero BTC, these experiments gave the dissociation constant for BTC binding to the phosphoryl-enzyme adduct, and the dephosphorylation rate constant in the presence of saturating BTC. The results are summarized in table 2.

Aging was determined separately, in the absence of substrate. The results are shown in table 3. The aging reactions of G117H all followed first-order time courses for more than three half-lives. The spontaneous reactivation rate constants were at least 20-fold faster than their respective aging rate constants, therefore use of spontaneous reactivation to determine the fraction of unaged enzyme remaining at each time point, did not bias the measurements. The results are shown in table 3.

Phosphorylation rate constants were calculated rather than measured directly. This is because the classical method for measuring phosphorylation rate constants was unworkable. In the classical method, OP is mixed with enzyme and the mixture is sampled periodically to determine the amount of uninhibited enzyme remaining (Main, 1979). Preliminary experiments indicated that with G117H the rate constant for dephosphorylation was about the same as that for phosphorylation. This meant that when the concentration of inhibitor was high enough so that phosphorylation could compete with dephosphorylation, the rate of inhibition was too fast to measure. But, when the concentration of inhibitor was lowered, the rate for dephosphorylation became large relative to the rate for phosphorylation, and the fraction of inhibited G117H became too small to measure. Consequently, we turned to calculation.

For this calculation, we used the maximum fraction of covalently inhibited G117H, together with the rate constant for dephosphorylation in the absence of substrate (measured separately). This analysis is conceptually similar to that used by Froede and Wilson (1984) to determine the fraction of acetyl-enzyme present during turnover.

The fraction of covalently inhibited G117H was determined by diluting the inhibited enzyme into a solution containing substrate, and measuring the residual activity. Dilution and competition from substrate dissociated the non-covalently inhibited complex, leaving only the covalently inhibited form. The maximum fraction of covalent complex was determined by increasing the concentration of OP until no further increase in the extent of inhibition was detected.

Calculations were based on the general mechanism for turnover of OP (Main, 1979) diagrammed in scheme 2.

The expression for the time dependent change in the concentration of the covalent complex for this mechanism can be derived following the general principles described by Strickland et al. (1975).

[C] is the concentration of covalent complex. Et is the total concentration of BChE. [OP] is the concentration of OP. k_app is the apparent rate constant for phosphorylation. Kd is k_-1/k₁, k_phos is the phosphorylation rate constant, and k_dephos is the dephosphorylation rate constant.

The inhibition reaction was allowed to reach equilibrium before the fraction of covalent complex was measured. The time required to reach equilibrium was determined from preliminary experiments. At equilibrium, the exponential term becomes small relative to unity. When the concentration of OP is saturating, [OP]»Kd and the concentration of covalent complex is maximal. Then, equation 4 can be reduced and re-arranged into equation 5.

[C]_max/Et is the maximum fraction of covalent complex.

discussion

Advantages of this screening assay. Following the progressive inhibition of BTC turnover by echothiophate is a simple and sensitive method for evaluating mutants of BChE for OP-hydrolase activity. The inability of echothiophate to completely inhibit BTC hydrolysis is highly suggestive of echothiophate hydrolysis by the mutant. The full time course of progressive inhibition provides an immediate, visual depiction of the level of residual activity. With a time course of 30-45 minutes, the assay is relatively rapid. The assay can be performed on sub-nanomolar levels of candidate enzyme—where 0.1 nanomolar (nM) wild-type BChE yields a readily detectable rate of product formation in the Ellman assay, when using 0.1 M BTC at pH, 25 °C. The assay can be performed on crude enzyme preparations, since BTC is highly selective for BChE. Thus, it is suitable for testing mutants made via transient transfection, without purification. In contrast, direct turnover of echothiophate is not a suitable tool for mutant screening. Though turnover of echothiophate would provide definitive evidence for OP-hydrolase activity, the rates for echothiophate turnover are too slow to be practical. Lockridge et al. (1997) have shown that G117H, the most reactive of the BChE mutants, turns over echothiophate with a rate constant of 0.75 min^-1. Three µM G117H were required to give reliable rates of product formation. In addition, use of crude preparations for echothiophate turnover creates a real potential for interference from A-esterases, such as paraoxonase. Direct turnover is more suited for confirmation of candidate mutants, which have been purified in micromolar concentrations.

Validity of the assay. Resistance of BChE to inhibition by OP can theoretically take two forms. Either the reactivity toward OP can be reduced, making inhibition more difficult, or reactivation of the inhibited enzyme can be enhanced, making recovery from inhibition faster. The screening assay employed in this work tested for the latter form of resistance, enhancing the reactivation of the inhibited enzyme.

The reactivation rate was used to calculate the rate of dephosphorylation of the active site serine. This calculation assumed that the active site serine became phosphorylated. Since OP was not used as a substrate, but only as an inhibitor, the assay leaves open the possibility that the OP bound somewhere other than to the active site serine and that dissociation from this noncovalent binding site accounted for the return of BChE activity. The assay described in this report does not rule out this possibility. Additional experiments have to be performed, after the screening assay is complete, to confirm OP hydrolase activity. For example, OP hydrolase activity for G117H BChE was demonstrated by Millard et al. (Millard et al., 1995; Millard et al., 1998) by measuring consumption of OP substrate. Lockridge et al. (1997) measured release of thiocholine from echothiophate and release of para-nitrophenol from paraoxon to demonstrate OP hydrolase activity of G117H. Claudianos et al. (1999) measured hydrolysis of a radiolabeled OP, C14-chlorfenvinphos, by the sheep blowfly carboxylesterase mutant G117D ortholog. The advantage of the present assay is that only small amounts of enzyme are required. This makes it possible to screen many mutants produced by transient expression in 293T or COS cells.

Mechanistic interpretation of the dephosphorylation enhancement promoted by G117H. The results indicate that position-117 plays a critical role in enhancing reactivation of echothiophate-inhibited BChE. There was a histidine at this position in six out of thirteen mutants that showed significant dephosphorylation rate constants. Two of the remaining seven reactive mutants had an acidic group at this position.

Mutation G117H was originally conceived as a means of introducing a nucleophilic residue into BChE, at a position which was geometrically favorable for activating water to attack the face of the phosphoryl-adduct opposite the position of the serine ligand (Broomfield et al., 1995). Jarv had earlier proposed that phosphoryl-adducts of serine esterases were stable because their active sites were not capable of activating water for attack on the phosphorus at this position (Jarv, 1984; Jarv, 1989). The 70,000-fold increase in the dephosphorylation rate constant upon introduction of histidine at position-117 supports the original hypothesis. Increase in the dephosphorylation rate constant upon substitution of acidic groups into this position (G117D and G117E) further supports the proposal, because the major feature which histidine, aspartate, and glutamate have in common is their ability of serve as a hydrogen-bond acceptor for water.

It is interesting to note, that a naturally occurring, homologous mutation (G137D) has been reported for carboxylesterase in OP resistant strains of both the blowfly (Lucillia cuprina) and the housefly (Musca domestica) (Newcomb et al., 1997; Claudianos et al., 1999). The authors in this case suggested that the increase in OP hydrolase activity was due to aspartate-137 acting as a base to orient a water molecule for attack on the phosphorylated enzyme intermediate (Newcomb, 1997), a hypothesis in agreement with the present results.

Four of the five remaining mutants that showed enhanced dephosphorylation rate constants, also carried a histidine. For those mutants, the histidine was at position-286. As was the case with position-117, inspection of the 3-dimensional structure of human BChE suggested that a histidine at position-286 might be favorably positioned to activate a water molecule for attack on the phosphoryl-adduct. Enhancement of dephosphorylation by L286H was 300-fold less than for G117H, but the mere existence of an enhancement supports the principle.

Dephosphorylation, phosphorylation and aging with G117H. The phosphorylation rate constants for G117H (4.1 min^-1 with diisopropylfluorophosphate and 2.4 min^-1 with echothiophate) are within an order of magnitude of those for wild-type BChE (11 min^-1 with diisopropylfluorophosphate (Main and Iverson, 1966) and 0.23 min^-1 with echothiophate (Broomfield et al., 1995). The t_1/2 values of aging for G117H (0.68 hour with the dimethylphosphoryl-adduct, 5.5 hour with the diethylphosphoryl-adduct (Lockridge et al., 1997), and 0.46 hour, with the diisopropylphosphoryl-adduct) are less than 10-fold lower than the corresponding values for wild-type BChE (3.9 hour with the dimethylphosphoryl-adduct (Worek et al., 1999), 11.6 hour with the diethylphosphoryl-adduct (Masson et al., 1997), and 0.96 hour with the diisopropylphosphoryl-adduct (Masson et al., 1997). Allowing for the differences in experimental conditions, the differences in phosphorylation and aging between G117H and wild-type BChE are minor. On the other hand, the dephosphorylation rate constants for G117H (0.23 min^-1 with the dimethylphosphoryl-adduct, and 1.4 min^-1 with the diethylphosphoryl-adduct) are respectively, 200- and 87,500-fold faster than those for wild-type BChE (0.0013 min-1 with the dimethylphosphoryl-adduct (Worek et al., 1999), and 0.000016 min^-1 with the diethylphosphoryl-adduct (Davison, 1955). Thus the primary effect of introducing a histidine into position-117 of BChE has been to increase the rate constant for dephosphorylation.

It is noteworthy that the dephosphylation rate constants for G117H are highly dependent upon the nature of the adduct. There is a 6-fold difference in rate constant between the dimethylphosphoryl-adduct (0.23 min^-1) and the diethylphosphoryl-adduct (1.4 min^-1). The dephosphylation rate constants for the highly toxic nerve agents are substantially slower: sarin (methyl, O-isopropylphosphonyl-adduct; 0.018 min^-1), VX (methyl, O-ethylphosphonyl-adduct; 0.07 min^-1), and soman (methyl, O-pinacolylphosphonyl-adduct; <0.00005 min^-1) (Millard et al., 1995; Millard et al., 1998).

Significance. The significance of these findings is in understanding the mechanism of OP hydrolase activity in a BChE mutant. The G117H mutant is a rare example of an enzyme that was designed to have a new catalytic activity. The design relied on a hypothesis for how the active site serine could be dephosphorylated. This report supports the original hypothesis for the mechanism of dephosphorylation. An understanding of the mechanism of action could be useful for the design of additional OP hydrolase mutants. A second significant outcome of this work is the introduction of an assay that allows screening for apparent OP hydrolase activity.

Figures

tables

references

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Broomfield, C. A., Millard, C. B., Lockridge, O., and Caviston, T. L. (1995) in Enzymes of the Cholinesterase Family (Quinn, D. M., Balasubramanian, A. S., Doctor, B. P., and Taylor, P., Eds.) pp. 169–175, Plenum Press, New York, NY.

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