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J. Biol. Chem., Vol. 277, Issue 45, 43301-43308, November 8, 2002

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Inhibitors of Different Structure Induce Distinguishing Conformations in the Omega Loop, Cys⁶⁹-Cys⁹⁶, of Mouse Acetylcholinesterase^*

Jianxin Shi, Zoran Radi, and Palmer Taylor

From the Department of Pharmacology, University of California, San Diego, La Jolla, California 92093

Received for publication, May 6, 2002, and in revised form, August 9, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We have shown previously that association of reversible active site ligands induces a conformational change in an omega loop ( loop), Cys⁶⁹-Cys⁹⁶, of acetylcholinesterase. The fluorophore acrylodan, site-specifically incorporated at positions 76, 81, and 84, on the external portion of the loop not lining the active site gorge, shows changes in its fluorescence spectrum that reflect the fluorescent side chain moving from a hydrophobic environment to become more solvent-exposed. This appears to result from a movement of the  loop accompanying ligand binding. We show here that the loop is indeed flexible and responds to conformational changes induced by both active center and peripheral site inhibitors (gallamine and fasciculin). Moreover, phosphorylation and carbamoylation of the active center serine shows distinctive changes in acrylodan fluorescence spectra at the  loop sites, depending on the chirality and steric dimensions of the covalently conjugated ligand. Capping of the gorge with fasciculin, although it does not displace the bound ligand, dominates in inducing a conformational change in the loop. Hence, the ligand-induced conformational changes are distinctive and suggest multiple loop conformations accompany conjugation at the active center serine. The fluorescence changes induced by the modified enzyme may prove useful in the detection of organophosphates or exposure to cholinesterase inhibitors.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Acetylcholinesterase (AChE)¹ plays a pivotal role in neurotransmission by terminating the action of neurotransmitter, acetylcholine, at neuromuscular junction and other cholinergic synapses (1-3). AChE is one of most efficient enzymes known with hydrolysis of its natural substrate reaching diffusion-controlled limits. Inhibitors of AChE target two sites in the active site gorge: an active center at the base of a narrow gorge 20 Å in depth and a peripheral site at the gorge rim (4). At the active center, a residue triad (Ser²⁰³-Glu³³⁴-His⁴⁴⁷) promotes acyl transfer and hydrolysis of the substrate, whereas Trp⁸⁶ at the gorge base primarily stabilizes choline moiety of the substrate through a cation- interaction. Active site inhibitors block substrate binding either by associating with the tryptophan in the choline binding site (tacrine and edrophonium) or by reacting irreversibly with catalytic serine (carbamates and organophosphates). Peripheral site inhibitors, such as propidium and gallamine, inhibit catalytic activity through both steric blockade and allosterically altering catalytic efficiency of the active center residues (4-8).

To elucidate the conformational changes associated with mechanistically distinctive inhibitors, we developed a means for physically monitoring the conformation of purified mouse AChE by site-directed labeling with an environmentally sensitive fluorophore, acrylodan. Six single cysteine mutants were prepared for acrylodan conjugation (Fig. 1). Three were on the Cys⁶⁹-Cys⁹⁶ omega loop ( loop) flanking the active site gorge: L76C near the tip of the loop, and E81C and E84C on the outer surface not lining the gorge. Two residues on the opposing face of the gorge, H287C and Y124C, were selected, solvent exposure of which would be expected to be occluded by bound ligands that extend to the outer reaches of the gorge. A final residue, A262C, on a distal disulfide loop and whose temperature coefficient (B factor) would indicate flexible loop movement (9, 10), was selected as a control region. This residue is not anticipated to be influenced by ligand-induced changes in conformation. Our previous study showed a bathochromic emission shift of acrylodan conjugated at  loop residues 76, 81, and 84 upon binding of inhibitors, such as tacrine, edrophonium, huperzine A, and m-(N,N,N-trimethylammonio)trifluoromethyl acetophenone, that interact with Trp 86 in the choline binding site (11). Acrylodan fluorescence is exquisitely sensitive to dipole moment of the surrounding solvent or macromolecular milieu (12-14). A bathochromic shift reflects exposure to solvent around the fluorophore. This pattern likely results from a concerted movement in the Cys⁶⁹-Cys⁹⁶  loop upon binding of reversible inhibitors.

View larger version (83K): [in this window] [in a new window]   Fig. 1.   Panel A, locations of introduced cysteines for fluorophore modification of mouse AChE. Residues 76, 81, and 84 are at the tip (residue 76) and outer portion (residues 81 and 84) of the  loop. Residues 124 and 287 are on an opposing face of the gorge and make up part of the peripheral anionic site. Residue 262 is on a small distal disulfide loop showing a large thermal factor in the crystal structure. White surface at the base of active site gorge shows the ethyl moiety of diethylphosphoryl-AChE when conjugated to serine 203 at the base of the active center gorge. Panel B, expanded view of the acrylodan side chain at the 81-position determined from a energy-minimized structure of mouse AChE (43). The aminonaphthalene moiety of acrylodan resides between the side chains of Asp131 and Leu463.

Because a conformational change in the  loop induced by ligand is not reflected in the crystal structures of the AChEs studied to date (9-10, 15-18), and steady state catalysis by  loop mutant AChEs yielded minimal evidence for the loop being involved in the catalytic cycle (19, 20), we developed a means to measure directly conformation and solvent exposure in and around the active center gorge. In this study, we investigate the conformational changes reflected in acrylodan fluorescence for peripheral site inhibitors and for a congeneric series of carbamates and organophosphates that react covalently with the active center serine. Fluorescence measurements, combined with kinetics of inhibitor association, reveal a linkage between inhibition and a conformational change in the  loop. Ligand conjugation at the active center and association at the peripheral site induce distinctive conformational changes in the loop. Because the character of the spectral changes is dependent on chirality and dimensions of the ligand as well as its site of association, the  loop exhibits considerable flexibility in the solution conformations of AChE.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Inhibitors and Substrates-- Acetylthiocholine iodide, 5,5'-dithiobis(2-nitrobenzoic acid), DFP, dithiothreitol, physostigmine, gallamine, neostigmine, and paraoxon were purchased from Sigma-Aldrich. Acrylodan was obtained from Molecular Probes (Eugene, OR), echothiophate was obtained from Ayerst Laboratories (Philadelphia, PA), and DDVP was obtained from Bayer Inc. (West Haven, CT). Rivastigmine was obtained as the commercial product (Exelon) from Novartis. Fasciculin 2 (purified from the venom of Dendroaspis angusticeps) was a gift of Dr. Pascale Marchot (University of Marseille, Marseille, France). Drs. Yacov Ashani and Bhupendra P. Doctor (Walter Reed Army Research Center, Washington, DC) kindly provided 7-[[(methylethoxy)phosphinyl]oxyl]-1-methylquinolinium iodide (MEPQ) and procainamide-linked Sepharose CL-4B resin. The chiral organophosphonate enantiomers, (S_p)-dimethylbutyl methylphosphonothiocholine ((S_p)-DMBMP-TCh), (S_p)-cycloheptyl methylphosphonothiocholine, (S_p)-isopropyl methylphosphonothiocholine, and (R_p)-dimethylbutyl methylphosphonothiocholine ((R_p)-DMBMP-TCh) were kindly provided by Dr. Harvey Berman (State University of New York, Buffalo, NY).

Expression, Mutagenesis, and Purification of mAChE-- Mouse AChE was produced by transfection of expression plasmid (pCDNA3, Invitrogen, San Diego, CA) containing an encoding cDNA where the AChE sequence was terminated at position 548. The plasmid was transfected into human embryonic kidney (HEK293) cells. Cells were selected with G418 to obtain stable producing cell lines, and AChE was expressed as a secreted soluble enzyme in serum-free media (21). Mutant enzymes were generated by standard mutagenesis procedures, and cassettes containing the mutation were subcloned into pCDNA3 (21). Nucleotide sequences of the cassettes were confirmed by double-stranded sequencing to ensure that spurious mutations were not introduced. Affinity chromatography permitted one-step purification of AChE. From 4-6 liters of media, mutant and wild type enzyme were purified in quantities ranging between 5 and 25 mg, as previously described (22-24). Purity was ascertained by SDS-PAGE and by specific activity determination. The cysteine-substituted enzymes show kinetics of acetylcholine hydrolysis similar to wild type enzyme (11).

Acrylodan Labeling-- Mutant enzymes were pretreated with 0.25 mM dithiothreitol for 30 min at room temperature to ensure reduction of the introduced cysteine. Excess dithiothreitol was removed by use of a G-50 Sephadex spin column (Roche Molecular Biochemicals) equilibrated in 10 mM Tris-HCl, 100 mM NaCl, 40 mM MgCl₂, pH 8.0. Conditions for acrylodan labeling and stoichiometry estimates have been described previously (11). Stoichiometry of labeling of the various preparations, estimated from a comparison of enzyme concentration by protein (280 nm) to acrylodan (360-380 nm) absorbance, ranged as follows: L76C, 0.7-1.0; E81C, 0.77-1.0; E84C, 0.77-1.0; Y124C, 0.78-1.0; A262C, 0.69-0.92; and H287C, 0.78-1.0. Specificity of labeling was assessed by comparison of areas under the fluorescence emission curves for acrylodan-treated mutant and wild type enzymes. Specific labeling for each mutant was: L76C, 72-85%; E81C, 81-92%; E84C, 85-93%; Y124C, 83-92%; A262C, 77-93%; H287C, 70-82%.

Enzyme Inhibition-- Picomolar concentrations of enzyme in 0.01% bovine serum albumin and 0.1 M sodium phosphate buffer, pH 7.0, were reacted with covalent inhibitor in the absence of substrate at 25 °C. Typically, four inhibitor concentrations were used. Inhibition was monitored by measuring residual enzyme activity by removal of aliquots during the course of the reaction. Bimolecular rate constants (k_i) were determined by the plot of pseudo first order rate constant (k_obs) against inhibitor concentration (25).

Spectrofluorometric Assays-- Steady state emission spectra were measured at room temperature using a Jobin Yvon/Spex FluoroMax II spectrofluorometer (Instrument S.A., Inc., Edison, NJ) with the excitation and emission bandwidths set at 5 nm. The excitation wavelength for acrylodan was set at 359 nm, and emission was monitored between 420 and 600 nm. Spectral changes in the presence of irreversible inhibitors were determined by allowing the reaction of the acrylodan-labeled enzymes to proceed until 99% inhibition was achieved. In the case of inhibitors with chromogenic leaving groups, the inhibited enzyme was passed through a G-50 Sephadex spin column (Roche Molecular Biochemicals) to remove the leaving group. Quantum yield changes in presence of MEPQ and paraoxon were determined by measuring the concentration of labeled enzyme by tryptophan emission and area of acrylodan fluorescence emission curve before and after organophosphate conjugation. Association of echothiophate and neostigmine with acrylodan-labeled E81C and E84C was assessed from the kinetics of change in fluorescence at 470 and 477 nm, respectively, following addition of a stoichiometric excess of inhibitor at several concentrations. Data were fitted to a single exponential approach to equilibrium. Bimolecular rate constants (k_i) were determined by the plot of pseudo first order rate constant (k_obs) against inhibitor concentration (25). Association of rivastigmine with acrylodan-labeled E81C was monitored from the kinetics of the increase in fluorescence at 460 nm. To ensure the observed change in fluorescence upon rivastigmine association was caused by carbamoylation and not reversible binding of rivastigmine, the enzyme was reacted with the fluorescent carbamoylating agent, M7C, to ascertain the concentration of residual reactive serines (26).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Kinetics of Organophosphorate and Carbamate Inhibition-- We determined the bimolecular rate constants (k_i) for echothiophate, neostigmine, and rivastigmine with unmodified and modified mAChE (Table I). For the wild type, E81C, and E84C mutant enzymes, the constants (k_i) were obtained from measurements of enzyme activity, whereas changes in fluorescent signal were used to monitor reaction with the acrylodan-modified enzyme. A typical example of monitoring of the fluorescence change is shown in Fig. 2. The data in Table I show that substitution of cysteine at the 81-position does not affect the carbamoylation and phosphorylation rates, whereas the modification at the 84-position causes a 3-4-fold reduction in rate. Upon modification of the introduced cysteine with acrylodan, reaction rates are reduced 3-4-fold compared with wild type following conjugation at the 81-position, whereas the reduction is 40-50-fold upon conjugation at the 84-position. It is important to note that the magnitude of these reductions in carbamoylation or phosphorylation rates by mutation and conjugation at each position is nearly the same, despite the inhibitors differing in their reactivity by a few orders of magnitude. In the case of rivastigmine, we measured the reaction rates by competition with excess M7C (26) and achieved similar kinetics of inhibition. This indicates that the spectral shift produced by rivastigmine likely reflects a conformational change induced by progressive carbamoylation rather than formation of a reversible complex.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Association of echothiophate with E84C acrylodan-modified AChE. Panel A, fluorescence emission spectra of acrylodan-labeled E84C AChE following addition of excess echothiophate. Echothiophate forms the diethylphosphoryl enzyme leading to a bathochromic shift and reduction of fluorescence quantum yield. Excess echothiophate (2 µM) was added to 94 nM AChE, and fluorescence spectra were taken at following time points: 0, 1, 11, 26, and 43 min. Panel B, time course of the fluorescence changes. Initial E84C acrylodan-modified AChE concentration was 190 nM. Excess echothiophate was added, and decrease in fluorescence signal at 477 nm was monitored using an ISA Jobin Yvon-Spex Fluoromax fluorometer. The three echothiophate concentrations were 3.5 (), 5.0 (), and 8.0 () µM. Control enzyme samples, to which buffer rather than echothiophate was added, did not show decreases in fluorescence emission over the time intervals measured.

Effect of Achiral Organophosphorates on Acrylodan Emission Spectra-- Organophosphates readily phosphorylate the active site serine (27), presumably generating a pentavalent trigonal bipyramidal intermediate before dissociation of leaving group. The resulting phosphorylated complex resembles the tetrahedral transition states of acylation and deacylation of the trigonal esters. The diethylphosphoryl conjugate at the active site serine formed by reaction with echothiophate produces very little perturbation at positions 76, 262, and 287, consistent with their positions being well removed from the phosphorylation site (Table II). A bathochromic emission shift is observed at position 84, although of smaller magnitude when compared with the shift induced by other ligands (Tables II-V). Interestingly, large hypsochromic shifts and enhancements of quantum yield are observed at positions 81 and 124. This pattern appears to be unusual, because the reversible active center ligands studied previously (11) and the dimethylphosphoryl conjugate formed from DDVP, which yields a phosphoryl serine with one methylene group shorter than echothiophate, confer little shift at position 124 and a large bathochromic shift at position 81. 

To confirm that the chromic shift is a result of conjugation of diethylphosphoryl group, not the retention of the thiocholine leaving group in the gorge, we examined the effect of paraoxon conjugation on chromic shift. Following reaction with the active site serine, paraoxon produces the same diethylphosphoryl conjugate as echothiophate. However, its leaving group is a neutral aromatic moiety rather than the cationic moiety of echothiophate. Because a similar spectral shift follows paraoxon conjugation, the conformational change is induced by the conjugated phosphorate, rather than being influenced by binding of residual leaving group.

If we extend additional methylene units to the diisopropyl phosphoryl conjugate formed by DFP, we observe a chromic shifts at the 81- and 84-positions similar to the diethylphosphoryl conjugate. However, the hypsochromic shift at the 124-position becomes slightly smaller for the diisopropyl phosphoryl conjugate. Measurements were made immediately after reaction to preclude aging (i.e. spontaneous loss of an alkoxy moiety rendering an anionic conjugate) of the diisopropyl phosphoryl moiety (28).

Effect of Chiral Organophosphonates on Acrylodan Emission Spectra-- To compare phosphoryl and phosphonyl conjugates of similar dimensions, racemic MEPQ was used to generate an ethyl methylphosphonyl conjugate. Kinetic studies show an enantiomeric preference of MEPQ, where presumably the S_p enantiomer reacts ~10-fold faster than the R_p enantiomer.² Hence, reaction with a stoichiometric excess of MEPQ should ensure one enantiomer covalently reacts preferentially with the enzyme. No discernable emission changes are observed at residues 262 and 287. Similar to DDVP, a bathochromic shift is observed for acrylodan at both positions 81 and 84. A moderate hypsochromic shift is observed at the 124-position, and very small change at the 76-position.

Table III also shows the changes in acrylodan emission for a series of S_p methylphosphonates with increasing alkoxy substituent dimensions. Because the absolute stereochemistry of the methylphosphonates is known (29), the chiral S_p methylphosphonates will direct their phosphonyl oxygen toward the oxyanion hole, the small methylphosphonyl moiety will be directed to the acyl pocket, and the more bulky alkoxy group directed to choline binding site (30). For the three S_p enantiomers, very little or no change in emission maxima for acrylodan at positions 124, 262, and 287 is discerned. Similar to reversible active site ligands that interact with choline binding site (11), bathochromic shifts are observed at the  loop positions with the largest shift at E84C, an intermediate value at E81C, and only small change at L76C. The ethyl methylphosphonyl conjugate, which contains the smallest alkoxy moiety among S_p conjugates, induces the smallest bathochromic shift at the 81- and 84-positions.

R_p alkyl methylphosphonates react far more slowly with the enzyme than the S_p enantiomers (30), and we use formation of the (R_p)-3,3-dimethylbutyl methylphosphonyl enzyme as an example. Formation of initial reversible complex can be detected by an immediate reduction in quantum yield of acrylodan at 81 with little change in emission maximum (Fig. 3). This is followed by a progressive hypsochromic shift that reflects the covalent reaction with the active center serine. The isoemissive point at 510 nm, evident through the course of the slow reaction, likely reflects the presence of two species (i.e. the reversible DMBMP-TCh ... AChE complex and the conjugated DMBMP-AChE being the dominant species in the progressive reaction). The resulting hypsochromic shift of conjugated acrylodan to 459 nm markedly contrasts with the S_p enantiomer with its bathochromic shift in emission spectrum. These two enantiomers provide the critical clue for linking fluorescence emission maxima at the 81-position to the characteristics of structural perturbations of the  loop.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Fluorescence emission spectra of acrylodan-labeled E81C AChE following addition of (Rp)-dimethylbutyl methylphosphonothiocholine (Rp-DMBMP-TCh). We observed a reduction in quantum yield immediately following addition of (Rp)-DMBMP-TCh, followed by a progressive hypsochromic shift and enhancement in quantum yield. Equivalent concentrations of enzyme (300 nM) were present for all measurements. A stoichiometric amount of (Rp)-DMBMP-TCh (340 nM) was added, and fluorescence spectrum was taken at following time points: 0, 1, 2, 8, 20, 53, 74, 89, 100, 115, and 150 min. The large shift for E81C reveals a isoemissive point at 510 nm, indicative of two (reversible DMBMP-TCh ... AChE complex and conjugated DMBMP-AChE) discrete species in the progressive reaction. Acrylodan-labeled E81C AChE free in solution (solid line), reversibly bound with (Rp)-DMBMP-TCh (dashed line), and covalently conjugated DMBMP-AChE (dotted line). (Rp)-DMBMP-AChE yields an emission maximum of 459 nm, a difference of 51 nm from the (Sp)-DMBMP-AChE (Table III).

Effect of Carbamates on Acrylodan Emission Spectra-- Formation of a carbamoyl serine conjugate of AChE affords an alternative means for forming a relatively stable modified enzyme conjugate (27). Kinetic studies and crystallographic evidence show the carbamoyl oxygen of the covalent conjugate directed toward the oxyanion hole, and the alkyl carbamoyl group pointing toward the acyl pocket (25, 31, 32). Similar to the tetrahedral phosphoryl and phosphonyl conjugates, the trigonal carbamoylated enzymes produce very little perturbation at positions 76, 262, and 287 (Table IV). The monomethyl (physostigmine), dimethyl (neostigmine), and ethylmethyl (rivastigmine) carbamoyl conjugates all produce a small hypsochromic shift and enhancement in acrylodan quantum yield at residue 124. All carbamates, similar to the organophosphates, produce 18-23-nm bathochromic shifts and decreases in quantum yield at position 84. Consistent with the organophosphate series, dimethylcarbamoyl AChE formed from neostigmine and ethylmethylcarbamoyl AChE formed from rivastigmine both produce significant hypsochromic shifts of acrylodan conjugated at position 81. The smaller methylcarbamoyl modification produces little change in E81C spectrum.

Influence of Fasciculin Capping on the Spectrum of Phosphorylated and Carbamoylated AChEs-- Because fasciculin is known to interact at the rim of the active center gorge of AChE (33-35) and can cap the gorge with a conjugated ligand at the base of the gorge (26, 36), we examined the acrylodan spectra of the conjugated enzymes after fasciculin addition. Here again, the most informative position to analyze is 81. Irrespective of whether conjugation at the active center causes a hypsochromic or bathochromic shift, the fasciculin complex yields an emission maximum of 510 nm (Table V, Fig. 4). Thus, fasciculin association dominates over the conformational changes induced by the phosphorylating or carbamoylating agents at the active center of AChE.

Effect of the Peripheral Site Inhibitor, Gallamine, on Acrylodan Emission-- Similar to fasciculin (11), addition of gallamine produces substantial hypsochromic shifts and enhancements in quantum yield with acrylodan conjugated at both the 124- and 287-positions, and a change of smaller magnitude at 76-position (Tables V and VI). This reflects an increase in hydrophobicity experienced by the conjugated fluorophores at the gorge entry upon gallamine binding. Addition of gallamine produces bathochromic shifts of 9 nm at position 84 and 14 nm at position 81. Compared with other AChE inhibitors, whether reversible or covalent, gallamine produces the smallest bathochromic shift seen at residue 84 (Tables II-VI).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We have used a structure-based approach to design a biosensor that responds to phosphorylation and carbamoylation of mAChE. Site-directed placement of an environmentally sensitive fluorophore, acrylodan, offers a sensor that not only responds to covalent conjugation of AChE, but also distinguishes AChE inhibitors based on chirality and molecular dimensions. Because the crystallographic structures (15-18) and the steady state kinetic studies (19, 20) have not revealed conformational changes in the  loop, studies that employ physical parameters to measure conformation in solution take on increased significance.

Acrylodan is known to show large Stokes shifts with differences in dielectric constant of the solvent. This presumably results from its excited state exhibiting an increased dipole moment that, in turn, is stabilized by solvents of higher dielectric constant (12-14). The increase in the emission maxima (bathochromic shift) likely reflects the acrylodan side chain becoming exposed to the solvent.

Loop Substitutions at 81 and 84-- We previously showed large bathochromic shifts for acrylodan side chains at 81 and 84 upon association of reversible active site ligands. Both of these residues are well removed from the active center serine (Fig. 1) and should be influenced only allosterically by the bound or conjugated ligand at the active center. We have attributed the enhanced solvent exposure to increased strain on the  loop resulting from ligand-induced closure of the gorge (11). However, the differences we see here between acrylodan labeling at the 84- and 81-positions suggest a greater degree of conformational flexibility in the  loop than can be ascribed simply to two conformational states.

Catalytic and inhibition parameters are differentially affected by cysteine substitution and acrylodan modification at the 81- and 84-positions. Our previous studies showed that substitution of cysteine at 84 had a greater effect on steady state catalytic parameters than the substitution at 81. Nevertheless, considering that AChE is highly refined for catalytic efficiency and the catalytic enhancement over H₂O catalysis of the ester is ~10¹⁴-fold (3), the acrylodan-substituted enzymes remain highly efficient. Moreover, the magnitude of the reduction in phosphorylation or carbamoylation rate resulting from acrylodan conjugation appears to be independent of the reactivity of the carbamoylating or phosphorylating agent (Table I).

The analysis of the changes in emission maxima at the 84-position show that all conjugating ligands and peripheral site ligands (Tables II-VI) induce a bathochromic shift in emission similar to ligands that bind reversibly at the choline subsite of the active center. Hence, all ligands appear to enhance solvent exposure of the acrylodan side chain at the 84-position. An ordering of the emission maxima at the 84-position for the S_p methylphosphonyl enzymes shows the greatest shift with the larger ligands. This trend is also evident for S_p methylphosphonyl conjugates at the 81-position. Thus, if the magnitude of the shift reflects fractional gorge opening and closing, then the larger ligands promote gorge closure or shift the equilibrium of conformations toward a closed gorge state.

The spectral changes seen at the 81-position appear to be the most discriminating with respect to the conjugated ligand, and here our structure-activity analysis reveals a clear trend. Ligands that conjugate to the active center serine and have the appropriate dimensions or chirality so as to fit into and not perturb the acyl pocket all cause bathochromic shifts in emission spectrum. This applies to the dimethylphosphoryl conjugate, all of the S_p methylphosphonyl conjugates and perhaps to the methylcarbamoyl conjugate. It should be noted that all of these compounds would allow for insertion of the phosphoryl and carbamoyl oxygen in the oxyanion hole formed by hydrogen bonds donors from amide backbone hydrogens at Gly¹²¹, Gly¹²², and Ala²⁰⁴ without deforming the acyl pocket (31).

By contrast, the diethyl and diisopropyl phosphoryl conjugates and the corresponding R_p phosphonyl-AChE derivatives cannot stabilize their phosphoryl or phosphonyl oxygen in the oxyanion hole unless the alkoxy moiety perturbs or moves out of the acyl pocket. Direct evidence for this comes from the crystallographic structure of the DFP-AChE that reveals significant perturbation of the two phenylalanines in the acyl pocket (18).

Additionally, long-standing prior investigations of substrate and inhibitor specificity permit a similar deduction. Over 50 years ago, Augustinsson (36) found that propionylcholine is an effective substrate for AChE, whereas butyrylcholine is not. This finding, when viewed with contemporary structures, suggests that the limits of acyl pocket tolerance occur at the propionyl to butyryl juncture. Thus, a dimethylphosphoryl conjugate would extend linearly from the serine hydroxyl a similar distance to the transition state for formation of propionyl serine. A diethylphosphoryl conjugate would have dimensions similar to the transition state for butyryl serine. A methylcarbamoyl chain would also be similar to the propionyl fits, whereas the dimethyl or ethylmethyl amino substitutions would impart additional steric constraints (32). Likewise, other early studies showed that associations of reversible inhibitors to AChE conjugated with phosphorylating or sulfonylating agents were unimpeded with smaller modifying groups, but sterically hindered with larger modifying groups (37, 38).

The limitations on acyl pocket dimensions position the conjugated ligand to alter potentially both the acyl pocket loop defined by residues Trp²⁸⁶-Ser²⁹⁸ (39) and the neighboring  loop Cys⁶⁹-Cys⁹⁶. Constraints on acyl pocket dimensions appear to affect conformation of both loops. Direct perturbation of the acyl pocket side-chain positions have been observed with DFP conjugation and the formation of its aged product (18). Additionally, by not fitting in the acyl pocket, the conjugated alkyl groups will reside elsewhere in the gorge, therein influencing  loop conformation and the extent of gorge closure as we observed for the side-chain 81-position. Morel and collaborators (39) have proposed various cross-gorge interconnectivities of side chains based on mutagenesis and thermal denaturation experiments.

Residues in the Active Center Gorge in Apposition to the  Loop-- Residues 124 and 287 lie across the gorge from the  loop with H287C at the rim of the gorge and Y124C, residing below the rim and within the gorge interior (Fig. 1). We previously showed a lack of spectral shift at 124 and 287 sites with binding of reversible active site inhibitors (11). Residue 287 is also not affected by any of the conjugating inhibitors. Similar to these reversible active site inhibitors, the larger chiral S_p organophosphonates produce little change in acrylodan emission maxima at the 124-position. By contrast, echothiophate, paraoxon, DFP, and MEPQ all cause a substantial hypsochromic shift and enhancement in quantum yield. Because of the small size of these alkyl moieties in phosphoryl or phosphonyl derivatives, they are unlikely to interact directly with 124, although they may affect solvent structure in the gorge. An increase of one methylene unit in phosphoryl conjugate from dimethylphosphoryl (DDVP) to diethylphosphoryl (echothiophate and paraoxon) gives a spectral change suggestive of solvent exclusion around the 124-position. A much smaller but clear hypsochromic shift is observed for carbamates, perhaps reflecting the different geometry in which the carbamoyl moieties position themselves in the gorge.

Interpretation of the basis of the changes in emission for acrylodan at the 124-position is likely to be complicated by three imposing factors. First, acrylodan at the 124-position can be expected to reside well within the gorge and ligands affecting H₂O structure in the gorge may affect its environment. Second, this side chain could be influenced by ligands occupying the oxyanion hole formed in part by amide hydrogen donors from Gly¹²¹ and Gly¹²². Third, perturbation of the acyl pocket may indirectly influence the position of the position 124 residue. The crystallographic structure of aged DFP conjugate reveals that the isopropyl group of DFP causes a displacement of acyl pocket loop that includes peripheral site residue Trp²⁸⁶ (18). Although Tyr¹²⁴ is not an acyl pocket loop residue, it is in close apposition with Trp²⁸⁶. Similar to DFP, the ethyl group of echothiophate and paraoxon could also cause a movement in the acyl pocket loop (Trp²⁸⁶-Ser²⁹⁸). This conformational change coupled with solvent exclusion upon ligand binding may lead to the substantial hypsochromic shift and enhancement of quantum yield at the 124-position.

Allosteric Effect of Peripheral Site Inhibitors-- Gallamine, a tris-quaternary ligand that binds at the peripheral site, induces a distinctive hypsochromic shift at the 124- and 287-positions. This pattern correlates remarkably with the first series of peripheral site ligand complexed mAChE solved recently (40). The crystallographic structure shows the aromatic pyrogallol moiety of gallamine in - stacking interaction with Trp²⁸⁶, and van der Waals contact with Tyr¹²⁴. Furthermore, bathochromic shifts observed at the 81- and 84-positions reflect a linkage between the binding at the peripheral site and allosteric conformational change in the  loop.

The marked decrease in fluorescence intensity and bathochromic shifts at the position 81 and 84 residues seen with fasciculin capping of covalently modified enzymes reveal a distinct conformation that the  loop adopts in fasciculin-AChE complex when compared with the various serine-modified enzymes. The dominance of conformation change induced by fasciculin binding likely reflects the large molecular dimensions of the peptidic toxin. When bound, ~30% of the fasciculin molecule is buried in the complex, giving rise to a van der Waals contact area of 1100 Å (9).

Acrylodan-conjugated Enzyme as a Biosensor for Inactivation of Acetylcholinesterase-- The surprisingly large and discriminating spectral shifts produced by close congeners of the organophosphates offer an opportunity for detection of organophosphate or carbamate exposure. With the fluorophore intrinsically conjugated to the protein target, reagent addition is not required for detection. Remote sensing of organophosphate exposure would be particularly valuable for oxon forms of the organophosphate insecticides (malathion, parathion, diazonin, and chlorpyrifos) that react directly with AChE as well as the more insidious nerve agents (sarin, soman, VX, cyclosarin, and tabun), where cumulative conjugation could be monitored (2, 41, 42). Moreover, by using multiple wavelength detection, the individual inhibitors can be distinguished. For example, maloxon and methylparaoxon forming the dimethylphosphoryl enzyme can be distinguished from paraoxon and chlorpyrifos oxon using dual detection at 510 and 477 nm for acrylodan conjugated at the 81-position. Inhibitions by R_p and S_p dimethylbutyl methylphosphonates yield conjugates that show emission maxima at 459 and 510 nm, respectively. The S_p methylphosphonates show smaller differences in fluorescence emission between them, but monitoring a combination of site-directed acrylodan-modified AChEs may distinguish individual methylphosphonate conjugates.

	FOOTNOTES

^* This work was supported by United States Public Health Service Grants GM-R37-18360 and ES10337 and Department of Army Medical Defense Grant 17-1-8014 (to P. T.), and by National Institutes of Health Training Grant GM07752 (to J. S.)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 858-534-1366; Fax: 858-534-8248; E-mail: pwtaylor@ucsd.edu.

Published, JBC Papers in Press, August 24, 2002, DOI 10.1074/jbc.M204391200

² Z. Radi, unpublished observation.

	ABBREVIATIONS

The abbreviations used are: AChE, acetylcholinesterase; mAChE, mouse acetylcholinesterase; MEPQ, 7-[[(methylethoxy)phosphinyl]-oxyl]-1-methylquinolinium iodide; DDVP, O,O-dimethyl O-(2,2-dichlorovinyl)phosphate; DFP, diisopropyl fluorophosphate; DMBMP, 3,3-dimethylbutyl methylphosphonyl; TCh, thiocholine; acrylodan, 6-acryloyl-2-dimethylaminonaphthalene; M7C, N,N-dimethylcarbamoyl N-methyl-7-hydroxyquinolinium.

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