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We have examined the specificity of planar carboxyl and tetrahedral phosphonyl esters for mouse cholinesterases and have delineated the orientation of these ligands in the enzyme active center. The approach involved altering acyl pocket dimensions by site-specific mutagenesis of two phenylalanines and varying ligand size and enantiomer presentation. Substrate catalysis rates by wild type acetylcholinesterase (AChE) of acetyl-, butyryl-, and benzoylthiocholine diminished with increasing size of the acyl moiety. In contrast, substitution of the acyl pocket phenylalanines giving the mutants F295L and F297I of AChE yielded more efficient catalysis of the larger substrates and a specificity approaching that of butyrylcholinesterase. Extension from planar substrates to enantiomerically pure organophosphonates allowed for an analysis of enantiomeric selectivity. We found that AChE reactions are 200-fold faster with the Sp than the Rp enantiomer of of cycloheptyl methylphosphonyl thiocholine. Upon the acyl pocket size being enlarged, the Rp enantiomer became more reactive while reaction with the Sp enantiomer was slightly reduced. In fact, the F297I mutant displayed inverted stereospecificity. A visual correlation with the kinetic data has been developed by docking the ligands in the active site. Upon placement of the phosphonyl oxygen in the oxyanion hole and the leaving group being directed out of the gorge, the Rp, but not the Sp, enantiomer engendered steric hindrance between the alkoxyl group and the acyl pocket. Replacing F297 with Ile accommodated the bulky alkoxyl group of the Rp isomer in the acyl pocket, allowing similar orientations of the phosphonyl oxygen and the leaving group to the Sp isomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Organophosphates inactivate acetylcholinesterase by reacting covalently with the active center serine. We have examined the reactivation of a series of resolved enantiomeric methylphosphonate conjugates of acetylcholinesterase by two oximes, 2-pralidoxime (2-PAM) and 1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4'-carbamoyl-1-pyridinium) (HI-6). The S(p) enantiomers of the methylphosphonate esters are far more reactive in forming the conjugate with the enzyme, and we find that rates of oxime reactivation also show an S(p) versus R(p) preference, suggesting that a similar orientation of the phosphonyl oxygen toward the oxyanion hole is required for both efficient inactivation and reactivation. A comparison of reactivation rates of (S(p))- and (R(p))-cycloheptyl, 3,3-dimethylbutyl, and isopropyl methylphosphonyl conjugates shows that steric hindrance by the alkoxy group precludes facile access of the oxime to the tetrahedral phosphorus. To facilitate access, we substituted smaller side chains in the acyl pocket of the active center and find that the Phe295Leu substitution enhances the HI-6-elicited reactivation rates of the S(p) conjugates up to 14-fold, whereas the Phe297Ile substitution preferentially enhances 2-PAM reactivation by as much as 125-fold. The fractional enhancement of reactivation achieved by these mutations of the acyl pocket is greatest for the conjugated phosphonates of the largest steric bulk. By contrast, little enhancement of the reactivation rate is seen with these mutants for the R(p) conjugates, where limitations on oxime access to the phosphonate and suboptimal positioning of the phosphonyl oxygen in the oxyanion hole may both slow reactivation. These findings suggest that impaction of the conjugated organophosphate within the constraints of the active center gorge is a major factor in influencing oxime access and reactivation rates. Moreover, the individual oximes differ in attacking orientation, leading to the presumed pentavalent transition state. Hence, their efficacies as reactivating agents depend on the steric bulk of the intervening groups surrounding the tetrahedral phosphorus.

We show here with a congeneric series of Rp- and Sp-alkoxymethyl phosphonothiolates of known absolute stereochemistry that chiral selectivity in their reaction with acetylcholinesterase can be described in terms of discrete orientational and steric requirements. Stereoselectivity depends on acyl pocket dimensions, which govern leaving group orientation and a productive association of the phosphonyl oxygen in the oxyanion hole. Overall geometry is consistent with a pentavalent intermediate where the attacking serine and leaving group are at apical positions. Oxime reactivation of the phosphonylated enzyme occurs through a similar associative intermediate presumably forming an oxime phosphonate. The oximes of differing structure show distinct angles of attacking the phosphate where the attack angles and access to the phosphorus are constrained in the sterically impacted gorge. Hence, efficacy of oxime reactivation is dependent on both oxime and conjugated phosphonate structures.

Single site acetylcholinesterase (AChE) mutants, F295L and F297I, were inhibited with enantiomeric Sp- and Rp- cycloheptyl (CHMP), isopropyl (iPrMP), and dimethylbutyl (DMBMP) methylphosphonyl thiocholine. The resulting conjugates were subjected to reactivation with 2-(hydroxyiminomethyl)-1-methylpyridinium methiodide (2-PAM) and 1 -(2′ -hydroxyiminomethyl-1′ -pyridinium)-3 -(4″-carbamoy1-1″-pyridinium)-2 -oxapropane dichloride (HI6). Rates of reactivation obtained for mutants were compared to wildtype AChE. HI6 was able to reactivate all conjugates except for the Rp- CHMP conjugates and Rp- DMBMP-F297I conjugate. The F295L mutant exhibited a 6–8 fold faster reactivation rate compared to wildtype for Sp- CHMP and Sp- DMBMP conjugates, while only a 2–3 fold difference was seen for the Sp- iPrMP conjugate. The Rp- DMBMP-F295L conjugate showed biphasic behavior upon reactivation with HI6. The F297I mutant only showed a 2–3 fold higher reactivation rate for all three Sp- enantiomeric conjugates compared to wildtype. The Rp- conjugates that underwent reactivation were slow and only slightly modified by these mutations. 2-PAM was able to reactivate all conjugates except for Rp- CHMP-AChE and Rp- CHMP-F295L. The F295L mutant did not show an enhancement in reactivation rates compared to wildtype for both Sp- and Rp- conjugates. The F297I mutant showed a 8–10 fold faster reactivation rates for the Sp- conjugates while no enhancement was seen with the Rp- conjugates. HI6 proved to be a more potent reactivator. Its maximum rate of enhancement (kmax) of the deacylation step is anywhere from 10–100 fold faster than 2-PAM. HI6 shows a greater enantiomeric selectivity than 2-PAM in reactivation. Kinetic analysis reveals that acyl pocket dimensions play a major role in controlling the reactivation of organophosphonyl conjugates.

Through site-specific mutagenesis, we examined the determinants on acetylcholinesterase which govern the specificity and reactivity of three classes of substrates: enantiomeric alkyl phosphonates, trifluoromethyl acetophenones, and carboxyl esters. By employing cationic and uncharged pairs of enantiomeric alkyl methylphosphonyl thioates of known absolute stereochemistry, we find that an aspartate residue near the gorge entrance (D74) is responsible for the enhanced reactivity of the cationic organophosphonates. Removal of the charge with the mutation D74N causes a near equal reduction in the reaction rate constants for the Rp and Sp enantiomers and exerts a greater influence on the cationic organophosphonates than on the charged trimethylammonio trifluoromethyl acetophenone and acetylthiocholine. This pattern of reactivity suggests that the orientation of the leaving group for both enantiomers is directed toward the gorge exit and in apposition to Asp 74. Replacement of tryptophan 86 with alanine in the choline subsite also diminishes the reaction rates for cationic organophosphonates, although to a lesser extent than with the D74N mutation, while not affecting the reactions with the uncharged compounds. Hence, reaction with cationic OPs depends to a lesser degree on Trp 86 than on Asp 74. Docking of Sp and Rp cycloheptyl methylphosphonyl thiocholines and thioethylates in AChE as models of the reversible complex and transition state using molecular dynamics affords structural insight into the spatial arrangement of the substituents surrounding phosphorus prior to and during reaction. The leaving group of the Rp and Sp enantiomers, regardless of charge, is directed to the gorge exit and toward Asp 74, an orientation unique to tetrahedral ligands.

International audience; The availability of a crystal structure and comparative sequences of the cholinesterases has provided templates suitable for analyzing the molecular bases of specificity of reversible inhibitors, carbamoylating agents and organophosphates. Site-specific mutagenesis enables one to modify the structures of both the binding site and peptide ligand as well as create chimeras reflecting one type of esterase substituted in the template of another. Herein we define the bases for substrate specificity of carboxylesters, the stereospecificity of enantiomeric alkylphosphonates and the selectivity of tricyclic aromatic compounds in the active center of cholinesterase. We also describe the binding loci of the peripheral site and changes in catalytic parameters induced by peripheral site ligands, using the peptide fasciculin.