Natilie A. Hosea

Structural bases for the specificity of cholinesterase catalysis and inhibition.

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.

Analysis of cholinesterase inactivation and reactivation by systematic structural modification and enantiomeric selectivity.

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.

Reactivation of Enantiomeric Organophosphonyl Conjugates of Acetylcholinesterase Mutants, F295L and F297I by Mono- and Bis-Quarternary Oximes

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.