ASPARTATE-74 AS A PRIMARY DETERMINANT IN ACETYLCHOLINESTERASE GOVERNING SPECIFICITY TO CATIONIC ORGANOPHOSPHONATES

Through site-specific mutagenesis, we examined the determinants on ace tylcholinesterase which govern the specificity and reactivity of three classes of substrates: enantiomeric alkyl phosphonates, trifluorometh yl acetophenones, and carboxyl esters. By employing cationic and uncha rged pairs of enantiomeric alkyl methylphosphonyl thioates of known ab solute stereochemistry, we find that an aspartate residue near the gor ge entrance (D74) is responsible for the enhanced reactivity of the ca tionic organophosphonates. Removal of the charge with the mutation D74 N causes a near equal reduction in the reaction rate constants for the R(p) and S-p enantiomers and exerts a greater influence on the cation ic organophosphonates than on the charged trimethylammonio trifluorome thyl acetophenone and acetylthiocholine. This pattern of reactivity su ggests that the orientation of the leaving group for both enantiomers is directed toward the gorge exit and in apposition to Asp 74. Replace ment of tryptophan 86 with alanine in the choline subsite also diminis hes the reaction rates for cationic organophosphonates, although to a lesser extent than with the D74N mutation, while not affecting the rea ctions with the uncharged compounds. Hence, reaction with cationic OPs depends to a lesser degree on Trp 86 than on Asp 74. Docking of S, an d R(p) cycloheptyl methylphosphonyl thiocholines and thioethylates in AChE as models of the reversible complex and transition state using mo lecular dynamics affords structural insight into the spatial arrangeme nt of the substituents surrounding phosphorus prior to and during reac tion. The leaving group of the R, and S, enantiomers, regardless of ch arge, is directed to the gorge exit and toward Asp 74, an orientation unique to tetrahedral ligands.