| Previous studies found that acetylcholinesterase (AChE) was stereoselectivity inhibited by isomalathion, a product of thermal/photochemical isomerization of the widely used organophosphorus (OP) pesticide malathion. It was demonstrated kinetically that (1R)- and (1 S)-stereoisomers inactivate the enzyme by different mechanisms. The goal of this dissertation was to test the hypothesis that inactivation of AChE and the structurally related serine hydrolases butyrylcholinesterase (BChE) and cholesterol esterase (CholE), proceeds by different mechanisms for the (1R)- versus (1S)-stereoisomers of isomalathion. Specific aims, involving the use of kinetics, mass spectrometry (MS) and molecular modeling, were devised and completed to address the hypothesis.; AChE, BChE and CholE were stereoselectively inhibited by isomalathion with the following order of potency: (1R,3R) > (1R,3S) ≥ (1S,3 R) > (1S,3S). Enzymes inactivated by (1R)-isomalathions reactivated readily, but those modified by (1S)-isomers were refractory toward reactivation.; Using a peptide mass mapping approach with matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF-MS) and post-source decay analysis, it was demonstrated that the reaction of electric eel AChE with the (1S,3 S)-stereoisomer proceeds with loss of thiomethyl and subsequent expulsion of diethyl thiosuccinate to yield an aged enzyme. Analysis of equine BChE peptides using the same MS-based technique revealed that inhibition of this enzyme by (1R)-isomalathion occurs with loss of diethyl thiosuccinate, but inactivation of equine BChE by (1S)-isomers proceeds with loss of thiomethyl followed by rapid aging.; Results of molecular modeling experiments of (1R)-isomalathions in the Torpedo AChE active site predicted that diethyl thiosuccinate was positioned as the primary leaving group for (1R)-isomers. In contrast, (1S)-isomalathions were oriented with thiomethyl as the primary leaving moiety.; These results demonstrate that (1R)- and (1 S)-stereoisomers inactivate AChE, BChE and CholE with a difference in primary leaving groups. These data also indicate that the aging pathway previously thought to occur via an SN1 process, proceeds via an SN2 reaction with scission of a P–S bond for AChE and BChE modified by isomalathion isomers.; Future work would involve the use of isomalathion analogs and H 218O to test the mechanisms postulated in this thesis or development of MS-based approaches to detect biomarkers of exposure to OP compounds. |
