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Nuclear magnetic resonance spectroscopy and enzyme kinetic investigation of acetylcholinesterase inhibition by organophosphates and reactivation by oximes

Perkins, Joe (2019) Nuclear magnetic resonance spectroscopy and enzyme kinetic investigation of acetylcholinesterase inhibition by organophosphates and reactivation by oximes. Doctor of Philosophy (PhD) thesis, University of Kent,. (KAR id:81231)

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Abstract

Organophosphates (OPs) are a class of compounds that have been predominantly used as insecticides but also as chemical warfare agents. They covalently inhibit the enzyme acetylcholinesterase (AChE) which plays a key role in terminating muscle contractions. Due to the toxic nature of these compounds, methods are needed that can assess the threat posed by new OPs and inform on the effectiveness of medical countermeasures. In this project the recombinant expression and purification of a mutated isoform of AChE (AChE-d) was optimised to allow in vitro kinetic assays to be carried out on OP inhibition of AChE-d and the reactivation of the OP-AChE-d conjugates by oximes. The isotopic labelling of AChE-d was optimised resulting in 15N uniformly labelled AChE-d being produced. Heteronuclear 2D NMR spectra were collected of labelled AChE-d inhibited by OPs. This data was analysed along with the reactivation data to determin if OPs with similar reactivation rates caused similar peaks to shift. Saturation transfer difference (STD) NMR spectra were collected on an AChE-d mutant that could not be phosphorylated by OPs. The inhibition rates of OPs did not correlate with their toxicity. The reactivation data for AChE-d differed from data collected using human AChE. The NMR spectra of OP inhibited AChE-d indicated that reactivation resistant OPs did cause similar peak shifts but further investigation is needed to confirm this due to the low resolution/signal of the NMR spectra. STD data indicated that phosphorylation was the rate limiting step in the inhibition reaction of an OP with AChE-d.

Item Type: Thesis (Doctor of Philosophy (PhD))
Thesis advisor: Robinson, Gary
Thesis advisor: Williamson, Richard
Subjects: Q Science
Divisions: Divisions > Division of Natural Sciences > Biosciences
Funders: Organisations -1 not found.
SWORD Depositor: System Moodle
Depositing User: System Moodle
Date Deposited: 14 May 2020 12:10 UTC
Last Modified: 19 Dec 2022 19:35 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/81231 (The current URI for this page, for reference purposes)

University of Kent Author Information

Perkins, Joe.

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