The Australian sheep blowfly, Lucilia cuprina, has evolved
resistance to organophosphate insecticides via a single point
mutation in a carboxylesterase (E3). In addition to their use as
insecticides, organophosphates have been synthesised as chemical
warfare agents, posing a threat to humans. Thus, an efficient
organophosphate detoxification agent, such as E3, has potential
value as a prophylactic that could break down organophosphates
before they cause intoxication. The work in this thesis aims at
understanding the mechanism of organophosphate catalysis by E3 to
reveal how insecticide resistance evolved and to evaluate whether
E3 could be a useful prophylactic to prevent organophosphate
poisoning.
The mechanism by which the naturally occurring mutant
E3-Gly137Asp catalyses the hydrolysis of organophosphates, as
predicted through quantum-cluster calculations, is presented
here. Whereas the initial phosphorylation of the active site
serine (Ser218) occurs in the mutant the same way as in the
wild-type enzyme, the results presented here suggest that the
enzyme plays two key roles in the second dephosphorylation step.
First, the new Asp137 residue in the active site acts as a
general base in the initial nucleophilic attack of a water
molecule on the phosphorylated serine. Second, the catalytic
histidine residue of E3 (His471) acts as a general acid in the
dephosphorylation step, donating a proton to the departing
phosphodiester. Additionally, the role of the oxyanion in
lowering activation energy barriers was identified. The accuracy
of computational methods for the prediction of turnover rates of
enzymes is assessed. Despite the limitations present in the
methods used, practically useful predictions were achieved.
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The in silico binding of a range of different substrates, both
carboxylester and organophosphate, to E3 is analysed in the light
of experimental data. Predictions are made about possible natural
substrates and the potential uses of E3 to degrade
organophosphate chemical warfare agents. This is additionally
investigated through the use of X-ray crystallographic data.
Molecular dynamics simulations have been used to investigate the
effects of substrate presence and mutations on the sampling of
different rotamers of amino acids within the active site of E3.
The implications of these results on enzyme engineering are
discussed. It is suggested that the E3- Gly137Asp mutation might
be detrimental to the catalytic activity of E3, owing to its
sampling of non-productive conformations. The additional
mutations that have accumulated in a laboratory-evolved mutant of
E3 (Gly137Asp/Lys306Met/Met308Val/Ser470Gly), which has further
enhanced catalytic activity, have apparently reduced the sampling
of non- productive states.
Altogether, this work has utilized a range of computational
techniques, from ligand docking to molecular dynamics simulations
and quantum chemical simulations, to generate several novel
insights into the catalytic mechanism of the recently evolved
organophosphate-degrading E3- Gly137Asp mutant from L. cuprina.
As part of a larger research program, many of these hypotheses
have been tested, and supported, through enzyme kinetics and
protein X-ray crystallography. The new knowledge that has been
gained from this work will hopefully aid in the further
improvement of this enzyme as an organophosphate detoxification
agent and in better understanding how insecticide resistance can
evolve