Abstract
Adenosylcobalamin (AdoCbl) serves as a reservoir for the 5’-deoxyadenosylradical, which is generated in enzyme by the homolytic cleavage of a Co-C bond and
harnessed to initiate radical reactions by abstracting a hydrogen from the substrate. How
these enzymes increase the rate of Co-C bond cleavage by an estimated 12 orders of
magnitude, whether the 5’-deoxyadenosyl radical exists as a metastable or transient
intermediate and how the first steps of the reaction are coupled are key unresolved
questions.
The Co-C bond breaking and hydrogen abstraction steps were modelled in AdoCbl
dependent glutamate mutase with MD simulations, adiabatic mapping and umbrella
sampling simulations using a novel empirical valence bond (EVB) potential, which was
calibrated to high level ab initio and DFT calculations. This potential was found to
compare favourably with the results of QM/MM calculations. Hydrogen bonding with the
protein stabilises the dissociated 5’-deoxyadenosyl radical and induce conformational
change, guiding the C5’ radical centre towards the substrate hydrogen to be abstracted.
The heme dioxygenase enzymes Indoleamine 2,3-dioxygenase (IDO) and
tryptophan 2,3-dioxygenase (TDO) catalyse the first step in the metabolism of
L-tryptophan (L-Trp) by insertion of both atoms of heme-bound O2 into the substrate. In
an attempt to improve understanding of the differences in substrate binding and reactivity
between these enzymes, molecular dynamics (MD) simulations, MM/PBSA binding free
energy calculations and reaction modelling with hybrid quantum mechanics/molecular
mechanics (QM/MM) adiabatic mapping calculations were performed. Starting with
crystal structures for a bacterial TDO (XcTDO) and human IDO (hIDO), reactivity and
binding of IDO, TDO and the H55A mutant TDO with L-Trp, D-tryptophan (D-Trp) and
1-methyl-L-tryptophan (1-Me-L-Trp) were investigated.
Differences in experimental KMs were partially rationalised by analysis of
substrate-protein interactions and calculated binding free energies. Although the
calculated barriers were unable to rank correctly the active systems, they were able to
predict whether a particular system was active, slightly active or inactive. Differences in
reactivity were related to the varying ability of the systems to position optimally the
substrate in relation to the heme-bound O2.
| Date of Award | 6 Jan 2015 |
|---|---|
| Original language | English |
| Awarding Institution |
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| Supervisor | Jeremy N Harvey (Supervisor) & Adrian J Mulholland (Supervisor) |