Modelling the reactivity of glutamate mutase and heme dioxygenase enzymes
von Glehn, Patrick #
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 correctly rank 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 optimally position the substrate in relation to the heme-bound O2.
Adenosylcobalamin (AdoCbl) serves as a reservoir for the 5’-deoxyadenosyl radical, 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 to QM/MM calculations. Hydrogen bonding with the protein was found to stabilise the dissociated 5’-deoxyadenosyl radical and induce conformational change, guiding the C5’ radical centre towards the substrate hydrogen to be abstracted.