Thermodynamics of hinge bending in β-Phosphoglucomutase

This thesis is primarily concerned with the characterisation of domain reorientation and hinge-bending motions in the conformational landscape of β-phosphoglucomutase (βPGM). βPGM comprises two domains connected by a flexible hinge region. The catalytic cycle involves dynamical exchange between an open, inactive conformation – to which the substrate can bind and from which the product is released – and a closed, catalytically competent conformation. X-ray crystallography has been used successfully to describe key structures along the reaction coordinate of βPGM. A multidisciplinary study, combining molecular dynamics computer simulations with small angle x-ray scattering and hydrodynamic measurements, is described to establish the conformational landscape in solution. Several simulation artefacts are described highlighting the importance of recent forcefield optimisations in the study of collective motions in proteins. The experimentally validated MD ensemble is more open than the crystal structures and is stabilised by burial of the Y19 sidechain in a hydrophobic pocket within the hinge region. This mechanism may serve to stabilize the substrate-free, open conformer, facilitating product release. In accordance with the phosphodianion-driven enzyme activation framework, domain closure is stimulated by recruitment of an inert phosphodianion group to the distal site. Using a combination of NMR, x-ray crystallography and steady state kinetics, a communication pathway between the distal phosphodianion binding site and the hinge has been characterised. This involves a hydrogen bonding relationship between a pair of carboxamides (N77 and N118) which in turn modifies the hydrogen bonding relationships within the 70s helix, and the backbone torsions of I84.