The Role of Enzyme Dynamics in Catalysis by β-Phosphoglucomutase

Abstract

This thesis primarily concerns the use of nuclear magnetic resonance (NMR) spectroscopy to study enzyme dynamics. Recent improvements in NMR and other biophysical methods have allowed the detailed study of protein dynamics, and have led to much speculation as to their involvement in the catalytic prowess of enzymes. β-Phosphoglucomutase (βPGM) is a phosphoryl transfer enzyme, and must therefore bring one of the slowest chemical reactions in nature onto a biologically-relevant timescale. As well as being exceptionally proficient catalysts, phosphate transfer enzymes are useful objects of study through the use of metal fluorides as ground-state and transition-state analogues which allow for the capture of conformations which relate to various stages along the reaction coordinate. Together then, βPGM and metal fluorides allow for a critical analysis of the role of enzyme dynamics in catalysis.

Formation of the transition state analogue (TSA) complex of βPGM captured by using MgF3- ions to mimic the transferring phosphate allows the study of both protein & TSA NMR signals. It is demonstrated that there are two conformational exchange processes occurring on the catalytic timescale: one which is coincident in rate to catalysis and therefore likely plays a role in catalytic turnover by βPGM, and another which is implicated in the folding stability of the protein. These processes are characterised in detail, and attempts to perturb them by mutation are made. The role of dynamics occurring on a faster timescale in βPGM is also explored.

It is concluded that in βPGM and all other enzymes, dynamics on the timescales most amenable to study by NMR (ms-μs and ns-ps timescales) are not directly involved in the chemical step. Instead they may be involved in allowing the enzyme to traverse the complex energy landscape from substrate to product, a role of equal importance.