Enzymes are powerful biological catalysts that provide rate accelerations of up to 1019 times the rate of the uncatalysed reaction in water by lowering the activation energy of the reaction. Enzymes also have an extraordinary specificity for reacting substrates, which can distinguish between groups with similar structural and chemical properties. Studies of enzyme structure and mechanism have been motivated by the wish to understand how enzymes exhibit such behaviour. Along with the protein tertiary structure, dynamics have also been shown to be important in determining the function of a given protein. Proteins experience motions on various timescales, from several seconds for large scale domain rearrangements to picosecond and femtosecond side- chain rotations and bond vibrations. However, it is not clear how motions on these different timescales are coupled to one other and much effort has been dedicated to understand how complex internal dynamic motions results in such efficient and highly selective enzyme catalysis. In this study we aim to characterise fast timescale dynamics and mechanistic details of phosphoglycerate kinase (PGK) during its catalytic trajectory. PGK (45 kDa) is an enzyme which generates ATP during the first step of glycolysis and it consists of two domains, an N-domain and a C-domain separated by a central ï¡-helix. It catalyses the reaction between ADP and 1,3-bisphosphoglycerate, generating ATP and 3-phosphoglycerate as products. We used NMR relaxation experiments to quantify backbone dynamics of human PGK and Geobacillus stearothermophilus PGK along their catalytic trajectories. Our results indicate that interdomain communication is present for both PGK homologues upon binding of either substrate. The backbone dynamics of the ligand bound domain are perturbed and are propagated to the neighbouring domain. Furthermore, we utilised X-ray crystallography experiments to understand the mechanistic details of the active site of PGK when bound to native substrates. Catalytically competent crystals of PGK in a closed conformation have been observed that reveal the details of active site interactions.