Regulation studies on human pyruvate kinases

Human pyruvate kinase performs the last step in glucose glycolysis in all cells and organisms and can be a key regulator of glycolytic flux. Pyruvate produced by PYK is transported into the mitochondria to fuel the TCA cycle, which enables the production of ATP; the main energy source of the cell. Human PYK contains four isoforms: M1 (found in muscle, heart and brain), M2 (in foetal cells and tumours), L (liver), and R (red blood cells) PYK. M2PYK plays a crucial role in tumour cell proliferation; by down-regulating metabolic flux, upstream metabolites can be used for protein and DNA synthesis. Reprogramming the metabolism of fast proliferating cells is called the ‘Warburg effect’. The biological relevance of the different isoform activities is also discussed. For example RPYK in red blood cells is exposed to slowly altering metabolite concentrations, especially after intestinal absorption in plasma and RBCs uptake some of the metabolites. This thesis describes biochemical and biophysical studies of human M1PYK, M2PYK, LPYK, and RPYK. PYK is allosterically regulated by a range of metabolites. A comparative enzyme kinetics study of the four isoforms was performed to examine the mechanisms of activation and inhibition of these small molecule regulators, including all 20 amino acids and the thyroid hormone T3. The redox state of the environment was also found to be an important regulator of PYK activity. All four PYK isoforms were successfully expressed and purified. Interestingly, only M2PYK and RPYK were strongly regulated by amino acids and metabolites. We also found that the redox state regulates the activity of all four PYK isoforms as well as the sensitivity of M2PYK in response to natural regulators. These studies also confirmed the dissociation of tetrameric PYK into inactive monomers as an important mechanism of regulation, particularly for M2PYK activity. Nuclear magnetic resonance (NMR) and Small-angle X-ray scattering (SAXS) studies were performed to investigate the conformational behaviour of PYK isoforms in solution and to compare the effects of ligand binding. NMR data of all four isoforms reveal a conserved binding mechanism between isoforms and specific amino acids. SAXS data of all four isoforms demonstrate that ligands affect tetramerisation of PYK isoforms