• Exploring the B. hominis TPI-GAPDH fusion enzyme glycolytic activity and stability

      McNerney, Daniel (University of Bedfordshire, 2018-12-17)
      Protist parasites cause deadly diseases and huge financial losses to crops. Eukaryotic organisms share similar cell structure and function. This makes novel drug targets difficult to find, and treating protist diseases very challenging. Studies have demonstrated a novel mitochondrial targeted glycolytically active TPI-GAPDH fusion enzyme as a potential drug target. This enzyme has been located in a variety of pathogenic Stramenopiles, including B. hominis and P. infestans. Additionally, the mitochondrial targeting of the enzyme suggests a retention of glycolysis originating from the mitochondrial precursor endosymbiont. This study explores the glycolytic function and enzyme stability of the TPI-GAPDH fusion protein when compared to its segregated subunits. Enzymatic assays demonstrated that GAPDH functions more effectively as a part of the intact fusion enzyme, and that GAPDH may compete for GAP with TPI. This is contrary to the canonical understanding that TPI is a perfect diffusion rate-controlled enzyme that is not rate-limiting. Additional assay data suggests that L-GAP may interact with GAPDH as a competitive inhibitor of glycolytic function. Higher initial rates of enzymatic activity were observed by GAPDH and TPI-GAPDH when isomerically pure D-GAP was used as substrate compared to DL-GAP. Lastly, thermal shift data suggests that the TPIGAPDH fusion enzyme may be far less stable than its subunits. TPI may have a destabilising effect on the GAPDH moiety of the fusion enzyme resulting in dissociation at approximately 25°C lower temperature than GAPDH by itself. Incubation of GAPDH and TPI subunits appeared to stabilise TPI and show potential evidence of protein-protein binding.