Date of Award
12-2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Genetics and Biochemistry
Committee Chair/Advisor
Cheryl Ingram-Smith
Committee Member
Lukasz Kozubowski
Committee Member
Kerry Smith
Committee Member
Lesly Temesvari
Abstract
Entamoeba histolytica is a water- and food-borne intestinal parasite that causes amoebiasis and liver abscess in ~100 million people each year leading to ~100,000 deaths. This amitochondriate parasite lacks many metabolic pathways including the tricarboxylic acid cycle and oxidative phosphorylation, and cannot synthesize purines, pyrimidines, or most amino acids. As a result, E. histolytica is presumed to rely on its modified pyrophosphate (PPi)-dependent glycolytic pathway for ATP production during growth on glucose. This pathway relies on a PPi-dependent rather than ATP-dependent phosphofructokinase (PFK) and thus has a net production of three ATP per glucose. However, in addition to the one PPi-dependent PFK, the E. histolytica genome encodes three putative ATP-dependent PFKs, (designated as EhPFK1, EhPFK2, and EhPFK3). I have recombinantly produced and purified EhPFK2 and EhPFK3 to analyze their enzymatic activities and regulation. Both enzymes displayed cooperative kinetics instead of Michaelis-Menten kinetics with respect to the two substrates fructose 6-phosphate (F6P) and ATP. Kinetic analysis showed that EhPFK2 is the more efficient enzyme compared to EhPFK3. Various ligands such as AMP that have been shown to regulate PFKs in other organisms have been tested to analyze their effects on E. histolytica PFK activities. Specifically, I identified phosphoenolpyruvate (PEP), PPi, and citrate as inhibitors, with PEP being the most potent, and CoA is a potent activator, differentiating EhPFK2 and EhPFK3 from the canonical PFK. I have shown experimentally and through structural model predictions that PEP, PPi, and citrate each bind at different allosteric sites. In addition, these inhibitors had different effects with respect to F6P and ATP substrate binding. The gene encoding PPi-dependent PFK is highly expressed during standard trophozoite growth in E. histolytica as well as in the reptile pathogen Entamoeba invadens. RNAseq studies in E. invadens indicate that one of its two genes encoding putative ATP-dependent PFK is strongly upregulated during excystation. The differences in enzymatic activity and regulation suggest that the four PFKs play different metabolic roles.
Recommended Citation
Cho, Jin, "Biochemical and Kinetic Analysis of Phosphofructokinase in the Eukaryotic Human Pathogen Entamoeba histolytica" (2023). All Dissertations. 3468.
https://open.clemson.edu/all_dissertations/3468