Date of Award
Doctor of Philosophy (PhD)
Michael J. Knapp
Robert M. Weis
Hydroxylase, oxygenase, PHD2, Prolyl
Prolyl Hydroxylase Domain 2 (PHD2) has been identified as a key oxygen sensor in humans along with Factor Inhibiting Hypoxia Inducible Factor (FIH). As such PHD2 and FIH play critical roles in myriad pathways of medical relevance by hydroxylation of their target substrate hypoxia inducible factor (HIF), a transcription factor responsible for the regulation of over 100+ genes. With such critical roles in human physiology the ability to selectively regulate these two enzymes could potentially lead the way for novel therapeutic treatments of a vast array of disease states from cancer to myocardial infarction. We report on three classes of iron chelators which show promise for independent regulation of the HIF hydroxylases. Compounds representing the pyrones/pyridinones, pyridines and catechols were tested and found to have differential impacts on PHD2 and FIH under the same experimental conditions. The mode of inhibition is the result of binding to the active site iron and is supported by UV-visible and electroparamagnetic resonance spectroscopy. PHD2 at the current time does not have a well resolved mechanistic understanding regarding its catalytic cycle and subsequent rate determining steps. We have employed pH, solvent isotope, and X-ray absorption studies in an effort to gain further understanding regarding PHD2's overall mechanism. Our data support that dissociation of an iron(II)-OH2 bond centered about the active site contributes to a portion of the overall rate determining steps in the catalytic reaction of PHD2 that activates oxygen and ends with the production of hydroxylated substrate.
Flagg, Shannon Coates, "Selective Inhibition and Mechanistic Studies of the Human O2 Sensor, Prolyl Hydroxylase Domain 2 (PHD2)" (2011). Open Access Dissertations. Paper 460.