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Author ORCID Identifier
Open Access Dissertation
Doctor of Philosophy (PhD)
Year Degree Awarded
Month Degree Awarded
The hypoxic response is a vast and complex system, delicately designed through evolution to allow our tissues to rapidly adapt and survive fluctuations in pO2. HIF1α, the master regulator of oxygen, is tightly controlled through oxygen-dependent, post-translational hydroxylation via PHD2 and FIH1.The aberrant stabilization of HIF1α is an adverse consequence of disease states, but can also occur under normoxia in the presence of ROS or in iron depletion. The extensive transcriptional network regulated by HIF1α makes the HIF pathway an attractive therapeutic target, particularly through the inhibition of the hydroxylases, PHD2 and FIH, however the active site chemistry is mechanistically similar to many enzymes in the Fe(II)/αKG dependent dioxygenase family. We address the mechanistic questions through the direct measurement of the hydrogen atom transfer (HAT) step using isotopically labeled peptide substrate to ascertain the macroscopic and microscopic kinetic isotope effect. Steady state kinetic assays exhibit ix a large KIE on kcat as a result of enzymatic uncoupling, which indicated that the HAT is partially rate limiting. We estimated a large primary KIE on the rate of HAT, providing experimental evidence that FIH follows the canonical mechanism within its family of enzymes. We demonstrated structural dependence of HAT on the steric interaction with nearby tyrosine-102. The steady-state kinetics of Y102X variant series diminished with decreasing bulk, and primary KIE observations provided large values for Y102F and Y102H, suggesting protein dynamics of the Tyr-102 are critical for positioning the substrate to undergo HAT. The cofactor relationship between ascorbate and FIH has previously shown redox sensitivity on enzymatic activity, but direct evidence of oxidative stress on enzyme stability is limited. We proposed an additional pathway to FIH-inactivation by exploring redox sensitivity on enzyme activity, and how its inhibition is accelerated through irreversible enzyme autohydroxylation through the treatment of H2O2. Finally, we begin to explore how HIF1α stabilization can be regulated using ionophores, which can differentially deliver or deplete intracellular iron through the labile iron pool in cells. A methodology for characterizing pseudohypoxic induction through the LIP using hinokitiol can provide a pathway to identifying other iron chelating compounds exhibiting prospective ionophore properties.
Mingroni, Michael A., "PRIMARY KINETIC ISOTOPE EFFECT FOR SUBSTRATE HYDROXYLATION AND IRON BINDING REGULATION IN FACTOR INHIBITING HIF-1" (2021). Doctoral Dissertations. 2256.