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Doctor of Philosophy (PhD)
Year Degree Awarded
Month Degree Awarded
Michael J. Knapp
Lila M. Gierasch
Michael J. Maroney
Stephen J. Eyles
Biochemistry | Chemistry | Laboratory and Basic Science Research
inhibiting HIF-1 (FIH-1) modulates the master regulator of hypoxia
sensing, hypoxia inducible factor-1 (HIF-1), by transcriptional repression making it an
attractive potential target for treatment of hypoxia-related diseases. Given that similar
enzymes are present within the cell and that they have other important physiological
roles, defining the therapeutic window by which it can be selectively targeted becomes an
issue. Consequently, it’s necessary to have a deeper understanding of the substrate
interactions in FIH-1 that contributes to catalysis as this is one avenue that can be
explored for future therapeutic investigations. The overall goal of this dissertation is to
gain kinetic and dynamic insights into the substrate interactions and catalysis of FIH-1
that can be used in the future to improve its selective inhibition over the related prolyl
hydroxylase domain enzymes (PHDs). The term dynamic here loosely refers to the
conformational flexibility of the enzyme.
An initial screen of inhibitor compounds that mimic the cosubtrate, αKG, was
performed with the goal of teasing out compound properties that might be useful for
selectively inhibiting FIH-1 over the PHD isoform studied in the lab, PHD2, and vice
versa. Results from steady state kinetic assays supported by EPR spectroscopy showed
that planar compounds are more effective at inhibiting PHD2 than FIH-1. One or two
compounds belonging to the pyrone/pyridinone framework may prove promising for
future development of selective FIH-1 inhibitors.
The succeeding studies were done in an effort to uncover unique substrate
interactions of FIH-1 that might provide further useful information for future selective
inhibition studies. Firstly, the effect of α-ketoglutarate (αKG) binding on the primary
substrate interactions of FIH-1 was tested. Initial observations in the lab, as well as
previous reports on the effect of αKG binding on the stability of similar enzymes, led us
to hypothesize that αKG binding induces conformational changes in FIH-1 which are in
turn required for the optimal binding of the primary substrate HIF-1α/C-terminal
transactivation domain (CTAD). To address this, fluorescence and thermal shift
experiments in addition to global hydrogen/deuterium exchange and limited proteolysis
coupled to mass spectrometry were performed. The data strongly implies a more tightly
folded structure of FIH-1 when bound to αKG. This is corroborated by the observations
that for the ternary enzyme-metal-αKG complex, (1) it is more thermally stable, (2) it is
less prone to solvent and protease attack, and (3) it apparently exhibits higher affinity for
the substrate HIF-1α/CTAD compared to apo or metal-bound form of the enzyme. The
data allowed for the conception of a model in which at least two conformational states of
the enzyme exist in equilibrium depending on the presence of αKG. The more rigid
structure likely primes the enzyme for optimal substrate binding via pre-formation of the
binding site, the configuration of which is tightly coupled to efficient turnover.
Lastly, the role of a protein loop on the substrate interactions and catalysis of
FIH-1 was tested. Based on (1) a previous computational study indicating that a loop
containing residues 100-110 of FIH-1 (which we called the 100s loop) becomes less
flexible in the presence of substrate and (2) an analysis of the crystal structure revealing
the FIH-1 loop residue Tyr102 stacked on top of the substrate target residue Asn803, we
hypothesized that the 100s loop via Tyr102 is involved in substrate binding and turnover.
The results from steady state kinetic assays combined with UV-vis and EPR spectroscopy
as well as succinate quantitation experiments performed on loop mutants indicates that
(1) the contribution of the loop residue Tyr102 to substrate binding is mainly through
steric interaction and that (2) this steric interaction ensures the tight coupling of substrate
binding to turnover via proper positioning of the substrate target residue for subsequent
Martin, Cristina B., "Kinetic and Dynamic Insights into the Substrate Interactions and Catalysis of Factor Inhibiting HIF-1 (FIH-1)" (2016). Doctoral Dissertations. 655.