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Document Type

Campus-Only Access for Five (5) Years

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

Year Degree Awarded

2016

Month Degree Awarded

May

First Advisor

Michael J. Knapp

Second Advisor

Lila M. Gierasch

Third Advisor

Michael J. Maroney

Fourth Advisor

Stephen J. Eyles

Subject Categories

Biochemistry | Chemistry | Laboratory and Basic Science Research

Abstract

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

hydroxylation.

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