Defense Date

2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biochemistry

First Advisor

John C Hackett

Abstract

The Cytochromes P450 superfamily are ubiquitous throughout life and are present in some viruses. Humans encode for 57 of these enzymes that are involved in the metabolism of ~90% of all chemicals. These membrane-bound monooxygenase enzymes utilize a heme protoporphyrin at their active site to activate molecular oxygen and oxidize their substrates through an intermediate deemed nature's most powerful oxidizer. They are a powerful system at our disposal yet understanding of their structure and ligand binding mechanisms in the presence of a lipid bilayer remains poorly understood. The focus of this work is on Cytochrome P450 3A4 (CYP3A4). We set out with the intention of establishing a monodisperse membrane-bound CYP3A4 system. Using Small-Angle X-ray and Neutron Scattering (SAXS/SANS) we systematically investigated the effect that anionic lipid had on the membrane mimetic known as Nanodiscs. Nanodiscs resemble a small, elliptical patch of lipid bilayer circumscribed by a membrane scaffold protein that allows precise control over the lipid composition. We found that incorporation of anionic lipid POPG aides in maintaining a monodisperse system. We inserted CYP3A4 into these nanodiscs and characterized monodisperse CYP3A4 in solution by SAXS/SANS. CYP3A4 has a large active site that exhibits a plasticity that gives rise to its infamous ligand binding promiscuity. An enzyme at the heart of the detoxification process that binds and metabolizes so many different chemical structures must have a highly coordinated and efficient binding mechanism. More than a century-old debate of whether conformational selection or induced fit dominates the binding mechanism landscape still remains unresolved. We aimed to elucidate how monodisperse and membrane-bound CYP3A4 binds three ligands: azamulin, retapamulin and mibrefadil by using UV-vis equilibrium binding titrations, stopped-flow kinetics and global fitting of the kinetic rate constants. Our findings are strongly supportive of the presence of an induced fit mechanism which states that the enzyme undergoes a conformational change following ligand binding. We were fortunate to directly observe this mechanism in the binding of mibrefadil through a spectroscopically observable intermediate. Unequivocal evidence for the presence an IF binding mechanism in CYP3A4 creates fertile ground to explore whether IF is associated with the xenobiotic P450s.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

8-12-2021

Available for download on Tuesday, August 11, 2026

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