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Global deconvolution of heterotropic cooperativity in cytochrome P450 3A4

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Title: Global deconvolution of heterotropic cooperativity in cytochrome P450 3A4
Author(s): Frank, Daniel J.
Director of Research: Sligar, Stephen
Doctoral Committee Chair(s): Sligar, Stephen
Doctoral Committee Member(s): Gerlt, John; Clegg, Robert; Fratti, Rutilio
Department / Program: Biochemistry
Discipline: Biochemistry
Degree Granting Institution: University of Illinois at Urbana-Champaign
Degree: Ph.D.
Genre: Dissertation
Subject(s): cooperativity cytochrome P450 3A4 drug-drug interactions
Abstract: Cytochrome P450 3A4 (CYP3A4) plays a central role in xenobiotic metabolism, and is of critical importance to both human health and the pharmaceutical industry. Its ability to interact with multiple molecules of the same substrate, or multiple substrates, leads to complex non-Michaelis kinetics, called “homotropic” and “heterotropic” cooperativity respectively. Significant progress has been made towards understanding the enzyme’s complex kinetics by work in our laboratory to isolate the enzyme in Nanodiscs. This provides a homogenous, monodisperse, native-like environment, where the monomeric enzyme is biophysically characterized in the absence of detergent micellar mixtures or liposomal systems which are reported to lead to enzyme heterogeneity and obfuscate its kinetic behavior. Three distinct observable properties which CYP3A4 displays as a result of its reaction cycle are: heme iron spin state, NADPH oxidation rate, and product formation rate. Measuring these three observables as a function of substrate concentration and simultaneously fitting the data sets to a model results in a global analysis of the enzyme’s properties. It reveals the source of homotropic atypical kinetics is not due to any binding cooperativity between the substrates, but rather differences in magnitude of the functional properties from the various enzyme-substrate complexes in solution. To extend this analysis to better understand heterotropic interactions in the system, we generate an interaction surface based upon the linear combination of two substrates kinetic profiles, which corresponds to the absence of any specific heterotropic interactions between them. By comparing the observed behavior of the mixed substrate system to that of the model, we show how two commonly reported heterotropic substrates of CYP3A4 are actually not cooperative, and the observed cooperativity is due to differences in the amplitudes of the functional properties from the various binding intermediates in the system which give rise to the overall observed behavior of the enzyme.
Issue Date: 2011-05-25
URI: http://hdl.handle.net/2142/24520
Rights Information: Copyright 2011 Daniel Frank
Date Available in IDEALS: 2011-05-25
2013-05-26
Date Deposited: 2011-05
 

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