Dissertation
Mechanistic evaluation of N-dealkylation by cytochrome P450 using N, N-dimethylaniline N-oxides and kinetic isotope effects
Washington State University
Doctor of Philosophy (PhD), Washington State University
12/2009
DOI:
https://doi.org/10.7273/000005899
Abstract
Cytochrome P450 enzymes (P450) are a large family of heme-containing monooxygenases found throughout nature. Five isoforms in humans have gained the attention of the pharmaceutical industry due to their responsibility for the oxidation of the majority of pharmaceutical compounds. Due to unconstrained active sites which permit multiple orientations of a given substrate and an arsenal of oxidations attributed to the enzyme, the drug-metabolizing P450s are promiscuous enzymes. Development of predictive models for the oxidation of a given compound by P450 is a major goal for drug development. Since a drug may be oxidized in several ways, prediction of P450-mediated metabolism requires understanding the mechanisms of the possible oxidations that can be performed. This work is focused on evaluating the mechanism of N-dealkylation by P450s using N-oxides as competent models of the native P450 oxidant. N-dealkylation is a common form of metabolism of drug compounds. P450s frequently opt for N-dealkylation over other potential oxidations due to its apparent ease. Recent work has argued a hydrogen atom abstraction mechanism contingent upon a heme-centered iron-oxene, termed Cmpd I. In this work, we used anilinic N-oxides as surrogate oxygen donors in attempts to directly form Cmpd I. The ability to donate only a single oxygen atom and N-dealkylation rate measurements permitted us to exclude formation of other oxidants in favor of Cmpd I. Further, products formed from the mechanistic probe N-cyclopropyl-N-methylaniline excluded a single electron transfer method. When considered together with previous observations of similar KIEs for the native and N-oxide-supported systems, we have concluded that both systems follow a hydrogen atom transfer mechanism originating from a Cmpd I species. We have also determined KIE profiles in an active site mutant implicated in switching P450 from Cmpd I to a "second oxidant." These profiles support contributions of this second oxidant to N-dealkylation in these mutants, suggesting a role in oxidations performed by P450
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Details
- Title
- Mechanistic evaluation of N-dealkylation by cytochrome P450 using N, N-dimethylaniline N-oxides and kinetic isotope effects
- Creators
- Kenneth M. Roberts
- Contributors
- Jeffrey P. Jones (Chair)William B Davis (Committee Member) - Washington State University, School of Molecular BiosciencesLisa M Gloss (Committee Member)ChulHee Kang (Committee Member) - Washington State University, Department of Chemistry
- Awarding Institution
- Washington State University
- Academic Unit
- School of Molecular Biosciences
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Publisher
- Washington State University
- Number of pages
- 146
- Identifiers
- 99901055132001842
- Language
- English
- Resource Type
- Dissertation