The role of P450-mediated reactions in cellular processes has made the understanding of P450 mechanism a focus of intense research. The knowledge of catalytic mechanisms is an integral part of the elucidation o enzyme specificity, an important topic in areas as diverse as drug design, bioremediation, and cancer risk estimation. Whereas evidence generally supports alternate mechanistic pathways describing Cytochrome P450-mediated substrate oxidation, it is still unclear what really determines the rate of product formation and hence the pathway of oxidation. To address this issue, we propose to employ a model system consisting of human P450 1A2 and various anisole and N,N-dimethylaniline derivatives and to determine the catalytic steps comprising the demethylation of these substrates. The design of this system is intended to simplify the reactions of interest by minimizing the products of oxidation and to enable an analysis of the steps of the catalytic cycle employing a variety of techniques. As a complement to this work, site- directed mutagenesis will be carried out at key regions implicated in alternate oxidative pathways. The resulting mutants will be characterized and analyzed in terms of the appropriate model catalytic mechanisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32GM019808-01
Application #
2767941
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1999-06-15
Project End
Budget Start
1999-01-01
Budget End
1999-12-31
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
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Guengerich, F Peter; Miller, Grover P; Hanna, Imad H et al. (2002) Diversity in the oxidation of substrates by cytochrome P450 2D6: lack of an obligatory role of aspartate 301-substrate electrostatic bonding. Biochemistry 41:11025-34
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Yun, C H; Miller, G P; Guengerich, F P (2000) Rate-determining steps in phenacetin oxidations by human cytochrome P450 1A2 and selected mutants. Biochemistry 39:11319-29