Regulation of carcinogen activation is affected by interaction of cytochrome P450 and cytochrome P450 reductase, as well as by other factors. We propose to define the factors which govern the interaction of reductase and P450 and thereby influence carcinogen activation. The factors include the structural domains of P450 and reductase, i.e. membrane binding domains, substrate binding domains, and the binding domains which interact through complementary charge pairing. We will define these domains through a series of chemical modifications of the proteins and a series of site directed mutagenesis studies. In each aspect, the aim of the study is to specify which amino acid residues in a particular domain govern the interaction of that domain with substrate, membrane, or pair protein and how these residues/domains act in concert to regulate carcinogen activation. In pursuit of these goals, we will define the amino acid residues of P450s responsible for charge pairing with critical carboxyl groups of the reductase. We will define the amino acid residues involved in substrate binding to P450 using a photoaffinity substrate analogue and site directed mutagenesis. We will define the NADPH binding region and the flavin binding regions of P450 reductase. These parameters will be evaluated for their role in overall control of substrate metabolism. For example, we will determine how substrate binding to P450 affects the interaction of P450 with reductase.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA053191-02
Application #
3197994
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1991-07-15
Project End
1995-06-30
Budget Start
1992-07-01
Budget End
1993-06-30
Support Year
2
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Type
Schools of Medicine
DUNS #
City
Houston
State
TX
Country
United States
Zip Code
77225
Cvrk, T; Strobel, H W (2001) Role of THR501 residue in substrate binding and catalytic activity of cytochrome P4501A1. Arch Biochem Biophys 389:31-40
Cvrk, T; Strobel, H W (2001) Role of LYS271 and LYS279 residues in the interaction of cytochrome P4501A1 with NADPH-cytochrome P450 reductase. Arch Biochem Biophys 385:290-300
Cvrk, T; Strobel, H W (1998) Photoaffinity labeling of cytochrome P4501A1 with azidocumene: identification of cumene hydroperoxide binding region. Arch Biochem Biophys 349:95-104
Yu, X C; Strobel, H W (1997) Hydroperoxide-mediated cytochrome P450-dependent 8-anilino-1-naphthalenesulfonic acid destruction, product formation and P450 modification. Mol Cell Biochem 167:159-68
Hodgson, A V; Strobel, H W (1996) Quantitation of FAD-dependent cytochrome P450 reductase activity by photoreduction. Anal Biochem 243:154-7
Yu, X C; Strobel, H W (1996) Interactions of 8-anilino-1-naphthalenesulfonic acid (ANS) and cytochrome P450 2B1: role of ANS as an effector as well as a reporter group. Mol Cell Biochem 162:89-95
Hodgson, A V; Strobel, H W (1996) Characterization of the FAD binding domain of cytochrome P450 reductase. Arch Biochem Biophys 325:99-106
Cvrk, T; Hodek, P; Strobel, H W (1996) Identification and characterization of cytochrome P4501A1 amino acid residues interacting with a radiolabeled photoaffinity diazido-benzphetamine analogue. Arch Biochem Biophys 330:142-52
Yu, X C; Liang, C; Strobel, H W (1996) Kinetics of substrate reaction in the course of hydroperoxide-mediated inactivation of cytochrome P450 1A1. Biochemistry 35:6289-96
Shen, S; Strobel, H W (1995) Functional assessment of specific amino acid residues of cytochrome P4501A1 using anti-peptide antibodies. Arch Biochem Biophys 320:162-9

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