The P450-dependent mixed-function oxidases found in almost all mammalian cells play a critical role in the metabolic activation and detoxication of many chemical carcinogens. The primary goals of the proposed research are to gain a better understanding of the structures of the active sites of several different forms of P450, to identify the critical amino acid residues in the P450 active sites involved in catalysis and substrate binding and to gain a better understanding of the mechanisms by which they catalyze the reactions involved in the metabolic activation and detoxication of chemical carcinogens and other toxic agents. In order to achieve these goals, the specific aims of this research proposal are: l. To investigate the mechanism-based inactivation of several purified isozymes of cytochrome P450 by 9-ethynylphenanthrene, to identify the amino acid(s) and peptide(s) at the active site modified during inactivation, and to determine the mechanism by which inactivation occurs; 2. To study the mechanism-based inactivation of P450 by 2- ethynylnaphthalene in order to identify the modified active site polypeptide(s) and amino acid residue(s) and determine the mechanism of inactivation; 3. To determine the structure-activity relationships and enzyme specificities for mechanism-based inactivation of P45Os by a series of polycyclic aromatic acetylenes; 4. To investigate mechanism-based inactivation of P450 by N-methylcarbazole in order to identify the modified amino acid residue(s) at the active site and ascertain the catalytic step(s) blocked by N-methylcarbazole; 5. To characterize P450 inactivation by N-benzyl-l-aminobenzotriazole, to identify the modified active site peptide(s) and amino acid residue(s) and the catalytic step(s) blocked by inactivation; 6. To investigate the mechanism-based inactivation of P450 by n-propylxanthate, to identify the modified active site peptide(s) and amino acid residue(s) and determine the mechanism of inactivation; 7. To investigate the metabolism of hypersensitive radical probes and determine substrate deuterium isotope effects for hydrocarbon hydroxylations by several P45Os in order to gain a better understanding of the mechanism(s) involved in P450-catalyzed hydrocarbon hydroxylations; and 8. To use site-specific mutagenesis of P450 2B1 and 2B2 to identify amino acid residues that play a critical role in substrate binding and catalysis by P450. The identification of critical amino acid residues involved in catalysis and the determination of their specific roles in P450-catalyzed reactions will prove to be extremely valuable for developing approaches for selectively modulating the catalytic activity of these enzymes. The results of these studies could provide information for developing methods to decrease the risk of developing cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
3R01CA016954-24S1
Application #
2879176
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Liu, Yung-Pin
Project Start
1981-09-01
Project End
2001-02-28
Budget Start
1998-03-01
Budget End
1999-02-28
Support Year
24
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Pharmacology
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Lin, Hsia-Lien; Zhang, Haoming; Hollenberg, Paul F (2018) Formation of Both Heme and Apoprotein Adducts Contributes to the Mechanism-Based Inactivation of Human CYP2J2 by 17?-Ethynylestradiol. Drug Metab Dispos 46:813-822
Lin, Hsia-Lien; Zhang, Haoming; Walker, Vyvyca J et al. (2017) Heme Modification Contributes to the Mechanism-Based Inactivation of Human Cytochrome P450 2J2 by Two Terminal Acetylenic Compounds. Drug Metab Dispos 45:990-999
Lin, Hsia-Lien; Zhang, Haoming; Kenaan, Cesar et al. (2016) Roles of Residues F206 and V367 in Human CYP2B6: Effects of Mutations on Androgen Hydroxylation, Mechanism-Based Inactivation, and Reversible Inhibition. Drug Metab Dispos 44:1771-1779
Walker, Vyvyca J; Griffin, Alisha P; Hammar, Dagan K et al. (2016) Metabolism of Anandamide by Human Cytochrome P450 2J2 in the Reconstituted System and Human Intestinal Microsomes. J Pharmacol Exp Ther 357:537-44
Snider, Natasha T; Walker, Vyvyca J; Hollenberg, Paul F (2016) Assay of Endocannabinoid Oxidation by Cytochrome P450. Methods Mol Biol 1412:227-36
D'Agostino, Jaime; Zhang, Haoming; Kenaan, Cesar et al. (2015) Mechanism-Based Inactivation of Human Cytochrome P450 2B6 by Chlorpyrifos. Chem Res Toxicol 28:1484-95
Calinski, Diane M; Zhang, Haoming; Ludeman, Susan et al. (2015) Hydroxylation and N-dechloroethylation of Ifosfamide and deuterated Ifosfamide by the human cytochrome p450s and their commonly occurring polymorphisms. Drug Metab Dispos 43:1084-90
Yoshigae, Yasushi; Sridar, Chitra; Kent, Ute M et al. (2013) The inactivation of human CYP2E1 by phenethyl isothiocyanate, a naturally occurring chemopreventive agent, and its oxidative bioactivation. Drug Metab Dispos 41:858-69
Lin, Hsia-lien; D'Agostino, Jaime; Kenaan, Cesar et al. (2013) The effect of ritonavir on human CYP2B6 catalytic activity: heme modification contributes to the mechanism-based inactivation of CYP2B6 and CYP3A4 by ritonavir. Drug Metab Dispos 41:1813-24
Kenaan, Cesar; Shea, Erin V; Lin, Hsia-lien et al. (2013) Interactions between CYP2E1 and CYP2B4: effects on affinity for NADPH-cytochrome P450 reductase and substrate metabolism. Drug Metab Dispos 41:101-10

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