We propose to continue investigation of the chemical mechanisms of several types of oxidation by cytochrome P-450 enzymes. In each case our approach will be to use substrates specifically designed to probe key aspects of the overall mechanism including both the rate-limiting and product-determining steps. To aid in interpretation of results, reactions of these substrates using non- physiological oxidants (e.g. cumene hydroperoxide, iodosyl benzene) with P-450, non-P-450 peroxidase enzymes, and purely chemical model systems will also be studied. Three major types of probes will be employed, both singly and in concert: 1) manipulation of the one electron redox potential of the substrates, 2) kinetic deuterium isotope effects and stable isotope tracer studies and 3) cyclopropyl groups for detection of odd electron intermediates. The P-450 catalyzed processes we will focus on include 1) benzylic hydroxylation, 2) N-dealkylation of arylamines, 3) olefin epoxidation and 4) heteroatom desarylation. Collectively these studies will address two important contemporary issues regarding P-450 mechanisms. One is the dichotomy between electron-abstraction vs. hydrogen atom abstraction mechanisms as a function of the chemical properties of individual substrates. The other potential involvement of quantum- mechanical tunnelling to account for some of the non-classically large deuterium isotope effects seen with some P-450 reactions involving H- abstraction mechanisms. Since this latter area is a relatively new aspect of enzymology in general, our unique approach to it may have broader significance beyond cytochrome P-450.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031910-07
Application #
3280336
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1985-04-01
Project End
1995-09-29
Budget Start
1992-09-30
Budget End
1993-09-29
Support Year
7
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Kansas Lawrence
Department
Type
Schools of Pharmacy
DUNS #
072933393
City
Lawrence
State
KS
Country
United States
Zip Code
66045
Lu, P; Alterman, M A; Chaurasia, C S et al. (1997) Heme-coordinating analogs of lauric acid as inhibitors of fatty acid omega-hydroxylation. Arch Biochem Biophys 337:1-7
Bambal, R B; Hanzlik, R P (1996) Effects of steric bulk and conformational rigidity on fatty acid omega hydroxylation by a cytochrome P450 4A1 fusion protein. Arch Biochem Biophys 334:59-66
Bambal, R B; Hanzlik, R P (1996) Active site structure and substrate specificity of cytochrome P450 4A1: steric control of ligand approach perpendicular to heme plane. Biochem Biophys Res Commun 219:445-9
Alterman, M A; Chaurasia, C S; Lu, P et al. (1995) Fatty acid discrimination and omega-hydroxylation by cytochrome P450 4A1 and a cytochrome P4504A1/NADPH-P450 reductase fusion protein. Arch Biochem Biophys 320:289-96
Chaurasia, C S; Alterman, M A; Lu, P et al. (1995) Biochemical characterization of lauric acid omega-hydroxylation by a CYP4A1/NADPH-cytochrome P450 reductase fusion protein. Arch Biochem Biophys 317:161-9
Rizk, P N; Hanzlik, R P (1995) Oxidative and non-oxidative metabolism of 4-iodoanisole by rat liver microsomes. Xenobiotica 25:143-50
Alterman, M A; Chaurasia, C S; Lu, P et al. (1995) Heteroatom substitution shifts regioselectivity of lauric acid metabolism from omega-hydroxylation to (omega-1)-oxidation. Biochem Biophys Res Commun 214:1089-94
Riley, P; Hanzlik, R P (1994) Electron transfer in P450 mechanisms. Microsomal metabolism of cyclopropylbenzene and p-cyclopropylanisole. Xenobiotica 24:1-16
Hall, L R; Hanzlik, R P (1991) N-dealkylation of tertiary amides by cytochrome P-450. Xenobiotica 21:1127-38
Hall, L R; Hanzlik, R P (1990) Kinetic deuterium isotope effects on the N-demethylation of tertiary amides by cytochrome P-450. J Biol Chem 265:12349-55

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