CYTOCHROME P450 SUBSTRATE SITING AND MOTION

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Rationalization and prediction of hydroxylation products of cytochrome P450 is crucial for understanding the mechanism of this enzyme and for understanding drug metabolism, but remains generally enigmatic. We propose studies of orientation of substrates in the enzyme pocket, and motion of substrates in the enzyme pocket, by new magnetic resonance methods. A longstanding hypothesis states that, counter to typical enzymological dogma, many substrates bound to the active site of (mammalian or prokaryotic) cytochrome P450 rotate rapidly on the timescale of enzymatic turnover; this putative motion might actually be reasonable and even needed for turnover, since the substrate pocket appears to be rather hydrophobic and chemically "featureless". If this motion does indeed occur, it would have important implications for understanding hydroxylation preferences. We have performed preliminary studies of substrate siting and motion for adamantane bound to the resting state of CP450cam, using deuterium magic angle spinning (MAS) SSNMR spectroscopy. These preliminary data support the hypothesis of extensive motion. On this preliminary basis we propose to conduct more detailed studies with medicinally relevant systems. In this preliminary study, competitive displacement and Curie-law temperature dependence of isotropic shifts were used to verify location at the active site. Simulation of deuterium spinning sideband intensities allowed us to determine the strengths of the electron-nuclear dipolar hyperfine coupling, the strength of the effective deuterium quadrupolar splitting (which serves as measure of local motion) and the Euler angles describing the mutual orientation of the dipolar and quadrupolar tensors. Simulations of analogous data from model compounds are reported in a recent publication; RMS agreements of approximately 5 percent are possible, and the simulations are used to determine distances to a precision of 0.5 Angstrom units up to a maximum "capture radius" of 7 Angstrom units, and angles were determined to within 20 degrees. Simulations of the data for enzyme-bound adamantane indicated an average metal-deuterium distance of 6.0 (+/- 0.2) Angstrom units and clear-cut evidence of a rapid high- symmetry motion. Computational and experimental improvements for analysis of the line-shape and spinning side-band intensities are proposed. We propose to characterize substrate motion for additional substrates (camphor, benzene, toluene, xylene, nicotine and warfarin), and an additional stable intermediate of the enzymatic cycle. Substrate geometry relative to the heme and substrate motion will be studies for the mammalian P450 enzyme using a membrane-mimic environment (DMPC/DHPC bicelles); information about substrate geometries is unavailable from crystallographic studies at present. Studies involving microbial detoxification enzymes with similar chemical mechanisms are also planned.