The objective of this application is to define the structural features that determine the substrate specificities of human microsomal cytochrome P450 enzymes. These proteins participate in the metabolism of structurally diverse drugs, environmental toxins and carcinogens, and provide a broad defense against chemical insults. Although these enzymes can metabolize a wide range of compounds, they exhibit distinct substrate profiles. Allelic variation can selectively alter metabolic capacity and limit safe exposure to compounds that are predominantly metabolized by a single P450. Species differences between orthologous enzymes indicate that thorough characterization of the human P450 enzymes will provide the most reliable means for predicting drug metabolism and the risks of chemical exposure. Structural characterization of rabbit 2C5 has provided insights regarding active site architecture and conformational changes that occur with substrate binding. The human 2C enzymes including four allelic variants of 2C9 will be characterized by x-ray diffraction studies. The influence of active site volume and topology on substrate selectivity, regio and stereo-specificity, competitive inhibition, catalytic competence, and drug-drug interactions that activate catalysis will be examined using site directed mutagenesis and panels of analogs for known substrates and inhibitors. The role played by a highly conserved aspartate residue will be analyzed. The extent and substrate dependence of P450 conformational flexibility will also be evaluated to determine dynamic active site characteristics that govern substrate recognition. These analyses will be extended to various family 2 P450 enzymes in order to develop a more comprehensive and coherent picture of P450 catalysis. This information will provide a practical basis for rational drug design and risk assessment. ? ? ?
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