Genetic deficiencies in the expression of human cytochromes P450 2C19 or 2D6 each occur in roughly 5-20% of individuals depending on racial background. Such deficiencies can lead to diminished capacities to metabolize drugs and other foreign compounds leading to undesirable side- effects. Additional evidence suggests that allozymic variation for these and related enzymes can selectively affect drug metabolism and that inter-individual variation in the expression of other human 2C enzymes also occurs. It is difficult to predict the affects of these alterations of activity and expression due to limited information regarding the spectrum of substrates metabolized by these enzymes. To enable a more detailed characterization of the human 2C and 2D P450s, we are developing means for the high-level expression of these enzymes in Escherichia coli. Preparations of these enzymes will be used to characterize the enzymic properties and to generate specific antibodies to characterize the expression of the human P450 2C enzymes. Information gleaned from epitope mapping studies will be used to generate specific anti-peptide antibodies and to define topological features of the 2C enzymes. The latter will be compared to models based on the structures of soluble prokaryotic P450s. Empirically based approaches to site-directed mutagenesis will be employed to map determinants of the catalytic specificities of the human 2C and 2D enzymes and to refine computer based models for their active sites as well as pharmacophore based models for their substrate specificity. Efforts will be made to crystallize the mammalian microsomal P450s for X-ray diffraction studies utilizing modifications that render the enzymes soluble while retaining their catalytic properties.
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