A major aim of the present application is to understand how the multi-substrate specificity of Class IIC cytochrome P450 enzymes is encoded in their primary and tertiary structures. The genetic diversity of the cytochrome P450 monooxygenases provides a critical defense against lipid soluble, toxic foreign compounds (xenobiotics) as well as an essential role in the metabolism of endogenous substrates such as steroids. Most hepatic P450s exhibit a distinct capacity to metabolize several different xenobiotics. Genetic studies indicate that P450IIC5 is unique among hepatic P450s, including those with highly similar amino acid sequences, in its catalysis of the 21-hydroxylation of progesterone. Yet, like several other hepatic P450s, it metabolizes carcinogens such as 2-acetylaminofluorene and benzo(a)pyrene. In order to identify segments of the primary structures which determine this multi-substrate specificity, we will identify segments of the amino acid sequence of P450IIC5, which when substituted into other class IIC P450s, generates efficient progesterone 21-hydroxylases. Reciprocal constructs will be tested to identify whether the substrate specificities of the other enzymes are conferred to IIC5. We will first express hybrid enzymes from chimeric cDNAs to localize these segments, and then employ oligonucleotide-directed mutagenesis to make single substitutions. This approach will define regions of linear sequence that determine substrate selectivity. In order to understand how these segments interact, we will obtain information on the topological organization of class IIC enzymes by mapping epitopes recognized by monoclonal antibodies prepared previously to class IIC P450s. Differences of amino acid sequence which disrupt binding will be mapped in chimeric enzymes. We anticipate identifying complex epitopes formed from the juxtaposition of distal segments of the amino acid sequence in the 3-dimensional structure of the enzyme. The results of epitope mapping studies and of identification of determinants of substrate selectivity will be modeled by computer on the basis of the 3-dimensional structure of a homologous enzyme, P450 cam. This model should be applicable to other microsomal P450s. Another major aim is the identification of how P450 enzymes become autoantigens in autoimmune chronic active hepatitis. We have previously shown that a polymorphic, human P450, IID6, is recognized by autoantibodies associated with this disease. We will map the epitope(s) recognized by autoantibodies to P450 IID6 in order to identify similarities with potential exogenous antigens. The potential association of a specific IID6 allele with the generation of the autoantibodies to P4501ID6 in this human liver disease will also be investigated. Defective alleles of this gene occur with a high frequency. If an association is established, it would more clearly identify the relation of IID6 to the autoimmune disease, and the characterization of the allele could provide mechanistic information on how autoantibodies arise to P450 IID6. These studies could also identify mutant alleles that contribute to deficiencies of human drug-metabolizing capacity.
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