Date of Graduation
Cytochromes P450 comprise a ubiquitous superfamily of heme-thiolate enzymes, considered among the most versatile biological catalysts known. Structure-function relationships of P450s are of particular interest to those attempting to elucidate such aspects of xenobiotic metabolism as substrate selectivity and regiospecific oxidations. We studied two members of the P450 1A subfamily, P450 1A1 and 1A2, found in highest abundance in lung and liver tissues, respectively. These enzymes may have an important link to cancer, as both are known to regiospecifically activate procarcinogenic substrates. To gain a better understanding of structure-function relationships in these enzymes, we applied a combination of site-directed mutagenesis, enzyme kinetics, and molecular modeling techniques to characterize the roles of key active site residues in substrate oxidation by P450 1A1. An amino acid residue at position 382 in both 1A1 and 1A2 enzymes was found to be a determinant of binding and oxidation of 7-alkoxyresorufins. Observations from molecular dynamics (MD) simulations indicated that side chains at position 382 made key contacts with the alkoxy moieties of the alkoxyresorufins, and thereby directly affect the ability of substrates to bind in catalytically productive orientations. This was confirmed in further studies with reciprocal active site mutants of P450 1A1 and 1A2, which demonstrated that only mutations at position 382 shifted substrate specificity from one enzyme to another. In later investigations, we successfully utilized molecular modeling methods to rationalize the observed stereo- and regiospecificities of substrate oxidation by P450 1A1. Enzyme substrates, arachidonic acid (AA), eicosapentaenoic acid (EA), benzo[a]pyrene (B[a]P) and B[ a]P-7,8-diols, were docked into the active site and subjected to molecular dynamics (MD) simulations. We developed an MD-based steric scoring method to evaluate the propensity of potential substrate oxidation sites to assume reactive orientations leading to products. Predicted in silico profiles were in general agreement with measured in vitro metabolite profiles, supporting the contribution of enzyme steric factors toward P450 1A1-mediated stereo- and regiospecific oxidation of substrates. The methodology provided in this study may be applied generally to determining the steric component of regioselective reactions in P450s.
Ericksen, Spencer Snyder, "Cytochrome P450 1A-substrate interactions: Determinants of substrate selectivity and stereo- and regiospecificity of oxidation." (2005). Graduate Theses, Dissertations, and Problem Reports. 8820.