The overall goal of this project is the elucidation of structure-function relationships for the mammalian cytochrome P450s. Such information will improve our understanding of the role of P450s in the metabolism of endogenous and environmental chemicals, and aid the development of specific P450 inhibitors. The research is focused on 1) structure of individual P450s, 2) interactions between P450s and other membrane proteins and 3) P450-substrate interactions. 1) A membrane topography study involving P450 forms 1A1, 2B1 and 2E1 revealed a common proteoly- tically sensitive region which corresponds to a predicted turn on the P450 surface. This finding is consistent with the concept of a common tertiary structure for different classes of mammalian P450s. Substrate binding experiments with the cleaved P450s showed that the structural integrity of this region is critical for binding of P450 2B1 to benzphetamine, but not for benzo[a]pyrene binding to P450 1A1. We are utilizing experimental data in conjunction with computer-assisted multiple sequence alignment and molecular modeling to develop a tertiary structure model of mammalian P450. 2) Our previous studies on quaternary structure of P450s have been extended to examine the specificity of the P450-cytochrome b5 interaction. Immuno-purification of P450 revealed binding of cytochrome b5 to P450 forms 2B1 and 2E1, but not 1A1. Multiple sequence alignment of P450s suggested several basic amino acids which may be important in binding to cytochrome b5. P450-P450 interactions were examined by using benzo[a]pyrene binding to P450 1A1 as an active site structural probe. While monoclonal antibody 1-7-1 to this P450 had no effect on binding, antibody-mediated cross-linking of P450s reduced binding. This result indicates that P450 activities may be modulated by quaternary interactions of P450s. 3) A monoclonal antibody to P450 2B1 was used to define the interaction of benzphetamine with this P450 in rat liver microsomes, in the presence of multiple P450s. Thermodynamic parameters for this reaction and membrane fluidity measurements both revealed a membrane phase transition at 20 degrees C. The data shows the influence of membrane structure on P450-substrate interactions.
