The ever-increasing number of mammalian cytochromes P-450 that have been identified, coupled with the known concurrent expression of very closely related forms (having >90% sequence identity) in a single tissue, has complicated efforts to rigorously purify and characterize individual forms of the enzyme. The realization that closely related forms that may copurify can have very different substrate specificities and catalytic activities has necessitated new approaches for the purification and characterization of these enzymes. These considerations, and, perhaps most importantly, the desire to undertake structure-function studies on these cytochromes through site-directed mutagenesis, has prompted my efforts to express these enzymes in heterologous hosts. To this end, the objective of the proposed research is the synthesis and purification of an individual cytochrome P-450 isoform from a heterologous expression system, with the subsequent examination of structure-function relationships through site-directed mutagenesis studies. Recent success in this laboratory with the expression of cytochrome P-450IIE1 in Escherichia coli has laid the groundwork for the proposed studies.
The specific aims of this research are: 1) to develop a purification procedure for cytochrome P-450IIE1 expressed in E. coli, based in part on the established procedure for isolating P-450lIE1 from rabbit liver, that will provide enzyme sufficiently clean for rigorous catalytic and mechanistic studies; 2) to identify the segment or segments responsible for binding the cytochrome to the endoplasmic reticular membrane by using deletion analysis and site-directed mutagenesis; 3) to identify those cytochrome P-450 residues likely to be involved in electron transfer between cytochrome P-450 reductase and the P-450 heme group by using site-directed mutagenesis with catalytic and spectroscopic assays, focusing initially on Trp-1 22 and Phe430.
| Porter, T D (1994) Mutagenesis at a highly conserved phenylalanine in cytochrome P450 2E1 affects heme incorporation and catalytic activity. Biochemistry 33:5942-6 |
