Probing structural and functional domains of human P450c17

Human P450c17 performs at least six chemical transformations, but this spec trum of activity is differentially regulated by structural changes and by r edox partner proteins. Furthermore, P450c17 isoforms from different species with similar to 90% amino acid identity exhibit markedly different relativ e rates for these transformations. Although this phenomenology has been rec ognized for nearly 20 years, the underlying chemistry and structural basis for these effects are poorly understood. We have constructed a structural m odel of human P450c17 using computational chemistry to understand informati ve, naturally occurring human mutations and to provide a rational basis for designing alterations in P450c17 that probe functional domains of the prot ein. We have mapped with considerable confidence key residues involved in t he interaction with redox partner proteins, including K89, R347, and R358, which form positive charges on the "proximal" surface of P450c17. Neutraliz ation of these charges selectively impairs 17, 20-lyase activity without la rge reductions in 17 alpha -hydroxylase activity or 17 alpha -hydroxypregne nolone binding. We are now directing our efforts to the identification of k ey residues in the active site that mediate the substrate specificity and c atalytic selectivity of human P450c17.