Human Steroid Metabolism by Cytochrome P450

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DESCRIPTION (provided by applicant): The cytochrome P450s represent one of the most prolific families of metalloproteins, with over 18,000 identified genes across the spectrum of life forms, being represented in all of the major branches in the evolutionary "tree-of-life". The function of these heme proteins can be classified into two major divisions. Operating in a catabolic role, the enzymes can functionalize un-activated chemical positions on a substrate, initiating degradation to provide fundamental foodstuffs or as a means of xenobiotic detoxification. Alternately, the same chemical transformations are used in anabolic pathways that play critical roles in the production of cellular signaling molecules such as the steroid hormones. In humans, steroid hormones are generated by P450 action on cholesterol in the ovaries, adrenals and testes. Due to the critical role of cytochrome P450 in human health, these enzyme systems have occupied NIH supported investigators for many decades. Despite this intense research effort, important mechanistic details remain unresolved. The major focus of our proposed work involves the cytochrome P450s (CYPs) operating in human steroid biosynthesis. Each enzyme under investigation sits at the critical branch point in the pathway that generates an important class of hormones. As such, these enzymes are important targets for pharmaceutical intervention in human disease. For example, P450 CYP17A1 is needed for the formation of both androgens and estrogens; mutations are associated with sex hormone deficiencies and human hypertension due to excess mineralocorticoid levels and patient variants are linked to an increase in prostate cancer risk. Inhibitors of this enzyme have recently reached the market as treatment for prostate cancer in men and androgen excess in women. CYP19A1 inhibitors are now the first line therapy in the treatment of estrogen-responsive breast cancer in post-menopausal women. Decreased activity of CYP19A1 has been suggested to play a role in polycystic ovary syndrome, which affects 10% to 20% of all women of childbearing age and leads to an increased risk of cardiovascular disease and Type II diabetes. Documenting the existence, electronic structure, stability and reactivity of P450 heme-oxygen intermediates is a prerequisite for developing therapeutics that can selectively modulate these steroidal biosynthetic processes in the treatment of human disease. Through the application of a broad array of biochemical and biophysical methods, applied in an integrated problem oriented research plan, we seek important new insights into the detailed catalytic mechanisms of the cytochromes CYP17A1 and CYP19A1.