The cytochromes P450 (cyts P450) are a ubiquitous superfamily of oxidases whose activity is influenced by a second protein, cytochrome b5 (cyt b5). The long-term goal of this project is to understand the structural and mechanistic basis of the modification of human cyt P450-mediated metabolism by cyt b5 in order to develop and employ strategies to modify the activity of these essential isozymes for human benefit. In order to understand the molecular mechanism by which cyt b5 influences oxidation by cyt P450 in vivo, one must understand the details of how cyt b5 interacts with cyt P450 and how it modifies the catalytic cycle of cyt P450 relative to cyt P450 reductase. There are more than 8,000 members of the cyts P450 superfamily. They are found in all kingdoms of living organisms and plants and are referred to as Mother Nature's blowtorch, due to their ability to oxidize a vast number of stable chemical entities. Humans possess 56 different cyts P450, many of which are essential for early development and life itself. Other human cyts P450 determine the toxicity, duration of action, and elimination of the vast majority of therapeutic agents, carcinogens, and environmental agents to which humans are exposed. Xenobiotic metabolizing cyts P450 are also responsible for the majority of drug-drug interactions and adverse drug reactions. A third group of cyts P450 are responsible for the biosynthesis or metabolism of essential endogenous compounds. This includes virtually all steroids (cholesterol, bile acids, estrogens, testosterone, cortisol, and vitamin D) and many lipids and eicosanoids. Cyts P450 exist in virtually every organ and tissue of humans. A second protein, cyt b5, modulates the biochemical mechanism and activity of selected cyts P450 in humans as well as animals. In fact, cyt b5 is essential for testosterone biosynthesis by a cyt P450 that is currently a therapeutic target for the treatment of prostate cancer. The knowledge gained from the proposed experiments will serve as the foundation for understanding the in vivo regulation of the catalytic mechanism of cyt P450. Although the cyt P450 interaction with cyt b5 has been intensely studied, significant gaps in our knowledge exist. The findings of the planned biochemical and structural experiments will also significantly enhance our ability to predict and eventually modify the routes of metabolism of a large number of environmental contaminants such as phthalates, bisphenol A, polychlorinated biphenyl (PCBs), and a vast number of currently used drugs, including chemotherapeutic agents, psychoactive compounds, and cardiovascular therapies. Knowledge of the molecular mechanism by which the activity of human cyts P450 can be modulated will also prove to be a tremendous asset in developing drugs and procedures to alter the large number of critical physiologic processes in which the 56 human cyts P450 participate. The results of the proposed studies will prove to be extremely valuable in designing less toxic and more specific therapeutic agents and prodrugs, especially chemotherapeutic agents and environmental contaminants.
The proposed studies on how the activity of three of the most important human drug metabolizing cytochromes P450 is affected by a second protein, cytochrome b5, will provide significant new information, not readily available by any others means, about how the activity of human cytochromes P450 is regulated. The proposed studies will also provide a better understanding of the structure and function of, not only drug metabolizing cytochromes P450, but also of cytochromes P450 that are important in the metabolism of endogenous compounds such as testosterone, estrogens, and eicosanoids. This information about the chemical reactivity of human cyts P450 in the presence of its different redox partners will facilitate the design and development of new drugs and enhance the ability of investigators to genetically engineer cyts P450 in bacteria and other organisms to produce compounds to enhance human welfare.
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