The cytochrome P450 (cyt P450) superfamily consists of more than 11,000 members. They are ubiquitous, being 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 exists in virtually every organ and tissue of humans. The cyts P450 are not self-sufficient but rather require interactions with other proteins in order to function. Cyt P450 reductase and cytochrome b5 (cyt b5), which provide electrons to cyt P450, are two proteins that support the activity of cyt P450. The long-term goal of this project is to understand the structural and mechanistic basis for the regulation of the activity of the membrane-bound microsomal cyts P450 by its redox partners, cyt P450 reductase and cyt b5. The short-term goals of this proposal, using both human and model cyts P450, are to understand the biochemical basis of how the redox partners of cyt P450 regulate its activity, substrate specificity, and catalytic mechanism. Experimental techniques, including site-directed mutagenesis, rapid quenching of cyt P450 activity by chemical means, and freezing, HPLC-mass spectrometry, and quantum mechanical/molecular mechanical calculations will be employed to elucidate how the activity of microsomal cyts P450 is regulated by its redox partners. Understanding how nature designs cyt P450 active sites is a fundamental question with implications for predicting and eventually modifying the routes of metabolism of a large number of environmental contaminants such as phthalates, bisphenol A, polychlorinated biphenyls (PCBs) and many 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 regulated 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 human cyts P450 participate, as well as in designing less toxic and more specific therapeutic agents and prodrugs, especially chemotherapeutic agents and environmental contaminants.
The proposed investigation of how the human enzymes responsible for drug metabolism, drug-drug interactions, blood clotting, and cholesterol levels function at a molecular level will provide new information that will prove to be an asset 1) in developing novel procedures to alter the large number of physiologic processes in which the 56 human cyts P450 participate and 2) in designing and synthesizing less toxic and more specific therapeutic agents and prodrugs, especially chemotherapeutic agents, and "greener" environmental contaminants.
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