Cytochrome P450 (P450) enzymes are the major catalysts involved in the metabolism of carcinogens, drugs, and steroids. Variations in the catalytic activities have a variety of effects in homeostasis and clinical practice. Continued studies on human P450s are proposed, with a focus on molecular understanding of function. (1) Of the 57 human P450 genes, 13 still have limited if any knowledge regarding function. We propose to establish sites of mRNA expression, express these """"""""orphan"""""""" P450s in heterologous systems, and examine their abilities to activate a wide variety of chemical carcinogens. In addition, several HPLC-mass spectrometry approaches will be used for identification of products and substrates, with tissue extracts as sources of substrates and the P450s as reagents. This part of the project is an effort towards understanding the functional genomics of human P450s. A related aspect is establishment of the roles of individual human P450s in morphine biosynthesis, for which strong evidence has been recently presented by others. (2) Comparisons of the kinetics of human P450s already studied in detail (1A2, 2A6, 2D6, 2E1, 3A4) will be done with several other P450s reported to have much higher rates of catalysis, with the goal of understanding which steps limit the (human P450) reactions. These studies will involve a variety of steady-state, pre-steady-state, and isotope effect approaches. (3) Kinetic analysis of multi-reaction P450s will be done, including P450s 51A1 (lanosterol 14a-demethylation), 19A1 (aromatase, oxidation of testosterone to 17p-estradiol), and 2A6 (oxidation of indoles), with a goal of defining the processivity of these systems. Several pre-steady-state and analysis approaches can be readily applied to the problem, with the goal of understanding the release of intermediates. (4) P450s 3A4 and 2A6 will be analyzed regarding hypotheses about cooperativity and induced fit in substrate binding and catalysis. The P450 3A4 work will focus on pre-steady-state kinetics of substrate binding, along with thermodynamic analysis of binding. The work on P450 2A6 induced fit will involve collaborative work on X-ray crystallography of mutants that have demonstrated expansion of the active site. These results should reveal whether a P450 has a fixed structure or can """"""""adapt"""""""" to individual substrates. Collectively, these studies have the goal of providing more understanding of the roles of human P450s in oxidation of drugs, carcinogens, and endogenous compounds and their potential contributions to cancer and other diseases and roles in disease treatments.
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