Cytochrome P450 2E1 is a key enzyme in the biotransformation of environmental chemicals. The long term objectives of this project are to understand the catalytic and physiological functions of this enzyme and its roles in environmental toxicology. Significant progress toward these goals was made during the previous grant period. The specific aims of this renewal grant are as follows: 1. To establish a heterologous expression system for 2E1 with the aims of obtaining purified 2E1 for catalytic studies and of conducting site- directed mutagenesis studies to elucidate structure-function relationships. 2. To define the structural features of substrates and inhibitors of 2E1 and to understand the active-site dynamics of this enzyme. These will be accomplished by studying the kinetic parameters (Km, Vmax, Ki) of 2E1- catalyzed reactions and by active-site modeling together with a site- directed mutagenesis approach. 3. To elucidate the normal physiological functions of 2E1 by overexpressing and inactivating (by a homologous recombination approach) the 2E1 gene in transgenic mice. The effects of these genetic manipulations on the survival, development, and physiological functions will be investigated. 4. To further characterize the physiological functions of 2E1 and its roles in the activation or detoxification of environmental chemicals. Both transgenic mice and other animals will be used to study the tissue distribution of 2E1, the physiological role of the 2E1-dependent gluconeogenesis from acetone, roles of 2E1 in the generation of peroxides and the metabolism of lipid hydroperoxides, and roles of 2E1 in the activation or detoxification of selected environmental chemicals. 5. To characterize the metabolic activation of acetaminophen by 2E1 and other enzymes, and to delineate its modulation by dietary chemicals such as flavonoids. Both in vitro and in vivo studies will be pursued in rodents and in humans. The proposed work is expected to contribute significantly to our understanding of the unique catalytic activities and physiological functions of 2E1 as well as its roles in the activation or detoxification of environmental chemicals and drugs.