The purpose of the proposed research is to gain a better understanding of the mechanism(s) by which the microsomal cytochrome P-450-dependent mixed-function oxidases catalyze the metabolic activation and detoxication of chemical carcinogens and to investigate their inactivation by suicide inactivators.
The specific aims of this proposal are to: 1) investigate the steady-state kinetics of peroxide-supported dealkylation reactions using several different purified isozymes of cytochrome P-450 in order to determine the mechanism(s) by which they catalyze these reactions and to see if different kinetic mechanisms are utilized by different forms of the enzyme; 2) investigate kinetic isotope effects on the cytochrome P-450-catalyzed O-demethylation reactions in order to elucidate the chemical mechanism(s) used by these enzymes to effect catalysis; 3) investigate substrate kinetic isotope effects and """"""""metabolic switching"""""""" to gain a better understanding of the formation of the activated enzyme intermediate; 4) perform photoaffinity labeling experiments in order to investigate the substrate-binding properties of the active site and identify polypeptide sequences which are involved in forming the substrate-binding site at the active site of the enzyme; and 5) to investigate the suicidal inactivation of cytochrome P-450 during the metabolism of N-methylcarbazole in order to identify amino acids and peptides which are at the active site of the enzyme and to gain a better understanding of the mechanism(s) by which these enzymes may be selectively inactivated. This enzyme system, which is present in most human tissues including liver, lung and pancreas, plays a crucial role in the metabolism of most carcinogens. The delineation of the manner in which this enzyme system functions and the elucidation of the mechanism(s) of suicidal inactivation of these enzymes could prove very helpful in developing strategies for the effective manipulation of the catalytic activity of these enzymes. These results could ultimately prove to be helpful in developing methods for the prevention of human cancer.
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