P450 enzyme metabolizes the oxidation of a variety of organic compounds in our body including drugs. The exact catalytic mechanism for mammalian P450 enzymes is not known because highly reactive substrates with tight binding affinities have not been discovered for these enzymes yet. The purpose of this grant is to find a substrate with such properties for the rabbit P450 enzyme, the model enzyme for human P450. The PI has recently reported that rabbit P450 binds butylated hydroxytoluene (BHT) with a very high affinity but only 35% of it converts into desired products. Several BHT derivatives will be tested for their binding affinity and improved catalysis over BHT. Advanced Gas-Chromatograph Mass Spectrometry will be applied to measure the amount of product produced from compounds with binding affinities similar to BHT. Spectroscopic techniques will be employed to identify the intermediates involved in catalysis for substrates which P450 finds them much more reactive than BHT.