| Because of their importance in drug metabolism, extensive efforts have been directed at characterizing P450s. However, despite significant advances in the crystallization of P450s, there are no crystal structures available for a mammalian P450. Therefore, only indirect methods can be used to obtain information about the active site architecture of mammalian P450s. One such method involves the use of compounds known as mechanism-based inactivators or 'suicide' inhibitors. These compounds undergo oxidation by the P450s to a reactive intermediate which covalently modifies the enzyme at the active site. Once this occurs, biochemical techniques can presumably be used to identify the peptides, or even amino acid residue(s), that become covalent adducted by the reactive intermediate, thus providing information about the active site. In this investigation, a general scheme was developed for the purification of P450s that were suitable for mass spectral analysis. Electrospray ionization mass spectrometry was used to detect and characterize the covalent adduction of tienilic acid to P450 2C9 and several furanocoumarins to P450s 2A6 and 2B1. In the course of these investigations, several furanocoumarins were found to fulfill the necessary criteria to be defined as mechanism-based inactivators of P450s 2A6 and 2B1. In addition, L-754,394 was further characterized as a mechanism-based inactivator of P450 3A4. In all cases, the ratio of inactivator to spectrally detectable P450 was less than 1:1 and a furanoepoxide was implicated as the reactive intermediate responsible for the observed inactivation. The reason for the lack of stoichiometry between inactivator and enzyme is postulated to be a result of an non-optimized reconstituted P450 system. Attempts to isolate the covalently modified peptides were met with difficulty due to the instability of the adduct. This result coupled with the known stability of various conjugates of the furanoepoxide intermediate suggests that the site of covalent adduction is at a Glu or Asp residue. It is presumed that the ester bond thus formed is stable in the intact P450, however, once the enzyme begins to denature, rapid hydrolysis of the label occurs. |