| Description |
3-Methylindole is an extremely pneumotoxic compound tat requires P450-mediated dehydrogenation to an electrophilic intermediate, 3-methyleneindoline, to elicit its toxicity. The pulmonary cytochromes P450 2F1 (human) and 2F3 (goat) exclusively catalyze the dehydrogenation reaction, without formation of oxygenated metabolites. The mechanism that govern selectivity between cytochrome P450-mediated oxygenation and dehydrogenation of 3-methylinodole have not elucidated. The hypothesis for these studies was that differentiation between P450-medicated 2,3-oxygenation and 3-methyl dehydrogenation of 3-methylindole is controlled by unique active site differences exhibited by pulmonary P450 enzymes. The mechanisms of the formation of the P450-meditaed oxygenation-dependent metabolites 3-methyloxindole and 3-hydroxy-3-methyloxindole were determined. An epoxide, 2,3-epoxy-3-methylindoline was demonstrated as an intermediated in the formation of both metabolites. 3-Methyloxindole retained P450-introduced oxygen at position 2, formed by a mechanism involving and "NIH" shift. 3-Hydroxy-3-methyloxindole retained P-450-introduced oxygen at position 3, and probably originated from the epoxide, which also formed the electrophilic intermediated 3-hydrosxy-3-methylindolenine. The presence of this intermediate was confirmed through characterization of novel intra-molecular thioether conjugate with thioglycolic acid. These data are consistent with formation of the precursor epoxide, that reacts by multiple ring-opening mechanisms. Site directed mutagenesis of cytochrome P450 2F3 was performed to identify active site residues that direct of participate in the dehydrogenation of 3-methylindole. Additionally, a 3-dimensional homology model was constructed, based on the structure of rabbit P450 2C5, for 2F3. When mutations were made to SR 1, the mutant P450 enzyme was not produced. Enzymes with mutations to SRS 4, 5 and 6 were successfully expressed in bacteria and their functions were characterized. The SRS 4 mutant enzyme was catalytically identical to the wild type, with regard to 30methylindole dehydrogenation, but a 3-methylindole-oxygenase function was introduced to 2F3 when mutations were made in either SRS 5 or 6. Analysis of the homology model indicated that the sites of mutation were proximal to the active site. These studies have provided specific novel methods to distinguish oxygenation vs. dehydrogenation of 3-methylindole, and have identified critical SRS residues in 2F3 that help direct the unique dehydrogenation mechanism of the pulmonary enzyme. |
