| Dehydrogenation reactions are relatively rare among P450-catalyzed reactions and have not been well characterized. Cytochrome P450 2F1 (human) and 2F3 (goat), are selectively expressed in pulmonary tissues and they both catalyze the dehydrogenation of the pneumotoxicant 3-methylindole (3-MI) to form the highly reactive 3-methyleneindolenine. Because of its role in the dehydrogenation and subsequent bioactivation of the pneumotoxicant 3-methylindole, both P450 2F1 and P450 2F3 are great candidates to gain understanding of the dehydrogenation reaction pathway. The central hypothesis of this dissertation work was that the specific mechanisms of dehydrogenation and bioactivation of 3-MI by pulmonary selective P450 2F1/2F3 are governed by the unique structural and catalytic properties of the enzyme active site. To test this hypothesis, we used several active site probes, including mechanism-based inactivation, site-directed mutagenesis, and kinetic isotope studies. Mechanism-based inactivation studies confirmed that 3-MI is indeed a mechanism-based inactivator of both 2F3 and 2F1. Site-directed mutagenesis studies were performed to identify amino acid residues of P450 2F3, that direct the substrate binding and catalysis, leading to dehydrogenation of 3-MI. Six conserved regions of all P450 enzymes have been identified as Substrate Recognition Sites (SRS 1-6), and mutations were chosen within these regions. SRS 5 (D362T) and SRS 6 (S476H) mutants increased both dehydrogenation and 3-methylindole oxygenase activity of the enzyme. SRS 6 (S477I) and SRS 2 (G214I and E215Q) mutants increased oxygenation, but completely abolished dehydrogenase activity. Analysis of the changes in the overall active site conformation and substrate binding orientation, using homology modeling and 3-MI docking studies, showed major alterations in substrate binding orientations for the S477I mutant, that were entirely consistent with the attenuation of the dehydrogenase function. Kinetic isotope studies were carried out to obtain a mechanistic understanding of the dehydrogenation chemistry. Intra-molecular kinetic deuterium isotope studies with 3-[2H 2-methyl]-indole, using purified 2F3, demonstrated an isotope effect (KH/KD) of 6.0. This relatively high intra-molecular deuterium isotope effect suggested that the dehydrogenation of 3-MI occurs through an initial hydrogen abstraction step. The studies presented in this dissertation have provided vital structural snd mechanistic information about the 2F family of enzymes and the dehydrogenation reaction chemistry. |
