Received for publication July 27, 2007.
Revised October 21, 2007.
Accepted for publication October 22, 2007.

Mechanism-Based Inactivation of Lung-Selective Cytochrome P450 CYP2F Enzymes

Abstract

3-Methylindole (3MI) is a pneumotoxin that requires P450-catalyzed metabolic activation (dehydrogenation), to an electrophilic methylene imine to elicit toxicity. Previous studies have shown that the human pulmonary cytochrome P450 enzyme, CYP2F1, and its goat analog, CYP2F3, catalyzed the dehydrogenation of 3MI. However, it was not known if the dehydrogenation product could bind to active site nucleophilic residues to inactivate these enzymes. Therefore, the purpose of this study was to determine if 3MI is a mechanism-based inhibitor of CYP2F3 and CYP2F1. The results showed that both enzymes were highly susceptible to 3MI-mediated suicide inactivation. The k_inact and the K_I for CYP2F3 were 0.09/min and 160 µM, respectively, and the approximate partition ratio was 220. Although CYP2F3 lost approximately 80% of its activity in 30 minutes, a concurrent loss of its reduced carbon monoxide complex was not observed, suggesting that the heme was not destroyed/modified during the inactivation. The exogenous nucleophile, glutathione, did not protect CYP2F1 from 3MI-mediated inactivation, suggesting that the reactive intermediate did not diffuse from the active site prior to inactivation events. Dialysis of 3MI-inactivated CYP2F3 did not restore activity, and alternate substrates protected CYP2F3. In addition, 3MI inhibited the 7-ethoxycoumarin deethylase activity of human CYP2F1 in a time- and concentration-dependent manner; the k_inact and K_I were 0.025/min and 49 µM, respectively. In conclusion, this study presents evidence that 3MI is a mechanism-based inhibitor of both CYP2F3 and CYP2F1, which are important enzymes in the bioactivation of pneumotoxicants like 3MI or 1,1-dichloroethylene, or carcinogens such as naphthalene, benzene and styrene.

Key words: cytochrome P450, lung cancer, lung cytochrome P450, P450 mechanism, pulmonary toxicology, reactive intermediate, suicide inhibition

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