Mechanism-Based Inactivation of Cytochrome P450 2B Enzymes: Metabolism of Triethylenethiophosphoramide by Rat P450 2B1 and Human P450 2B6
Cytochrome P450 2B isozymes belong to a large class of P450 enzymes, which are all members of the hemeprotein family. In humans, P450s serve to catalyze the metabolism of endogenous substrates as well as exogenous compounds such as drugs and chemical carcinogens. In a previous study using human liver microsomes and expressed P450s, triethytenethiophosphoramide (thioTEPA) was shown to inhibit the S-mephenytion N-demethylation activity of P450 2B6. However, the exact mechanism of activity loss was not characterized. In the present study, thioTEPA was found to inactivate rat 2B1 in a time-, concentration-, and NADPH-dependent manner suggesting mechanism-based inactivation. Mechanism-based inactivation is characterized by the metabolic conversion of a substrate to a reactive intermediate that covalently binds to the enzyme's active site resulting in a loss in enzymatic activity. The K1 of the pseudo-first order inactivation reaction was determined to be 38 uM, the rate of inactivation was 0.3 1/min and the t(l/2) was 2.5 minutes. Spectral and HPLC studies indicated that the P450 2B1 heme was not compromised by thioTEPA, suggesting that a thioTEPA reactive intermediate inactivated P450 2B I by binding to the apoprotein. It was also found that exogenous nucleophiles had a minimal effect on the rate of inactivation. In the presence of glutathione the rate of inactivation decreased by only ten percent. The turnover number, which is the number of times P450 2B1 metabolized a thioTEPA molecule before a reactive thioTEPA intermediate inactivates the enzyme, was determined to be approximately 166. Inactivation was irreversible under dialysis conditions. Inactivation of P450 2B1 by thioTEPA in the presence of 7-EC, an alternate substrate of P450 2B1, was reduced. Further studies showed that thioTEPA also inactivated human P450 2B6 in a time- and concentration-dependent manner with a KI of 80 uM, a k(inact) of 0.1 min(^-1) and a t(l/2) of 13.9 min.