INACTIVATION OF PURIFIED RAT-LIVER CYTOCHROME-P-450 2B1 AND RABBIT LIVER CYTOCHROME-P-450 2B4 BY N-METHYLCARBAZOLE

Metabolism of N-methylcarbazole by purified rat liver cytochrome P-450 2B1 or rabbit liver P-450 2B4 resulted in the inactivation of these e nzymes in a time-dependent, pseudo-first order manner as assayed spect rally by the decrease in the reduced CO spectrum at 450 nm. The inacti vation was saturable with respect to the concentration of N-methylcarb azole, and a K-l = 5.2 mu M and K-INACT = 0.14 min(-1) were determined for the inactivation of P-450 2B1. For P-450 2B4 inactivation, the K- l was 23 mu M and the K-INACT = 0.21 min(-1). There was no increase in the reduced CO spectrum at 420 nm accompanying the inactivation, and the slight loss of the P-450 heme prosthetic group, as determined by t he spectrum at 418 nm, was not sufficient to account for the loss of t he reduced CO spectrum at 450 nm. The metabolism of N-methylcarbazole by P-450 did not result in the formation of a metabolic intermediate c omplex, which could also be responsible for the loss of cytochrome P-4 50 activity. Loss of catalytic activity for further substrate metaboli sm was also observed after preincubation of enzyme with N-methylcarbaz ole and the loss of catalytic activity correlated with the loss of the reduced CO spectrum. Accompanying the loss of spectrally detectable P 450 2B1 and P-450 2B4 catalytic activity, there was an increase in th e NADPH oxidation rate. This increased rate persisted on subsequent ad dition of NADPH.