CATALYTIC SELECTIVITY AND MECHANISM-BASED INACTIVATION OF STABLY EXPRESSED AND HEPATIC CYTOCHROMES P450 2B4 AND 2B5 - IMPLICATIONS OF THE CYTOCHROME-P450 2B5 POLYMORPHISM

Cytochrome P450 (P450) 2B5 was recently found to be functionally disti nct from three other rabbit P450 2B forms, based on androstenedione hy droxylase activities. In this investigation, we examined the frequency of the P450 285-null phenotype and the functional consequences of pol ymorphic P450 2B5 expression in hepatic microsomes from phenobarbital- treated rabbits. Four of the 10 animals examined did not have detectab le levels of P450 2B5 mRNA and exhibited much lower microsomal androst enedione 15 alpha- and 16 alpha-hydroxylase activities. The 15 alpha-h ydroxylase activity was found to correlate (r = 0.91) with liver P450 2B5 mRNA. P450 2B4 and 2B5 were stably expressed in human kidney 293 c ells to further characterize substrate specificities and to investigat e mechanism-based inactivation by phencyclidine. P450 2B4 was 4-16-fol d more active than 2B5 towards benzphetamine, 7-ethoxycoumarin, methyl enedioxybenzene, and pentoxyresorufin. Benzyloxyresorufin O-debenzylas e activity was 160-fold higher for P450 2B4 than P450 2B5. Anti-P450 2 B4 IgG inhibited benzyloxyresorufin O-debenzylation nearly completely in untreated and phenobarbital-induced liver microsomes. Phencyclidine selectively inactivated P450 2B4, compared with 2B5, in both human ki dney 293 cell and liver microsomes. Poor inactivation of P450 2B5 by p hencyclidine was found to be a result of its low maximal rate constant . Results of this study establish the idea that the metabolic conseque nces of phenobarbital induction depend on the potential of animals to express functionally variant P450 2B forms. Furthermore, we conclude t hat one or more of the 11 amino acid differences between these highly related P450 forms are critical to their substrate specificities and s elective inactivation.