Significance of glycine 478 in the metabolism of N-benzyl-1-aminobenzotriazole to reactive intermediates by cytochrome P450 2B1

The effect of mutating Gly 478 to Ala in rat cytochrome P450 2B1 on the metabolism of N-benzyl-1-aminobenzotriazole was investigated. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of the wild-type enzyme was completely inactivated by incubating with 1 mu M BET. The G(478)A mutant, however, was not inactivated by incubating with up to 10 mu M BET. Whereas metabolism of BET by the wild-type 2B1 resulted in the formation of benzaldehyde, benzotriazole, aminobenzotriazole, and a new metabolite, the G(478)A mutant generated only the later. This metabolite was found by NMR, IR, and mass spectrometry to be a dimeric product formed from the reaction of two BET molecules. Two spectral binding constants, a high-affinity constant that was the same for both enzymes (30-39 mu M) and a low-affinity constant that was 5-fold lower for the mutant enzyme (0.3 mM vs 1.4 mM), were observed with BET. The apparent K-m and k(cat) values for the G(478)A mutant with BET were 0.3 mM and 12 nmol (nmol of P450)(-1) min(-1), respectively, Molecular modeling studies of BET bound in the active site of P450 2B1 suggested that a mutation of Gly 478 to Ala would result in steric hindrance and suppress oxidation of BET at the 1-amino nitrogen. When BET was oriented in the 2B1 active site such that oxidation at the 7-benzyl carbon could occur, no steric overlap between Ala 478 and the substrate was observed. Thus, this orientation of BET would be preferred by the mutant leading to oxidation at the 7-benzyl carbon and subsequent dimer formation. These findings indicate that a glycine 478 to alanine substitution in P450 2B1 altered the binding of BET such that inactivating BET metabolites were no longer generated.