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 th e 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 u p to 10 mu M BET. Whereas metabolism of BET by the wild-type 2B1 resul ted in the formation of benzaldehyde, benzotriazole, aminobenzotriazol e, 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 di meric product formed from the reaction of two BET molecules. Two spect ral 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 w ere 0.3 mM and 12 nmol (nmol of P450)(-1) min(-1), respectively, Molec ular modeling studies of BET bound in the active site of P450 2B1 sugg ested that a mutation of Gly 478 to Ala would result in steric hindran ce 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 c arbon 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 dim er formation. These findings indicate that a glycine 478 to alanine su bstitution in P450 2B1 altered the binding of BET such that inactivati ng BET metabolites were no longer generated.