Abstract

In this study metabolism of bupropion, efavirenz, and 7-EFC by CYP2B6 wild type (CYP2B6.1) and six polymorphic variants (CYP2B6.4 to CYP2B6.9) was investigated in a reconstituted system to gain a better understanding of the effects of the mutations on the catalytic properties of these naturally occurring variants. All six variants were successfully over-expressed in Escherichia coli, including CYP2B6.8 (the K139E variant) which previously could not be over-expressed in mammalian COS-1 cells (Lang et al., J. Pharmacol. Exp. Ther. 311:34-43, 2004). The steady-state turnover rates for the hydroxylation of bupropion and efavirenz and for the O-deethylation of 7-EFC showed that these mutations significantly alter the catalytic activities of CYP2B6. It was found that CYP2B6.6 exhibits 4- and 27-fold increases in the Km values for the hydroxylation of bupropion and efavirenz, respectively, and CYP2B6.8 completely loses its ability to metabolize any of the substrates under normal turnover conditions. However, when compared to CYP2B6.1, CYP2B6.8 retains 77% of its 7-EFC Odeethylase activity in the presence of tert-butyl hydroperoxide as an alternative oxidant, indicating that the heme and the active site are catalytically competent. Pre-steady-state measurements of the rate of electron transfer from CPR to CYP2B6.8 using stoppedflow spectrophotometry revealed that CYP2B6.8 is incapable of accepting electrons from CPR. These observations provide conclusive evidence suggesting that the chargereversal mutation in the K139E variant prevents CYP2B6.8 from forming a functional complex with CPR. Results from this work provide further insights to better understand the genotype-phenotype correlation regarding CYP2B6 polymorphisms and drug metabolism.