Roles of Residues F206 and V367 in Human CYP2B6: Effects of Mutations on Androgen Hydroxylation, Mechanism-Based Inactivation, and Reversible Inhibition.

The crystal structures of human CYP2B6 indicate that Phe206 and Val367 are in close proximity to the substrate binding site and suggest that both residues may play important roles in substrate metabolism and inhibitor binding. To test this hypothesis, we investigated the effects of mutating these residues to Ala on the regiospecificity of CYP2B6 for the metabolism of testosterone and androstenedione. For testosterone metabolism, 16β-OH-testosterone formation by the F206A mutant was <5% of the wild type (WT), whereas the V367A mutant exhibited a doubling of 16α-OH-testosterone formation with a 50% decrease in 16β-OH-testosterone formation compared with the WT. Significant alterations in the regiospecificity for androstenedione metabolism were also observed. To investigate the roles of these two residues in the metabolic activation of mechanism-based inactivators, tert-butylphenylacetylene (BPA) and bergamottin (BG) were used to test the susceptibility to inactivation. Although the rates of inactivation of both mutants by BG were not significantly decreased compared with the WT, the efficiency of inactivation by BPA of both mutants was more than an order of magnitude lower. Our results demonstrate that Phe206 plays a crucial role in determining the specificity of CYP2B6 for the 16β-hydroxylation of testosterone and androstenedione and that it also plays an important role in BG binding and mechanism-based inactivation by BPA. In addition, Val367 dramatically enhances the catalytic activity of CYP2B6 toward androstenedione and plays an important role in mechanism-based inactivation by BPA. The results presented here show the important roles of Phe206 and Val367 in interactions of CYP2B6 with substrates and inactivators/inhibitors and are consistent with the crystal structures.