Reduced Catalytic Activity of P450 2A6 Mutants with Coumarin: A Computational Investigation

Human cytochrome P450 2A6 is the major enzyme to catalyze coumarin 7-hydroxylation, and this enzyme also plays an important role in the metabolism of nicotine and other tobacco-specific compounds. Recent experimental data showed that the N297S and A481T mutants of P450 2A6 decreased the catalytic activity toward coumarin by about 4-fold and 10-fold, respectively. These two mutants also had about 30-fold decrease in binding affinity for coumarin when compared to its wild type. At present, however, how the mutations affect the enzymatic activity and/or the substrate binding remains unclear. In this study, a combination of molecular docking and molecular dynamics (MD) simulation was employed to investigate the above question. Our results demonstrated that the N297S mutation altered the hydrogen-bonding network mediated by a water molecule between the B'-C loop and the I helix and thus a shift of the B' helix/B'-C loop region, whereas the A481T mutation triggered the conformational changes of its adjacent residues including Phe209 and Phe280 via an indirect manner to affect the substrate binding. However, the mutations did not significantly alter the substrate binding orientation because the only polar residue 297 in the active site provided the hydrogen-bonding donor to guide the binding of coumarin. Both mutations perturbed the shape of "Phe-cluster" in the active site and thus weakened the interactions with coumarin. The calculated binding free energies were in agreement with the relative potency of the experimental binding affinities.