Figure 5.

Interaction of CYP2D6(dextromethorphan) with [¹⁵N]b₅ as determined by NMR and catalytic modulation of b₅ on CYP2D6-mediated metabolism of dextromethorphan. A, b₅ resonance intensity plot at a 1:1 (CYP2D6(DXM):[¹⁵N]b₅) ratio normalized to the free b₅ resonance intensity. The color code is as described in the legend to Fig. 2B. B, human soluble domain b₅ structure (PDB entry 2I96) with residues displaying differential broadening effects colored red, residues that are assigned in the NMR spectrum colored gray, and unassigned residues colored white. C, b₅ resonance intensity plots comparing the effects of line broadening between WT b₅ and b₅ mutants D65N and E49Q at a fixed 0.5:1 (CYP2D6(DXM):[¹⁵N]b₅) ratio. D, effect of WT b₅ and mutants on Michaelis–Menten kinetic parameters of CYP2D6-mediated O-demethylation of dextromethorphan. Each sample was generated in triplicate with S.D. illustrated by error bars. Steady-state kinetic constants below are shown with S.D. E, measurement of NADPH consumed (nmol of NADPH/min/nmol of CYP2D6) for CYP2D6 dextromethorphan reaction at a fixed dextromethorphan concentration of 1 mm. Samples were generated in duplicate with the S.D. illustrated by error bars. F, percent coupling of CYP2D6-mediated O-demethylation of dextromethorphan. Samples were generated in duplicate with the S.D. illustrated by error bars. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.