Figure 6.

Interaction of CYP2A6(pNP) with [¹⁵N]b₅ as determined by NMR and catalytic modulation of b₅ on CYP2A6 mediated metabolism of para-nitrophenol. A, b₅ resonance intensity plot at a 0.5:1 (CYP2A6(pNP):[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 (CYP2A6(pNP):[¹⁵N]b₅) ratio. D, effect of WT b₅ and mutants on Michaelis–Menten kinetic parameters of CYP2A6-mediated 2-hydroxylation of pNP. Each sample was generated in duplicate 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 CYP2A6) for CYP2A6 pNP reaction at a fixed pNP concentration of 1.6 mm. Samples were generated in duplicate with the S.D. illustrated by error bars. F, percent coupling of CYP2A6-mediated 2-hydroxylation of pNP. Samples were generated in duplicate with the S.D. illustrated by error bars. ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01.