Abstract
We have previously shown that Phe(120), Glu(216), and Asp(301) in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolizing enzyme (Paine, M. J., McLaughlin, L. A., Flanagan, J. U., Kemp, C. A., Sutcliffe, M. J., Roberts, G. C., and Wolf, C. R. (2003) J. Biol. Chem. 278, 4021-4027 and Flanagan, J. U., Maréchal, J.-D., Ward, R., Kemp, C. A., McLaughlin, L. A., Sutcliffe, M. J., Roberts, G. C., Paine, M. J., and Wolf, C. R. (2004) Biochem. J. 380, 353-360). We have now examined the effect of mutations of these residues on interactions of the enzyme with the prototypical CYP2D6 inhibitor, quinidine. Abolition of the negative charge at either or both residues 216 and 301 decreased quinidine inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation by at least 100-fold. The apparent dissociation constants (K(d)) for quinidine binding to the wild-type enzyme and the E216D and D301E mutants were 0.25-0.50 microm. The amide substitution of Glu(216) or Asp(301) resulted in 30-64-fold increases in the K(d) for quinidine. The double mutant E216Q/D301Q showed the largest decrease in quinidine affinity, with a K(d) of 65 microm. Alanine substitution of Phe(120), Phe(481),or Phe(483) had only a minor effect on the inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation and on binding. In contrast to the wild-type enzyme, a number of the mutants studied were found to be able to metabolize quinidine. E216F produced O-demethylated quinidine, and F120A and E216Q/D301Q produced both O-demethylated quinidine and 3-hydroxyquinidine metabolites. Homology modeling and molecular docking were used to predict the modes of quinidine binding to the wild-type and mutant enzymes; these were able to rationalize the experimental observations.
Highlights
Metabolism [2,3,4]
We have previously shown that Phe120, Glu216, and Asp301 in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolizing enzyme
Inhibition of CYP2D6 Mutants by Quinidine—We investigated the effects of mutations of the active-site residues Phe120, Glu216, Asp301, Phe481, and Phe483 on the inhibition of CYP2D6 activity by quinidine
Summary
Materials—Terrific broth, chloramphenicol, dithiothreitol, glucose 6-phosphate, NADPϩ, phenylmethylsulfonyl fluoride, sodium dithionite, cytochrome c, and quinidine were purchased from Sigma (Poole, UK). Quinidine Inhibition of Bufuralol 1Ј-Hydroxylation and Dextromethorphan O-Demethylation—Incubations were carried out in triplicate at 37 °C with shaking in 300 l of 50 mM potassium phosphate (pH 7.4) containing E. coli membranes equivalent to 10 pmol of CYP2D6 (wild-type or mutant), quinidine (0, 1, 10, or 100 M), an NADPHgenerating system (comprising 5 mM glucose 6-phosphate, 1 unit of glucose-6-phosphate dehydrogenase, and 1 mM NADPϩ), and bufuralol or dextromethorphan at concentrations equivalent to the Km of each sample. Quinidine Metabolism—To investigate quinidine metabolism, reaction mixtures consisted of 50 mM potassium phosphate (pH 7.4) containing 100 M quinidine, E. coli membranes equivalent to 10 pmol of CYP2D6 (wild-type or mutant) and an NADPH-generating system (as described above) in a total volume of 200 l. E. coli membranes containing wild-type or mutant CYP2D6 enzymes were diluted in 100 mM potassium phosphate buffer (pH 7.4) to a final concentration of 0.5 M cytochrome P450 and split into two matched black-walled quartz cuvettes. The docked energy of a solution that positioned the tethered groups farther than 4.5 Å apart was penalized, the size of the penalty being determined using a harmonic force constant of 5.0 kJ1⁄7molϪ11⁄7ÅϪ2
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