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Amino Acid Changes in Allelic Variants of CYP2D6 Alter Kinetics and Susceptibility to Inactivation
Author(s) -
Cho Christi W.,
Glass Sarah M.,
Osorio Victoria M.,
Sunden Kyle F.,
Fujiwara Rina,
Furge Laura Lowe
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.920.4
Subject(s) - dextromethorphan , cyp2d6 , metabolite , cytochrome p450 , chemistry , enzyme , drug metabolism , enzyme kinetics , biochemistry , mutant , active site , biology , pharmacology , gene
Cytochrome P450s (CYPs) are a superfamily of heme‐containing enzymes that are responsible for the metabolism of many xenobiotics and other small endogenous substrates. CYP2D6 is a major P450 enzyme studied during drug development due to its ability to metabolize ~20% of all pharmaceuticals. Moreover, there are over a 100 allelic variants of CYP2D6 resulting in a wide‐range of drug responses among individuals including cases of adverse drug events. The kinetics and susceptibility to inactivation of four allelic variants ‐ three with a series of distal mutations (*34, *17‐2, *17‐3) and one ultra‐metabolizer (*53), and one active‐site mutant (CYP2D6‐T309A), were investigated in the current study. The variants were expressed in E. coli and characterized with the prototypical substrates bufuralol and dextromethorphan. Decreased activity was observed for *34, *17‐2, and *17‐3 while the increased activity of *53 confirmed it as an ultra‐metabolizer. CYP2D6‐T309A displayed similar kinetic values as reference CYP2D6*1. A new abundant metabolite of dextromethorphan formed by *53 was identified using mass spectrometry. The inactivation of 2D6 variants by a known‐mechanism based inactivator, SCH66712, was investigated in a time‐ and concentration‐dependent manner for *1, *53, and T309A. Though *1 was inactivated by SCH66712, *53 and T309A were not susceptible to inactivation. These findings support the designation of T309A as the nucleophilic target for inactivation of CYP2D6 by SCH66712. The greatly decreased rate of inactivation of *53 suggests that inactivation may be dependent on time and positioning of inactivator in the active site. Computational methods using Molecular Dynamics and AMBER tools were used to gain insights into how individual amino acid changes cause alterations in structural stability and plasticity, and may lead to observed differences in reaction rates and susceptibilities to inactivation. Support or Funding Information Support: NIH 1R15‐GM086767‐02 and the F.W. and Elsie L. Heyl, Hutchroft, and Roger F. and Harriet G. Varney Endowments of Kalamazoo College.