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Cytochrome P450 1A2 forms catalytically relevant homomeric complexes
Author(s) -
Connick John Patrick,
Reed James R.,
Cheng Dongmei,
Cawley George F.,
Backes Wayne L.
Publication year - 2012
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.26.1_supplement.850.5
Subject(s) - homomeric , cyp1a2 , chemistry , biochemistry , biophysics , luciferase , cytochrome p450 , enzyme , protein subunit , biology , transfection , gene
Our laboratory and others have shown that one P450 enzyme can affect the catalytic function of other P450s. The goal of this study was to determine if CYP1A2 forms functionally relevant homomeric complexes. The catalytic activity of CYP1A2 was measured in reconstituted systems containing CYP1A2 and varying levels of NADPH‐cytochrome P450 reductase (CPR). Interestingly, a sigmoidal response was observed with respect to [CPR] instead of the hyperbolic curve expected for simple Michaelis‐Menten kinetics – the sigmoidicity increased with increasing [CYP1A2]. A physical interaction between CYP1A2 molecules was verified using bioluminescence resonance energy transfer (BRET). Varying ratios of Renilla luciferase‐ and GFP2‐tagged CYP1A2 were cotransfected into HEK293T cells and energy transfer from luciferase to GFP2 was measured. Cotransfection of the tagged CYP1A2 proteins with increasing amounts of unlabeled CYP1A2 decreased the BRET response, indicating a specific physical interaction. Taken together, these results show that CYP1A2 forms CYP1A2•CYP1A2 complexes that exhibit altered catalytic activity.