CYP2E1 active site residues in substrate recognition sequence 5 identified by photoaffinity labeling and homology modeling

• Published in: Archives of biochemistry and biophysics 2007-03, Vol.459 (1), p.59-69
• Main Authors: Collom, Samuel L., Jamakhandi, Arvind P., Tackett, Alan J., Radominska-Pandya, Anna, Miller, Grover P.
• Format: Article
• Language: English
• Subjects: Active site; Amino Acid Sequence; Amino Acids - chemistry; Azido; Binding Sites; Computer Simulation; CYP2E1; Cytochrome; Cytochrome P-450 CYP2E1 - chemistry; Cytochrome P-450 CYP2E1 - ultrastructure; Enzyme Activation; Labeling; Modeling; Models, Chemical; Models, Molecular; Molecular Sequence Data; P450 2E1; Photoaffinity; Photoaffinity Labels - analysis; Protein Binding; Protein Structure, Tertiary; Sequence Analysis, Protein - methods; Sequence Homology, Amino Acid
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2006.10.028

Despite its biological importance, our knowledge of active site structure and relevance of critical amino acids in CYP2E1 catalytic processes remain limited. In this study, we identified CYP2E1 active site residues using photoaffinity labeling with 7-azido-4-methylcoumarin (AzMC) coupled with a CYP2E1 homology model. In the absence of light, AzMC was an effective competitor against substrate p-nitrophenol oxidation by CYP2E1. Photoactivation of AzMC led to a concentration-dependent loss in CYP2E1 activity and structural integrity resulting from the modification of both heme and protein. The photo-labeling reaction degraded heme and produced a possible heme adduct. Probe incorporation into the protein occurred at multiple sites within substrate recognition sequence 5 (SRS-5). Based on a CYP2E1 homology model, we hypothesize AzMC labels SRS-5 residues, Leu363, Val364, and Leu368, in the active site. In addition, we propose a series of phenylalanines, especially Phe106, mediate contacts with the coumarin.