CYP2E1 active site residues in substrate recognition sequence 5 identified by photoaffinity labeling and homology modeling.
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.
Collom
Samuel L
SL
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 W. Markham St. Slot 516, Little Rock, AR 72205, USA.
Jamakhandi
Arvind P
AP
Tackett
Alan J
AJ
Radominska-Pandya
Anna
A
Miller
Grover P
GP
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D003198:Computer Simulation; D019392:Cytochrome P-450 CYP2E1 / Q000737:chemistry* / Q000648:ultrastructure*; D004789:Enzyme Activation; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019920:Photoaffinity Labels / Q000032:analysis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein / Q000379:methods*; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D019920:Photoaffinity Labels; D019392:Cytochrome P-450 CYP2E1