Comparative homology modeling of human cytochrome P4501A1 (CYP1A1) and confirmation of residues involved in 7-ethoxyresorufin O-deethylation by site-directed mutagenesis and enzyme kinetic analysis.
CYP1A1 homology models based on the CYP2C5 and a composite of CYP2C5, CYP2C8, and CYP2C9 X-ray crystal structures were compared to a model generated using the recently published coordinates of CYP1A2. The model using the CYP1A2 coordinates, CYP1A1-(1A2), gave near ideal stereochemical quality and was favored energetically. Docking studies identified the active-site residues potentially involved in binding of the prototypic CYP1A1 substrate 7-ethoxyresorufin. CYP1A1 mutants S122A, F123A, F224A, A317Y, T321G, and I386G were generated to explore the roles of these residues in 7-ethoxyresorufin binding and turnover, and generally confirmed the importance of aromatic interactions over hydrogen bonding in orientating 7-ethoxyresrufin in a catalytically favorable orientation. Although 7-ethoxyresorufin O-deethylation by CYP1A1 and several mutants exhibited substrate inhibition, it is unlikely that inhibition arises from the simultaneous binding of two substrates within the active-site given the geometry of the active site-cavity.
Lewis
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Department of Clinical Pharmacology, Flinders University, School of Medicine and Flinders Medical Centre, Bedford Park, Adelaide, SA 5042, Australia. ben.lewis@flinders.edu.au
Mackenzie
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Miners
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D003198:Computer Simulation; D019363:Cytochrome P-450 CYP1A1 / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010078:Oxazines / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D000596:Amino Acids; D010078:Oxazines; C007649:ethoxyresorufin; D019363:Cytochrome P-450 CYP1A1