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内容記述 |
Many cytochrome P450 (CYP) inhibitors bind to the heme iron and compete with a substrate to bind to the active site. Such competitive inhibition indicates that the substrate or the inhibitor can bind to the active site. There are also CYP noncompetitive inhibitors that likely bind to allosteric sites that are distant from the active site of the enzyme. Noncompetitive inhibitors are believed to form an enzyme-substrate-inhibitor (ESI) complex. A previous docking simulation showed that noncompetitive inhibitors are bound to an allosteric site of CYP3A4, but the simulation assumed that noncompetitive inhibitors bind to this allosteric site and not to the active site. To date, there has been no experimental structural evidence of an ESI complex formed by CYP enzymes, whereas over nine hundred CYP protein structures have been deposited in the Protein Data Bank. We performed biochemical, X-ray crystallographic, and computational analyses of CYP105A1 from Streptomyces griceolus. CYP105A1 can catalyze 2-step hydroxylation reactions of vitamin D3, and this enzyme also metabolizes 12 types of anti-inflammatory drugs including diclofenac. Enzyme inhibition assays were performed using diclofenac as the substrate and ketoconazole, lanoconazole, and miconazole as imidazole containing inhibitors. The inhibition assays showed that ketoconazole and miconazole act as competitive inhibitors, whereas lanoconazole acts as a noncompetitive inhibitor of CYP105A1. We determined the X-ray structure of an ESI complex comprising CYP105A1, diclofenac, and lanoconazole. This structure shows that lanoconazole binds to the heme iron and that diclofenac closely interacts with the bound lanoconazole. Furthermore, the 4' hydroxylation site of diclofenac is distant from the heme iron in the ESI complex. Quantum mechanical calculations indicate that Cl-π and electrostatic interactions stabilize the formation of the ESI complex. Based on these results, we propose a mechanism for cooperative inhibition between a substrate and an apparent noncompetitive inhibitor. |
