Cooperative inhibition in cytochrome P450 between a substrate and an apparent non-competitive inhibitor.

Abstract

Cytochrome P450 (CYP) enzymes bind a heme group that acts as a catalytic center. Inhibition mechanisms in CYP enzymes have been studied extensively by biochemical and structural analyses. Non-competitive inhibitors are generally believed to bind to allosteric sites remote from the active site to form enzyme-substrate-inhibitor (ESI) complexes. Docking simulations predict the binding sites of non-competitive inhibitors to CYP enzymes, but to date there has been no experimental structural verification of ESI complexes formed by CYP enzymes. We performed biochemical and structural analyses of CYP105A1 using the imidazole-containing inhibitors ketoconazole, lanoconazole, and miconazole. Spectroscopic analyses showed that ketoconazole and miconazole act as competitive inhibitors, whereas lanoconazole acts as a non-competitive inhibitor of CYP105A1. The obtained X-ray structures of enzyme-inhibitor (EI) complexes showed that lanoconazole can bind in various orientations to the heme iron compared with ketoconazole and miconazole. We also 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 but it is positioned distant from the heme group. Quantum mechanical calculations indicate that Cl-π and electrostatic interactions between diclofenac and lanoconazole, and electrostatic interactions between diclofenac and positively charged arginine residues, stabilize formation of the ESI complex. Based on these results, we propose a mechanism for cooperative inhibition between a substrate and an apparent non-competitive inhibitor.