Homotropic Cooperativity During The Mechanism‐Based Inactivation Of Cytochrome P450 2B4 By 9‐Ethynylphenanthrene

Abstract

We investigated the mechanism by which 9‐ethynylphenanthrene (9EP) inactivates cytochrome P450 2B4 (CYP2B4). Our results demonstrated that 9EP is a potent mechanism‐based inactivator (MBI) of CYP2B4 with a partition ratio and kinact of 0.2 and 0.25 min−1, respectively. More importantly the kinetics of the MBI exhibit homotropic cooperativity with a Hill coefficient of 2.5 and S50 of 4.5 μM. To the best of our knowledge, this is the first report of homotropic cooperativity in the mechanism‐based inactivation of P450s. A fully modified CYP2B4 was purified to homogeneity, and its structure was solved by X‐ray crystallography. Based on this crystal structure, 9EP is covalently attached to the Oγ of Thr 302 via an ester bond, resulting in inward rotation of Phe 297 and Phe 206. It is unlikely that the active site of CYP2B4 can accommodate two 9EP molecules. To explore other binding site(s) responsible for cooperativity, fluorescence quenching resulting from 9EP binding to CYP2B4 was investigated. These studies revealed two distinct binding sites for 9EP in CYP2B4. A high affinity site with a Kd of ~46 nM was observed in the modified CYP2B4, which likely arises from the binding of 9EP to a peripheral site. In contrast, a high affinity site as well as a low affinity site were associated with the binding of 9EP to the unmodified CYP2B4. These results suggest that 9EP binds to two distinct sites in CYP2B4 that function cooperatively.