Structure of Mammalian Cytochrome P450 2C5 Complexed with Diclofenac at 2.1 Å Resolution: Evidence for an Induced Fit Model of Substrate Binding,

Abstract

The structure of the anti-inflammatory drug diclofenac bound in the active site of rabbit microsomal cytochrome P450 2C5/3LVdH was determined by X-ray crystallography to 2.1 Å resolution. P450 2C5/3LVdH and the related enzyme 2C5dH catalyze the 4‘-hydroxylation of diclofenac with apparent K_m values of 80 and 57 μM and k_cat values of 13 and 16 min^-1, respectively. Spectrally determined binding constants are similar to the K_m values. The structure indicates that the π-electron system of the dichlorophenyl moiety faces the heme Fe with the 3‘- and 4‘-carbons located 4.4 and 4.7 Å, respectively, from the Fe. The carboxyl moiety of the substrate is hydrogen bonded to a cluster of waters that are also hydrogen bonded to the side chains of N204, K241, S289, and D290 as well as the backbone of the protein. The proximity of the diclofenac carboxylate to the side chain of D290 together with an increased binding affinity at lower pH suggests that diclofenac is protonated when bound to the enzyme. The structure exhibits conformational changes indicative of an adaptive fit to the substrate reflecting both the hydration and size of the substrate. These results indicate how structurally diverse substrates are recognized by drug-metabolizing P450 enzymes.