title: Structure of mammalian cytochrome P450 2C5 complexed with diclofenac at 2.1 A 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 A resolution. P450 2C5/3LVdH and the related enzyme 2C5dH catalyze the 4'-hydroxylation of diclofenac with apparent K(m) values of 80 and 57 microM 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 pi-electron system of the dichlorophenyl moiety faces the heme Fe with the 3'- and 4'-carbons located 4.4 and 4.7 A, 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.
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, MEM-255, La Jolla, California 92037, USA.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Wester
authors.forename: Eric F
authors.initials: EF
authors.lastname: Johnson
authors.forename: Cristina
authors.initials: C
authors.lastname: Marques-Soares
authors.forename: Sylvie
authors.initials: S
authors.lastname: Dijols
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Dansette
authors.forename: Daniel
authors.initials: D
authors.lastname: Mansuy
authors.forename: C David
authors.initials: CD
authors.lastname: Stout
mesh_terms: D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000737:chemistry* / Q000378:metabolism*; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D004008:Diclofenac / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D008861:Microsomes / Q000201:enzymology; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011817:Rabbits; D013255:Steroid 21-Hydroxylase / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014867:Water / Q000737:chemistry
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D002264:Carboxylic Acids; D014867:Water; D004008:Diclofenac; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D007501:Iron; C065407:CYP2C5 protein, Oryctolagus cuniculus; D000072467:Cytochrome P450 Family 2; D013255:Steroid 21-Hydroxylase