Cytochrome P450 2C9
Created by: Glen Hookey
Cytochrome P450 2C9 (CYP2C9) is a member of the larger cytochrome P450 family (CYP450s), that are represented in microorganisms, plants, and animals. In mammals, CYP450s are membrane-bound heme proteins responsible for the recognition and metabolism of various xenobiotics including drug molecules, environmental compounds, and pollutants.1 Specifically, CYP450s are liver-specific, lipid-anchored monooxygenases that complex with CYP reductase (CPR) to accept electrons for the oxidation of a wide range of water-soluble and lipo-soluble compounds.2 CYPs and their associated CYRs are anchored by a single N-terminus transmembrance anchor (residues 1-29, not pictured) in the endoplasmic reticulum membrane, with the catalytic domain facing the cytosol.2
Cytochrome P4502C9 (PDB ID: 1OG2) is composed of two identical subunits, alpha and beta, each consisting of 490 residues, characterized by sixteen alpha-helices and nine beta-sheets that surround a highly conserved heme moiety with a central iron atom.1 As determined by a mutational study on the affinity of CYP for CPR, CYP2C9:CPR complex formation is dependent on eight water-accessible (and therefore available for interaction) residues: Lys-121, Arg-125, Arg-132, Phe-134, Met-136, Lys-138, Lys-432, and Gly-442.3 The canonical substrate access channel opens at the B' helix and the F-G helix loop, leads between Phe-114 and Phe-476, and reaches the heme moiety and I-helix at its base.1 Differential access to heme group, and subsequent regioselectivity of the redox reaction, depends upon four core amino acids: Ala-297, Leu-362, Leu-366, and Thr-301.4
In humans, the relevance of CYP450s lies in the metabolism of clinical drugs. Five isoforms of CYP450s (CYP2C9, CYP1A2, CYP2C19, CYP2D6, and CYP3A4) contributed to the oxidative metabolism of over 90% of the drugs in clinical use in 2003.1 Three of these isoforms, 2C8, 2C9, and 2C19, are located in the same gene cluster on chromosome 10 and are expressed in the liver. Each is critical for hepatic funcation, as they are differentiated by structural features that confer selectvity for various substrates.5 The aforementioned protein of interest, CYP2C9, is involved in the oxidation of 16% of drugs in current clinical use, including the anti-coagulant drug, warfarin.1 While warfarin is a racemic mixture of R and S enantionmers, the S-form is five times more potent and is therefore more relevant when considering anti-coagulatory effect. CYP2C9 metabolizes only this S-enantiomer (Image 1).4,8
When S-warfarin interacts with the binding site, mobile surface elements of CYP2C9 open the previously closed channel leading to the internal heme group responsible for the redox reaction.4 In addition to ligand binding (S-warfarin interaction), two other factors influence the opening motions in CYP2C9: (i) the conformation of the FG loop, and (ii) the protein's association with the lipid membrane.2 At the active site, the largely aromatic warfarin molecule interacts with a hydrophobic binding pocket adjacent to the heme moeity.1 The substrate-bound enzyme conformation is characterized by a change in the orientation of Ile-99 and Phe-100, which act to open and close the active site, and conformational change in the B-C loop that allows Arg-108 to reside in the active site. A recent study specfically identifies Arg-108 as critical for CYP2C9's high affinity binding of warfarin. Substitution of phenylalanine for Arg-108 results in a near total loss of cytochrome function, suggesting the positively charged arginine residue complements the negative charge of warfarin's phenolate moiety to enhance active site binding.5 Additionally, an aromatic gate composed of three phenylalanine residues, Phe-100, Phe-114, and Phe-476 is suggested to lock enzyme substrates in place directly above the heme center.2
Current areas of study include polymorphic variants of the CYP450 isoforms, which have implications for the efficacy of drugs in individuals.1 In CYP2C9-specific research, recent investigation has focused on the advantages and consequences of combining drugs with warfarin. A 2008 study revealed that acute alcohol intoxication significantly reduces the metabolism of warfarin by CYP2C9 via allosteric interacions, leading to increased anticoagulant effects and the risk of hemorrhage.6 A 2009 study defined the structure-activity relationship of CYP2C9 with fluoxetine (Prozac), ibuprofen, naproxen, and metenamic acid and provided insights into the allosteric interactions responsible for the effects of combining certain drugs.7 With regard to the structure and function of the protein itself, two major questions continue to challenge researchers: (i) is there a large-scale conformational change in CYP2C9 upon substrate binding and (ii) are there differential access and egress channels for the various molecules that CYP2C9 metabolizes?3