Multiplicity and ligand selectivity

Within the human CYP1-3 families, some 23 functional forms of P450 can be identified.5 However, arguably only CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 are critical to drug metabolism, as these enzymes constitute the bulk of the hepatic P450 complement and are known to have wide substrate selectivities. Other extensively studied human P450s, such as CYP1A1 and CYP1B1, have predominantly an extrahepatic location and are not significantly involved in the metabolic clearance of drugs, although they may be important for localized bioactivation reactions that can lead to xenobiotic toxicity.8 Some newer members of the human P450 family, identified through large-scale sequencing projects, e.g., CYP2S1, CYP2U1, and CYP2W1, have unknown functions and have been referred to as 'orphan' P450s.

Diagnostic substrates and inhibitors for the major hepatic forms of human P450 are listed in Table 2. These ligands, together with commercially available recombinant forms of all the major human P450s, are invaluable tools for dissecting the participation of specific human microsomal P450 isoforms that contribute to a given metabolic pathway.9 Because highly specific substrates and inhibitors can be identified for certain P450s, it could be expected that each isoform would possess a unique active site architecture complementary to key hydrophobic, electrostatic, and steric features of their preferred ligands. Recent crystallization of mammalian forms of P450 is beginning to shed light on these interactions.

5.07.2.1.3 Structure

In general, P450s are 450-500-amino-acid (55-60 kDa) proteins with a completely conserved C-terminal cysteine residue that provides the thiolate coordinate bond to the iron atom which tethers the heme prosthetic group. Intracellularly, P450s reside in the soluble fraction of the cell (bacterial enzymes), the inner membrane of the mitochondrion (several steroid-metabolizing P450s), or the endoplasmic reticulum (drug-metabolizing P450s); targeting is dependent on the nature of the N-terminal leader sequence.10 Drug-metabolizing P450s contain a hydrophobic string of 20-30 amino acids that is inserted into the endoplasmic reticulum and anchors the enzyme to the membrane, with the bulk of the protein oriented towards the cytosol in a monotopic fashion.

Until 2000, P450 tertiary structure information was limited to the soluble bacterial forms of the enzyme (e.g., CYP101/P450cam and CYP102/P450 BM3). The availability of the first mammalian P450s structure, an N-terminally truncated form of rabbit CYP2C5 also engineered internally to limit additional membrane or oligomer interactions, confirmed that the global features of this enzyme evident for the early bacterial structures were conserved, viz., a heart-shaped protein with an alpha-helix-rich C-terminus and a beta-sheet-rich N-terminus.11 At the time of writing, crystal structures for human CYP2C9, CYP2C8, CYP3A4, and CYP2A6 have been solved.12-15 Notably, the structure for

Figure 1 Crystal structures of CYP3A4 and CYP2C9. (a) The overall structural fold of CYP3A4 and location of the heme prosthetic group (ball and stick). Secondary structure elements are represented as coils for helices and arrows for strands. (b) and (c) contrast the solvent accessible molecular surfaces of the active sites of CYP3A4 (green) and CYP2C9 (magenta). These structures highlight differences in volume and shape of P450 active sites. (Reproduced with permission from Williams, P. A.; Cosme, J.; Vinkovic, D. M.; Ward, A.; Angove, H. C.; Day, P. J.; Vonrhein, C.; Tickle, I. J.; Jhoti, H. Science 2004, 305, 683-686. Copyright (2004) AAAS.)

Figure 1 Crystal structures of CYP3A4 and CYP2C9. (a) The overall structural fold of CYP3A4 and location of the heme prosthetic group (ball and stick). Secondary structure elements are represented as coils for helices and arrows for strands. (b) and (c) contrast the solvent accessible molecular surfaces of the active sites of CYP3A4 (green) and CYP2C9 (magenta). These structures highlight differences in volume and shape of P450 active sites. (Reproduced with permission from Williams, P. A.; Cosme, J.; Vinkovic, D. M.; Ward, A.; Angove, H. C.; Day, P. J.; Vonrhein, C.; Tickle, I. J.; Jhoti, H. Science 2004, 305, 683-686. Copyright (2004) AAAS.)

CYP2C9 with the flurbiprofen bound shows an important salt bridge interaction between an active site Arg108 residue and the charged carboxylate of this nonsteroidal anti-inflammatory drug, which helps explain this isoform's relative selectivity for acidic substrates.16 A comparison of the active site of CYP3A4 and CYP2C9 is depicted in Figure 1. In some cases, ligands have been crystallized within the P450 active site in multiple conformations, highlighting the plasticity of mammalian P450 active sites. This is in accord with the long-held observations of the broad substrate specificity of these enzymes.11

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