Abstract
Human microsomal cytochrome P-450 2E1 (CYP2E1) monooxygenates > 70 low molecular weight xenobiotic compounds, as well as much larger endogenous fatty acid signaling molecules such as arachidonic acid. In the process, CYP2E1 can generate toxic or carcinogenic compounds, as occurs with acetaminophen overdose, nitrosamines in cigarette smoke, and reactive oxygen species from uncoupled catalysis. Thus, the diverse roles that CYP2E1 has in normal physiology, toxicity, and drug metabolism are related to its ability to metabolize diverse classes of ligands, but the structural basis for this was previously unknown. Structures of human CYP2E1 have been solved to 2.2 angstroms for an indazole complex and 2.6 angstroms for a 4-methylpyrazole complex. Both inhibitors bind to the heme iron and hydrogen bond to Thr303 within the active site. Complementing its small molecular weight substrates, the hydrophobic CYP2E1 active site is the smallest yet observed for a human cytochrome P-450. The CYP2E1 active site also has two adjacent voids: one enclosed above the I helix and the other forming a channel to the protein surface. Minor repositioning of the Phe478 aromatic ring that separates the active site and access channel would allow the carboxylate of fatty acid substrates to interact with conserved 216QXXNN220 residues in the access channel while positioning the hydrocarbon terminus in the active site, consistent with experimentally observed omega-1 hydroxylation of saturated fatty acids. Thus, these structures provide insights into the ability of CYP2E1 to effectively bind and metabolize both small molecule substrates and fatty acids.
Highlights
Cytochrome P-450 (P-450)3 is a superfamily of enzymes involved in monooxygenation of both endogenous and exoge
Protein Design and Structure Determination—Understanding the substrate selectivity of cytochrome P-450 2E1 (CYP2E1) as it plays roles in drug metabolism and both normal and pathophysiology has been hampered by the absence of experimental structural information about this enzyme
Absorbance spectra revealed that the purified CYP2E1 was in a mixed spin state, suggesting that in solution some CYP2E1 protein molecules have water bound to the sixth coordination site of the heme iron, but in other molecules the water is absent, and the heme iron is five-coordinate
Summary
Cytochrome P-450 (P-450) is a superfamily of enzymes involved in monooxygenation of both endogenous and exoge-. Among cytochrome P-450 enzymes, CYP2E1 is notable for this ability and the resulting toxicity [1] This activity is most substantial in the liver because CYP2E1 comprises over 50% of the hepatic cytochrome P-450 mRNA [2] and 7% of the hepatic cytochrome P-450 protein [3]. CYP2E1 has been connected with liver toxicity through two mechanisms: by the activation of substrates into reactive metabolites and potentially by generation of reactive oxygen species (ROS). Oxygen binding, and electron delivery are not closely coordinated during catalysis, diatomic oxygen can be converted to superoxide or hydrogen peroxide and released instead of being used to monooxygenate the substrate These ROS have been implicated in a range of damaging events including lipid peroxidation, protein oxidation, and DNA oxidation.
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