A TALE OF TWO ENZYMES: IDENTIFICATION OF AN UNKNOWN LIGAND BOUND TO CYTOCHROME P450 2A13 AND UNDERSTANDING SUBSTRATE SELECTIVITY OF CYTOCHROME P450 2E1

Abstract

Cytochrome P450 (CYP) is the predominate superfamily of enzymes responsible for Phase I metabolism of drugs and other xenobiotics. Understanding the structural reasons for the substrate selectivity of these enzymes is important for both pharmacological and toxicological reasons. Two isoforms of interest from this enzyme superfamily that are CYP2A13 and CYP2E1. Cytochrome P450 2A13 (CYP2A13) is a lung specific enzyme known to activate the potent tobacco procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) into two carcinogenic metabolites. CYP2A13 has been crystallized and X-ray diffraction experiments illuminated the structure of this enzyme, but with an unknown ligand present in the enzyme active site. This unknown ligand was suspected to be indole but a selective method had to be developed to differentiate among indole and its metabolites in the protein sample. We successfully modified a microbiological colorimetric assay to spectrophotometrically differentiate between indole and a number of possible indole metabolites in nanomolar concentrations by derivatization with p-dimethylaminocinnamaldehyde (DMACA). Further differentiation of indoles was made by mass spectrometry (HPLC-UV/vis-MS/MS) utilizing the chromophore generated in the DMACA conjugation as a UV signature for HPLC detection. The ligand in the crystallized protein was unambiguously identified as unsubstituted indole, which facilitated refinement of two alternate conformations of indole in the CYP2A13 crystal structure active site. Human cytochrome P450 2E1 (CYP2E1) is a xenobiotic metabolizing enzyme that is highly conserved among mammals. In addition to small molecular weight exogenous drugs like the analgesic acetaminophen and the volatile anesthetic halothane, CYP2E1 is also involved in endogenous fatty acid metabolism. To more fully understand the structural factors that contribute to the substrate selectivity of CYP2E1, it has been cocrystallized with two structurally different heme-binding compounds: indazole, a small molecular weight inhibitor and ω-imidazolyl-decanoic acid, a fatty acid analog. Comparison of the CYP2E1 structures shows that only small side chain movements are required for the accommodation of the much larger fatty acid analog. Rotation of the side chain of F298 causes a change in the active site volume from 190 Å3 in the indazole-bound structure to 440 Å3 in the ω-imidazolyl-decanoic acid-bound structure. Future work will be focused on cocrystal structures of CYP2E1 with both longer and shorter chain analogs to better understand the ability of the enzyme to metabolize a variety of fatty acids substrates.