Human Cytochrome P450 2E1: Functional Comparison to Cytochromes P450 2A13 and 2A6 by Melanie A. Blevins B.S., Graceland University, 2002 Submitted to the Department of Medicinal Chemistry and the Faculty of the Graduate School of the University of Kansas in partial fulfillment of the requirements for the degree of Master’s of Science. Dissertation Committee: Major Professor Committee Date Defended: April 24, 2008 The Thesis Committee for Melanie Blevins certifies that this is the approved Version of the following thesis: Human Cytochrome P450 2E1: Functional Comparison to Cytochromes P450 2A13 and 2A6 Committee: _______________________________ Chairperson* _______________________________ _______________________________ ii Abstract The cytochrome P450 (CYP) superfamily of enzymes plays the predominant role in human phase I xenobiotic metabolism. These enzymes participate in the metabolism of a greater part of the drugs in present clinical use and have been linked to the activation of carcinogens and other toxins. The CYP2 family, in particular, is known for it extensive Phase I metabolism of a majority of the xenobiotic compounds [1]. The goal of this project is to determine the structural foundation for the substrate selectivities of the CYP2A6 and CYP2A13 enzymes versus CYP2E1. Because these enzymes metabolize both common, as well as unique, small molecule substrates, it is likely that only few key residue-substrate interactions are responsible for those metabolic capabilities that differ between them. Amino acid residues in regions of the CYP2E1 protein likely to contact ligands and that differ between CYP2E1 and the CYP2A enzymes were examined by site-directed mutagenesis. The resulting mutated CYP2E1 proteins were characterized for their ability to hydroxylate the reportedly selective CYP2E1 substrates p-nitrophenol (pNP) [2] and chlorzoxazone (CZN) [3], but none showed significant differences in activity from the CYP2E1 wild type enzyme. iii However, in contrast to previous literature reports [4], both CYP2A6 and CYP2A13 were observed to metabolize both CYP2E1 substrates pNP and CZN with catalytic efficiencies equal to or greater than CYP2E1 (Table 1). These unexpected activities of the CYP2A enzymes with CYP2E1 substrates demonstrate that the human CYP2A and CYP2E enzymes are more functionally similar than previously believed. Table 1: pNP and CZN kinetic parameters for CYP2E1, CYP2A13, and CYP2A6. p-Nitrophenol Chlorzoxazone CYP450 kcat Km kcat/Km kcat Km kcat/Km (min-1) (µM) (µM-1min-1) (min-1) (µM) (µM-1min-1) 2E1 15.9 ± 0.4 75.8 ± 4.4 0.21 5.1 ± 0.4 105.5 ± 22.9 0.05 2A13 30.3 ± 1.3 62.7 ± 7.4 0.48 22.7 ± 1.1 64.8 ± 10.4 0.23 2A6 52.6 ± 2.4 135.8 ± 13.7 0.39 4.7 ± 0.3 100.7 ± 15.1 0.07 References 1 Rendic, S. and Di Carlo, F. J. (1997) Human cytochrome P450 enzymes: a status report summarizing their reactions, substrates, inducers, and inhibitors. Drug Metab Rev 29, 413-580 2 Koop, D. R., Laethem, C. L. and Tierney, D. J. (1989) The utility of p-nitrophenol hydroxylation in P450IIE1 analysis. Drug Metab Rev 20, 541-551 3 Peter, R., Bocker, R., Beaune, P. H., Iwasaki, M., Guengerich, F. P. and Yang, C. S. (1990) Hydroxylation of chlorzoxazone as a specific probe for human liver cytochrome P-450IIE1. Chem Res Toxicol 3, 566-573 4 Zerilli, A., Ratanasavanh, D., Lucas, D., Goasduff, T., Dreano, Y., Menard, C., Picart, D. and Berthou, F. (1997) Both cytochromes P450 2E1 and 3A are involved in the O- hydroxylation of p-nitrophenol, a catalytic activity known to be specific for P450 2E1. Chem Res Toxicol 10, 1205-1212 iv Acknowledgements I would like to thank all of the people who helped me along the way during my graduate career. I would like to acknowledge all of the guidance from Brian Smith and Lena Zaitseva in helping with the optimization of the CYP2E1 purification protocol. I would also like to thank Patrick Porubsky for the purification of NADPH-P450 oxidoreductase, CYP2A13, and CYP2A6. I would like to thank all of the past and present members of the Scott lab, Natash DeVore, Naseem Nikaeen, Eric Carrillo, Anu Metha, Agnes Walsh, Kathleen Meneely, and Linda Blake for their support and encouragement over the last few years. Thank you to my committee members Dr. David Benson and Dr. Sunil David for taking the time to review my research. A humongous thank you to my advisor, Dr. Emily Scott, for her continous patience, and guidance through out my graduate career. A special thank you to the NIH RR17708 and GM076343 grants for funding. Finally, I’d like to offer a huge thank you to my family and friends who have counseled me through my graduate career. You all have contributed greatly to my success here at the University of Kansas. Without all of your support I would have never made it this far. v Table of Contents Page Abstract iii Acknowledgements v List of Figures viii List of Tables x List of Schemes xi Chapter 1. Introduction to Cytochromes P450 1 Introduction 1 Xenobiotic Metabolism 2 Cytochromes P450 4 CYP450 Enzymes: Organization of Isozymes and Nomenclature 8 CYP450 Structure and Active Site Topography 10 The Catalytic Cycle 13 CYP450 2 Family: Substrate Overlap and Diversity 16 CYP2A6 19 CYP2A13 21 CYP2E1 23 Enzyme Kinetics 25 References 28 Chapter 2. Project Goals, Hypothesis, and Design 37 Project Goals and Hypothesis 37 Project Design 38 References 42 Chapter 3. Site-Directed Mutagenesis, Expression, and Protein Purification 44 Introduction 44 vi Methods 56 Site-directed mutagenesis 56 Purification of Plasmid DNA 59 Restriction Enzyme Digestion 60 Protein Expression 62 Protein Purification 63 Results 64 Conclusions 71 References 72 Chapter 4. Characterization of 2E and 2A Proteins Using Chlorzoxazone and p-Nitrophenol Hydroxylation Assays 73 Introduction 73 Methods 80 p-Nitrophenol Hydroxylation Assay 80 Chlorzoxazone Hydroxylation Assay 82 Results 83 p-Nitrophenol Metabolism Assay 84 Chlorzoxazone Metabolism Assay 90 Conclusions 94 References 97 Chapter 5. Conclusions 100 References 111 vii List of Figures Figures Page 1-1. Carbon monoxide difference spectrum 7 1-2. CYP450 enzyme heme moiety 7 1-3. Crystal structure of CYP2A13: substrate recognition sites 11 1-4. CYP450 enzymes involved in phase I metabolism 17 1-5. CYP2A6 substrates 20 1-6. CYP2A13 substrates 22 1-7. CYP2E1 substrates 24 1-8. Michaelis-Menten kinetics 27 2-1. Amino acid sequence alignment of CYP2E1, CYP2A13, and CYP2A6 40 3-1. Site-directed mutatgenesis strategy 46 3-2. QuikChange II site-directed mutagenesis strategy 49 3-3. Schematic of Ni-NTA metal affinity column chromatography 53 3-4. Schematic of ion-exchange column chromatography 55 4-1. The hydroxylation of p-nitrophenol 76 4-2. The hydroxylation of chlorzoxazone 78 4-3. Comparison of CYP450 p-nitrophenol activities 85 4-4. Comparison of Michaelis-Menten kinetics determined by both visible colorimetric and HPLC UV detection methods 87 4-5. Overlay of enzyme kinetics for p-nitrophenol metabolism by CYP2E1, CYP2A6, and CYP2A13. 89 4-6. Comparison of CYP450 chlorzoxazone activities 91 4-7. Overlay of enzyme kinetics for chlorzoxazone metabolism by CYP2E1, CYP2A6, and CYP2A13 93 viii 5-1. Overlay of crystal structures of CYP2E1 and CYP2A13 104 5-2. Crystal structure of CYP2E1: substrate recognition site 1 106 5-3. Crystal structure of CYP2E1: substrate recognition site 2 109 List of Tables Tables Page 1-1. Human CYP2 family enzymes and their known locations, reactions, and inducers 18 3-1. Thermal cycling parameters used for mutagenesis reactions 58 3-2. Restriction enzyme reaction conditions 61 3-3. The physical characteristics of designed oligonucleotides 65 3-4. Site-directed mutagenesis results 66 3-5. Characterization of purified CYP2E1 proteins by UV/Vis spectroscopy and CO difference spectra. 69 4-1. Comparison of p-nitrophenol activity determined by visible colorimetric and HPLC UV detection methods 87 4-2. p-Nitrophenol kinetic parameters determined for CYP2E1, 2A6, and 2A13 89 4-3. Chlorzoxazone kinetic parameters determined for CYP2E1, 2A6, and 2A13 93 4-4. Comparison between CYP2E1 mutant protein activities for p-nitrophenol and chlorzoxazone 96 5-1. p-Ntirophenol and chlorzoxazone kinetic parameters for CYP2E1, 2A13, and 2A6. 101 ix List of Schemes Schemes Page 1.1. The catalytic cycle of cytochromes P450 14 x Chapter 1 Introduction to Cytochrome P450 Enzymes Introduction Metabolism can be described as the chemical and physical processes occurring within a living organism, involved in the maintenance of life [1]. In humans, a large portion of metabolic energy is involved in energy production (catabolism) and protein and nucleic acid biosynthesis (anabolism). Nonetheless, xenobiotic metabolism plays a crucial role in preserving homeostasis. Because humans are continually exposed to a variety of foreign compounds, the metabolic conversion and subsequent removal of these often lipophilic and toxic compounds from the body is an important process. Over the years, the human body has evolved the ability to defend itself against lipophilic environmental toxins that otherwise persist in cells. The primary defense is the use of enzymes that metabolize these lipophilic foreign compounds into more polar molecules that can easily be excreted. Enzymes catalyze the majority of these chemical transformations in the liver and many other extrahepatic tissues, including the kidneys, brain, respiratory tract, and GI tract [2]. 1 Consideration of the metabolism of a drug is an integral part of developing a drug for clinical use. It is one of the four main pharmacological considerations when administering a drug: absorption, distribution, metabolism, and excretion [3]. If a drug is cleared too slowly or too quickly, the drug will not be maintained within the therapeutic window, causing treatment to fail due to either lack of therapeutic effect or associated toxicity [4]. It is also important that the drug be metabolically converted into metabolites that are relatively nontoxic.