| dc.description.abstract | The cytochrome P450 (P450) superfamily of mixed function oxidase enzymes catalyze the metabolism of a variety of endogenous and exogenous biochemicals, including steroids, fatty acids, vitamins, eicosanoids, drugs, pesticides, and toxins. P450-mediated oxidative metabolism often serves a beneficial role in the clearance of foreign compounds and the regulation of endogenous molecules, both of which are necessary for the maintenance of homeostasis. However, the reactions catalyzed by P450 enzymes also have the potential to promote disease and injury, whether it be in the activation of procarcinogens or by undermining the therapeutic efficacy of a drug. Altogether, these processes make P450 enzymes important subjects of interest for the prediction of chemical toxicology and the development of therapeutic agents. The focus of this thesis research is the characterization of the CYP26 and CYP2A families of P450 enzymes in an effort to contribute to an understanding of how substrates and inhibitors specifically interact with the individual enzymes within these families. The functional enzymes of the human CYP2A family include CYP2A6 and CYP2A13. CYP2A6 is primarily a hepatic enzyme, while CYP2A13 is mainly expressed within the respiratory tract. Though these two enzymes are primarily localized to different tissues within the body, they share many substrates in common as a result of their 94% amino acid sequence identity. These substrates include: nicotine, cotinine, para-nitrophenol, and coumarin. However, CYP2A13 appears to preferentially activate a number of procarcinogens, including the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), into reactive intermediates that can result in DNA adducts and the initiation or promotion of carcinogenesis. In order to evaluate the relative involvement of CYP2A6 activity versus that of CYP2A13 in the metabolism and/or activation of potentially harmful chemical agents in vivo, selective inhibitors are needed. A number of compounds were reported to selectively inhibit CYP2A6 before CYP2A13 was determined to be a functional member of the CYP2A family, or have simply not been tested against CYP2A13. This work examined such compounds including: phenethyl isothiocyante (PEITC), 4-dimethylaminobenzaldehyde (DMABA), 8-methoxypsoralen (8-MOP), tranylcypromine, tryptamine, pilocarpine, (S)-nicotine, (R)-(+)-menthofuran, and β-nicotyrine. The relative impact of these inhibitors on CYP2A6 and CYP2A13 function was evaluated by determining Ki values and modes of inhibition for each of the compounds against both enzymes, followed by the calculation of a selectivity factor. The results of these studies serve as the first explicit determination of the selectivity of these compounds for enzymes within the CYP2A family and indicate that only (R)-(+)-menthofuran and tranylcypromine demonstrate even a 10-fold preference for CYP2A6 inhibition over CYP2A13. This information can be used as a guide for the selection of inhibitors with the greatest potential for determining whether CYP2A6 or CYP2A13 is responsible for the metabolism or activation of procarcinogenic compounds, such as tobacco-derived NNK in the human respiratory tract. Collectively, the CYP26 family of P450 enzymes serve as important mediators of retinoic acid (RA) catabolism in the body. In humans, the CYP26 family of enzymes consists of three isoforms: CYP26A1, CYP26B1, and CYP26C1. As a result of RA's endogenous role in regulating cellular growth and differentiation, geometric isomers of RA such as all-trans-RA (atRA) and 13-cis-RA represent attractive targets for cancer therapy and the treatment of dermatological conditions. Unfortunately, RA resistance is often experienced in patients undergoing prolonged RA-based therapy. This failure has been suggested to be the result of up-regulation of P450 enzymes, particularly the enzymes of the CYP26 family, resulting in enhanced RA metabolism in vivo. As a result, a significant amount of work has been conducted for the development of chemical agents that inhibit P450-mediated metabolism of atRA, commonly referred to as retinoic acid metabolism blocking agents (RAMBAs). The development of safe and effective RAMBAs could be greatly facilitated by more detailed structural and functional characterizations of the CYP26 family of enzymes, since very little is known about these enzymes aside from their dominant role in atRA hydroxylation. However, the fact that the human CYP26 enzymes are membrane-bound proteins, a characteristic shared by all human P450 enzymes, represents a major challenge to the recombinant expression and purification of these enzymes in sufficient quantities to enable detailed biochemical and biophysical characterization studies. To address this challenge, a new method for the production of pure CYP26 enzymes was developed based on techniques that have proven successful in the expression and purification of other membrane-bound P450 enzymes in E. coli, while incorporating the use of detergents and stabilizing ligands specific to the CYP26 enzymes. The combination of these approaches led to the successful generation of mouse CYP26A1 and CYP26B1 proteins in yields of 200-400 nmol per 2.25 L of E. coli expression media and the ability to recover 20-50 nmol following a single chromatographic step. Unfortunately, this protein appeared to be inactive according to reduced carbon monoxide difference spectrum analysis and assays of RA metabolism. As a result, further optimization will be required to reach the original goal set forth to generate pure recombinant CYP26 enzymes for basic kinetic and structural determination studies. | |
