The metabolism and detoxication of potentially toxic xenobiotic molecules relies on the involvement of several membrane-bound enzymes including the cytochromes P-450, microsomal epoxide hydrolase and UDP-glucuronosyltransferase. Epoxide hydrolase catalyzes the trans-addition of water to epoxides that are often generated by the action of the cytochromes P-450 on unsaturated hydrocarbons. UDP-glucuronosyltransferase catalyzes the transfer of the clucuronyl group from UDP-glucuronate to hydrophobic molecules bearing nucleophilic functional groups such as -OH, -SH, -NH2 and - COOH. The net result of these reactions is to enhance the solubility, transport and excretion of the parent molecules.
The specific aims for the proposed project period are focused on three general areas of inquiry including the improvement of expression systems for the proteins, the elucidation of the mechanisms of action of the enzymes and preliminary investigations of the structure of the enzymes by X-ray crystallography and biochemical techniques.
The specific aims of the project with respect to microsomal epoxide hydrolase are: (I) the elucidation of the kinetic mechanism by pre-steady-state and steady- state techniques; (ii) the definition of the chemical mechanism of catalysis by site-directed mutagenesis and characterization of the ester intermedicate; (iii) evaluation of potential mechanism-based inhibitors of the enzyme; and (iv) production of X-ray diffraction-quality single crystals.
The specific aim for the investigations of UDP- glucuronosyltransferase include: (I) optimization of the expression of the phenol UDPGT in a baculovirus-based or bacterial systems and ; (ii) an exploration of the conformer specificity of the enzyme toward dihydrodiol substrates. These studies are anticipated to increase our understanding of the molecular details of the metabolism of environmental pollutants and drugs and eventually enhance our ability to predict metabolic scenarios for new, potentially toxic compounds.
| Armstrong, R N; Cassidy, C S (2000) New structural and chemical insight into the catalytic mechanism of epoxide hydrolases. Drug Metab Rev 32:327-38 |
| Armstrong, R N (1999) Kinetic and chemical mechanism of epoxide hydrolase. Drug Metab Rev 31:71-86 |
| Tzeng, H F; Laughlin, L T; Armstrong, R N (1998) Semifunctional site-specific mutants affecting the hydrolytic half-reaction of microsomal epoxide hydrolase. Biochemistry 37:2905-11 |
| Laughlin, L T; Tzeng, H F; Lin, S et al. (1998) Mechanism of microsomal epoxide hydrolase. Semifunctional site-specific mutants affecting the alkylation half-reaction. Biochemistry 37:2897-904 |
| Lacourciere, G M; Armstrong, R N (1994) Microsomal and soluble epoxide hydrolases are members of the same family of C-X bond hydrolase enzymes. Chem Res Toxicol 7:121-4 |
