The metabolism and detoxication of potentially toxic foreign (xenobiotic) molecules in eukaryotes relies on the involvement of several membrane- bound enzymes such as cytochrome P-450, epoxide hydrolase and UDP- glucuronosyltransferase that participate in coherent metabolic pathways. To truly comprehend the pathways for the metabolism and detoxication of xenobiotics it is necessary to have a basic understanding of the mechanism of action, substrate specificity and structure of each enzyme that participates or could participate in metabolism. The research program outlined in this proposal seeks to enhance that understanding with respect to two membrane-bound detoxication enzymes, epoxide hydrolase (EH) and UDP-glucuronosyltransferase (UDPGT). Microsomal EH catalyzes the trans-addition of water to epoxides that are often generated by the action of cytochrome P-450 on unsaturated hydrocarbons. UDPGT catalyzes the transfer of the glucuronyl group from UDP-glucuronate to hydrophobic molecules bearing nucleophilic functional groups such as - OH, -SH, -NH2 and -COOH. The net result of this reaction is to enhance the solubility, transport and excretion of the parent molecule.
The specific aims for the proposed project period focus on three principal areas of inquiry including the development of suitable expression systems for the two proteins, studies of the mechanism of action of the enzymes using the specifically designed substrates, kinetic, and isotopic labeling experiments, and investigations of the structure of the enzymes using physical and biochemical techniques.
The specific aims of the project with respect to epoxide hydrolase are: (i) the construction and optimization of bacterial or baculovirus-based expression systems to produce sufficient quantities of protein for mechanistic and structural studies; (ii) the use of nitrogen-containing substrate analogues (azaarene oxides and aziridines) and sterically hindered arene oxides to probe the mechanism of action and the role of desolvation in substrate recognition and catalysis; (iii) single-turnover isotopic labeling experiments to rule out or confirm the participation f an ester intermediate in catalysis; (iv) site specific mutagenesis to identify putative active site residues; (v) hydrodynamic studies to define the oligomeric state of the native enzyme and N-terminal truncated versions of the enzyme as a prelude to a search for x-ray diffraction-quality single crystals of EH.
The specific aims for the investigations of UDP- glucuronosyltransferase include (i) construction of a baculovirus-based expression system for the phenol UDPGT and characterization of the immature and mature protein products and various truncated proteins; (ii) a search for a suitable bacterial expression system; (iii) definition of the internal equilibrium constant in catalysis for selected substrates in an attempt to define an """"""""evolutionarily optimal"""""""" substrate and (iv) 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 enhance our ability to predict metabolic scenarios for new 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 |
