An investigation into the mechanism of action of the membrane-bound detoxication enzyme UDP glucuronosyltransferase is underway. The specific aims of the study are to i) elucidate the kinetic mechanism of the enzyme using alternative substrates ii) probe the structure of the transition state of the enzymatic reaction using specifically designed alternative substrates and linear-free energy relationships iii) determine the effect of membrane or phospholipid environment on the transition state of the reaction by observing their effects on linear-free energy relationships iv) probe the topology of the active site of the enzyme toward sterically hindered chiral phenols. Alternative glucuronosyl-donating substrate having similar geometries but with leaving groups (e.g. UDP, UMPCH2P, UMPCHFP, and UMPCF2P) of different pKa will be synthesized and used to probe the amount and importance of bond-breaking in the transition-state. Nucleophilicity of the aglycone will be varied by using the appropriately substituted phenol to examine the degree of bond-making in the transition state. The enzyme will be incorporated into different micellar or vesicular environments to determine the importance of membrane stereoselectivity of the enzyme and the equilibrium constant for glucuronidation of dihydrodiols is being determined in the reverse (deconjugation) reaction. Finally, chiral phenols of known stereochemistry will be used to determine the extent to which the aglycone binding site can recognize chiral substrate surfaces.
