Heterotropic modulation of sulfotransferase 2A1 activity by celecoxib: product ratio switching of ethynylestradiol sulfation.

Abstract

The major sulfated product of 17alpha-ethynylestradiol (EE) after incubations with 3'-phosphoadenosine-5'-phosphosulfate and recombinant human sulfotransferase 2A1 (SULT2A1), or liver cytosol, is the 3-O-sulfate of EE. However, when celecoxib is also present in the incubation, sulfation is switched (in a concentration-dependent manner) from the 3-O-position to the 17beta-O-position of ethynylestradiol. In incubations with recombinant SULT2A1, increasing concentrations of celecoxib decreased the Vmax of 3-O-sulfate product formation by 3- to 4-fold, with no major change in the Km value. For 17beta-O-sulfate formation, increasing concentrations of celecoxib resulted in an 8-fold decrease in the Km and a 7-fold increase in Vmax. Celecoxib not only modulated the regioselectivity of the enzyme, but also activated the enzyme such that total sulfated product exceeded product formation by the native enzyme, 3- to 4-fold (at 250 microM celecoxib). Finally, IC50 values obtained by varying celecoxib concentrations (0-250 microM) at fixed concentrations of EE showed that 3-O-sulfation was inhibited by celecoxib to the same extent, independent of the concentration of EE. In addition, the apparent kinetic constant for celecoxib (as measured by EE 17beta-O-sulfation) decreased 2-fold in the presence of high concentrations of EE, consistent with the potential for celecoxib to bind to either the enzyme-EE complex or to free enzyme. Taken as a whole, these data suggest that celecoxib is acting as a heterotropic modulator of SULT2A1 activity, most likely involving a separate noncompetitive binding site.