| pubmed-article:8814222 | pubmed:abstractText | Two cDNA clones of rat hepatic hydroxysteroid sulfotransferase (ST) (ST-40 and ST-20) were isolated and expressed in Escherichia coli cells. Several histidine residues in their coding regions are highly conserved in the ST superfamily, and histidine mutants were constructed by site-directed mutagenesis. The substitution of alanine or lysine for the histidine at position 98 in the ST-40 enzyme resulted in a loss of ST activities toward dehydroepiandrosterone (DHEA), androsterone (AD) and cortisol (CS). The mutation of histidine 98 into alanine abolished the specific binding to 3'-phosphoadenosine 5'-phosphate agarose, suggesting that the residue is located at a critical position in the 3'-phosphoadenosine 5'-phosphosulfate (PAPS) binding site. In the ST-20 enzyme, the replacement of histidine 98 with alanine also resulted in the loss of ST activity toward its preferential substrate, CS. In the ST-40 enzyme, the mutation at histidine 256 into alanine markedly reduced CS-ST activity, but DHEA-ST activity was not changed. Furthermore, selective decrease in CS-ST activity was also observed in the alanine mutant at lysine 254 or at asparagine 255 of the ST-40 enzyme. Kinetic analysis on the ST-40 and its mutant at asparagine 255 indicated that the Km value for CS was significantly increased in the mutant without any change in the Km values for 3'-phosphoadenosine 5'-phosphosulfate and DHEA. Inhibition studies demonstrated that DHEA-ST activity was competitively inhibited by AD, but not by CS in the ST-40 enzyme, whereas triethylamine, a noncompetitive inhibitor of hydroxysteroid ST, inhibited DHEA-ST activity in the ST-40 enzyme but did not inhibit CS-ST activity in either ST-40 or ST-20 enzymes. These data provide evidence that DHEA and CS bind to different sites, which probably function in a different manner in the ST-40 enzyme. | lld:pubmed |
