Carboxyl residues in the active site of human phenol sulfotransferase (SULT1A1)

The carboxyl-specific amino acid modification reagent, Woodward's reagent K (WK), was utilized to characterize carboxyl residues (Asp and Glu) in the active site of human phenol sulfotransferase (SULT1A1). SULT1A1 was purifie d using the pMAL-c2 expression system in E. coil. WK inactivated SULT1A1 ac tivity in a time- and concentration-dependent manner. The inactivation foll owed first-order kinetics relative to both SULT1A1 and WK. Both phenolic su bstrates and adenosine 3'-phosphate 5'-phosphosulfate (PAPS) protected agai nst the inactivation, which suggests the carboxyl residue modification caus ing the inactivation took place within the active site of the enzyme. With partially inactivated SULT1A1, both V-max and K-m changed for PAPS, while f or phenolic substrates, V-max decreased and K-m did not change significantl y. A computer model of the three-dimensional structure of SULT1A1 was const ructed based on the mouse estrogen sulfotransferase (mSULT1E1) X-ray crysta l structure. According to the model, Glu83, Asp134, Glu246, and Asp263 are the residues likely responsible for the inactivation of SULT1A1 by WK. Acco rding to these results, five SULT1A1 mutants, E83A, D134A, E246A, D263A, an d E151A, were generated (E151A as control mutant). Specific activity determ ination of the mutants demonstrated that E83A and D134A lost almost 100% of the catalytic activity. E246A and D263P, also decreased SULT1A1 activity, while E151A did not change SULT1A1 catalytic activity significantly. This w ork demonstrates that carboxyl residues are present in the active site and are important for SULT1A1 catalytic activity. Glu83 and E134 are essential amino acids for SULT1A1 catalytic activity.