Kinetic mechanism and identification of the active site tyrosine residue in Enterobacter amnigenus arylsulfate sulfotransferase

Bacterial arylsulfate sulfotransferase (ASST) catalyzes the transfer of a s ulfate group from a phenyl sulfate ester to a phenolic acceptor. The kineti c mechanism of Enterobacter amnigenus ASST was determined. Plots of 1/v ver sus 1/[substrate (A)] at different fixed substrate (B) concentrations gave a series of parallel lines. One of the reaction products, p-nitrophenol, in hibited the enzyme noncompetitively with respect to p-nitrophenyl sulfate, but competitively to alpha -naphthol. These results correspond to a ping po ng bi bi mechanism. By site-directed mutagenesis, we substituted each conse rved tyrosine residue with phenylalanine. Among the mutants, Y123F showed s everely reduced catalytic activity. We conclude that Tyr 123 is an essentia l active site residue. A mechanistic hypothesis is presented to account for these observations. (C) 2001 Academic Press.