Bacterial arylsulfate sulfotransferase (ASST) catalyzes the transfer of a sulfate group from a phenyl sulfate ester to a phenolic acceptor. The kinetic mechanism of Enterobacter amnigenus ASST was determined. Plots of 1/v versus 1/[substrate (A)] at different fixed substrate (B) concentrations gave a series of parallel lines. One of the reaction products, p-nitrophenol, inhibited the enzyme noncompetitively with respect to p-nitrophenyl sulfate, but competitively to alpha -naphthol. These results correspond to a ping pong bi bi mechanism. By site-directed mutagenesis, we substituted each conserved tyrosine residue with phenylalanine. Among the mutants, Y123F showed severely reduced catalytic activity. We conclude that Tyr 123 is an essential active site residue. A mechanistic hypothesis is presented to account for these observations.