Purification and reaction mechanism of arylsulfate sulfotransferase from Haemophilus K-12, a mouse intestinal bacterium

A novel type of sulfotransferase, arylsulfate sulfotransferase [EC 2.8.2.22], was purified to homogeneity from Haemophilus K-12, a mouse intestinal bacterium. The purified enzyme (M_r 290,000) is composed of four subunits (M_r 70,000). The best donor substrate was 4-methylumbelliferyl sulfate, followed by β-naphthyl sulfate, p-nitrophenyl sulfate (PNS), and α-naphthyl sulfate. The best acceptor substrate was α-naphthol, followed by phenol and resorcinol. The apparent K_m for PNS using phenol as an acceptor and that for phenol using PNS as a donor substrate were determined to be 0.095 and 0.71 mM, respectively. One of the reaction products, p-nitrophenol inhibited the enzyme noncompetitively with respect to PNS, but competitively with respect to α-naphthol. The K₁ values of PNP for PNS and α-naphthol were 0.89 and 0.12 mM, respectively. The other reaction product, α-naphthyl sulfate, inhibited the enzyme competitively with respect to PNS, but non-competitively with respect to α-naphthol. The K₁ values of α-naphthyl sulfate for PNS and for α-naphthol were 2.72 and 1.7 mM. These results suggest that the sulfate transfer reaction proceeds according to a ping pong bi bi mechanism.