Reaction product affinity regulates activation of human sulfotransferase 1A1 PAP sulfation

Abstract

Cytosolic sulfotransferase (SULT)-catalyzed sulfation regulates the activity of bio-signaling molecules and aids in metabolizing hydroxyl-containing xenobiotics. The sulfuryl donor for the SULT reaction is adenosine 3'-phosphate 5'-phosphosulfate (PAPS), while products are adenosine 3',5'-diphosphate (PAP) and a sulfated alcohol. Human phenol sulfotransferase (SULT1A1) is one of the major detoxifying enzymes for phenolic xenobiotics. The mechanism of SULT1A1-catalyzed sulfation of PAP by pNPS was investigated. PAP was sulfated by para-nitrophenyl sulfate (pNPS) in a concentration-dependent manner. 2-Naphthol inhibited sulfation of PAP, competing with pNPS, while phenol activated the sulfation reaction. At saturating PAP, a ping pong kinetic mechanism is observed with pNPS and phenol as substrates, consistent with phenol intercepting the E-PAPS complex prior to dissociation of PAPS. At high concentrations, phenol competes with pNPS, consistent with formation of the E-PAP-phenol dead-end complex. Data are consistent with the previously reported mechanism for sulfation of 2-naphthol by PAPS, and its activation by pNPS. Overall, data are consistent with release of PAP from E-PAP and PAPS from E-PAPS contributing to rate-limitation in both reaction directions.

Fig. 1

Initial velocity pattern obtained in the reverse reaction direction with PAP and pNPS as reactants. Rates were measured at pH 6.2 and 37 °C with PAP varied at the following concentrations: 5 μM (◇), 10 μM (△) and 20 μM (○). Points in the primary plot are experimental, while curves are theoretical based on a fit to Eq. (2). All rates are per mg protein.

Fig. 2

Saturation curves with pNPS as the sulfuryl donor in the reverse reaction direction. Rates were measured at pH 6.2 and 37 °C with PAP fixed at 0.1 mM and varying the concentration of pNPS as shown in the absence of alcohol (●), and in the presence of 0.1 mM 2-naphthol (△) or 0.1 mM phenol (○). Data were fitted using Eq. (1). All rates are per mg protein.

Fig. 3

Initial velocity pattern for activation by phenol. Rates were measured at pH 6.2 and 37 °C. The initial rate was measured as a function of pNPS with PAP fixed at 0.1 mM and phenol varied at the following phenol concentrations: 0 (●), 6.25 μM (□), 25 μM (▲), 100 μM (◇), 400 μM (△), 1.6 mM (○). Points are experimental, while the lines are based on a fit to Eq. (2). The line with closed circles is for zero phenol and was fitted separately using Eq. (1). All rates are per mg protein.

Scheme 1

Proposed ping pong mechanism for the phenol activation of PAP sulfation by human sulfotransferase 1A1.