Purification and Kinetic Characterization of a Dopamine-Sulfating Form of Phenol Sulfotransferase from Human Brain

The kinetic and biochemical properties of a purified, monoamine-sulfating form of phenol sulfotransferase (M-PST) from human brain are described. M-PST activity was separated and purified from phenol-sulfating activity by anion-exchange chromatography on DEAE-cellulose and subsequently purified on AffiGel Blue and Sephacryl S-200, routinely giving a final purification of over 20 000-fold, with approximately a 3% yield. The molecular weight of the active species, as estimated by gel filtration chromatography, was 250000. The purified enzyme was inhibited by NaCl (50% at 325 mM) and showi i an optimum for dopamine sulfation at pH 7.0. of the monoamine substrates examined, 4-methoxytyramine was the most extensively sulfated at 20 μM, while at higher substrate concentrations (200 μM), tyramine was the apparent preferred substrate. Kinetic analysis demonstrated that sulfation by M-PST proceeds via an ordered, bisubstrate reaction mechanism, where 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is the leading substrate. True Km values for dopamine and PAPS were 2.9 and 0.35 μM, respectively. The product inhibitor 3'-phosphoadenosine 5'-phosphate possessed a K1 of 0.07 μM, while the dead-end inhibitor ATP exhibited a Ki of 170 μM.