Purification and kinetic characterization of a phenol-sulfating form of phenol sulfotransferase from human brain

The identification of three forms of phenol sulfotransferase (PST) in human brain and the subsequent purification and kinetic characterization of a phenol-sulfating form of the enzyme are described. Two forms of PST which were capable of conjugating phenol and a third form which sulfated dopamine were resolved from one another using DEAE-cellulose chromatography. One of the phenol-sulfating forms (P_I-PST) was sub-sequently purified on Affi-Gel blue and Sephacryl S-200, giving a final purification of almost 390-fold, with an overall yield of approximately 5%. The purified enzyme was sensitive to NaCl and showed an optimum for phenol conjugation at pH 8.5. Kinetic analysis demonstrated that sulfation by P_I-PST proceeds via a sequential ordered, bisubstrate reaction mechanism, where 3′-phosphoadenosine-5′-phosphosulfate (PAPS) is the leading substrate. The true K_m and K_ia values for PAPS were both 0.35 μm, while the true K_m value for phenol was 2.8 μm.