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

• Published in: Biochemistry (Easton) 1985-05, Vol.24 (10), p.2477-2482
• Main Authors: Whittemore, Russell M, Pearce, L. Bruce, Roth, Jerome A
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - pharmacology; Analytical, structural and metabolic biochemistry; aryl sulfotransferase; Arylsulfotransferase; Biological and medical sciences; brain; Cerebral Cortex - enzymology; dopamine; Dopamine - analogs & derivatives; Dopamine - analysis; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Humans; Hydrogen-Ion Concentration; Kinetics; man; Molecular Weight; Osmolar Concentration; Substrate Specificity; Sulfurtransferases - isolation & purification; Sulfurtransferases - metabolism; Transferases
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00331a013

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 250 000. The purified enzyme was inhibited by NaCl (50% at 325 mM) and showed an optimum for dopamine sulfation at pH 7.0. Of the monoamine substrates examined, 4-methoxytyramine was the most extensively sulfated at 20 microM, while at higher substrate concentrations (200 microM), 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 microM, respectively. The product inhibitor 3'-phosphoadenosine 5'-phosphate possessed a Ki of 0.07 microM, while the dead-end inhibitor ATP exhibited a Ki of 170 microM.