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 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.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D015239:Arylsulfotransferase; D002540:Cerebral Cortex / Q000201:enzymology*; D004298:Dopamine / Q000031:analogs & derivatives* / Q000032:analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009994:Osmolar Concentration; D013379:Substrate Specificity; D013466:Sulfurtransferases / Q000302:isolation & purification* / Q000378:metabolism
C022807:dopamine 4-O-sulfate; C007558:dopamine 3-O-sulfate; D000255:Adenosine Triphosphate; D013466:Sulfurtransferases; D015239:Arylsulfotransferase; D004298:Dopamine