Whittemore R M, Pearce L B, Roth J A
Biochemistry. 1985 May 7;24(10):2477-82. 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.
本文描述了从人脑中纯化得到的单胺硫酸化形式的酚硫酸转移酶（M-PST）的动力学和生化特性。通过在DEAE-纤维素上进行阴离子交换色谱，将M-PST活性与酚硫酸化活性分离并纯化，随后在AffiGel Blue和Sephacryl S-200上进一步纯化，常规情况下最终纯化倍数超过20000倍，产率约为3%。通过凝胶过滤色谱估计，活性物质的分子量为250000。纯化后的酶受到NaCl抑制（325 mM时抑制50%），在pH 7.0时多巴胺硫酸化的活性最佳。在所检测的单胺底物中，4-甲氧基酪胺在20 μM时硫酸化程度最高，而在较高底物浓度（200 μM）下，酪胺是明显的首选底物。动力学分析表明，M-PST的硫酸化反应通过有序的双底物反应机制进行，其中3'-磷酸腺苷5'-磷酸硫酸酯（PAPS）是主要底物。多巴胺和PAPS的真实Km值分别为2.9和0.35 μM。产物抑制剂3'-磷酸腺苷5'-磷酸的Ki为0.07 μM，而终产物抑制剂ATP的Ki为170 μM。
