Sulfuryl transfer: The catalytic mechanism of human estrogen sulfotransferase
Sulfuryl transfer: The catalytic mechanism of human estrogen sulfotransferase
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DOI：
10.1074/jbc.273.18.10888
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发表时间：
1998-05-01
影响因子：
4.8
通讯作者：
Leyh, TS
中科院分区：
文献类型：
--
作者：
Zhang, HP;Varmalova, O;Leyh, TS
雌激素磺基转移酶（EST）催化硫酰基从3 '-磷酸腺苷5'-磷酸硫酸酯（PAPS）转移到17 β-雌二醇（E-2）。E-2的硫酸化阻止其结合并由此激活雌激素受体。EST的调节似乎与乳腺和子宫内膜的肿瘤发生有因果关系。在这项研究中，重组人EST的特点，并在配体结合和初始速率实验的转移反应的催化机制进行了研究。天然酶是35-kDa亚基的二聚体，在pH 6.3和T = 25 +/- 2 ℃时，转移至E-2的表观平衡常数为（4.5 +/- 0.2）× 10（3）。初始速率研究提供了反应的动力学常数并提出了顺序机制。E-2是部分底物抑制剂（Ki = 80 +/- 5 nM）。两个E-2每个EST亚基的结合表明，通过在变构位点的结合发生部分抑制。除了提供配体-酶复合物的解离常数外，结合研究还表明，每种底物独立地与酶结合，并且形成E. PAP. E2 S和E.PAP.E-2死端复合物。这些结果强烈建议一个随机Ri Ri机制与两个死端复合物。
Estrogen sulfotransferase (EST) catalyzes the transfer of the sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to 17 beta-estradiol (E-2), The sulfation of E-2 prevents it from binding to, and thereby activating, the estrogen receptor. The regulation of EST appears to be causally linked to tumorigenesis in the breast and endometrium. In this study, recombinant human EST is characterized, and the catalytic mechanism of the transfer reaction is investigated in ligand binding and initial rate experiments. The native enzyme is a dimer of 35-kDa subunits, The apparent equilibrium constant for transfer to E-2 is (4.5 +/- 0.2) x 10(3) at pH 6.3 and T = 25 +/- 2 degrees C. Initial rate studies provide the kinetic constants for the reaction and suggest a sequential mechanism. E-2 is a partial substrate inhibitor (K-i = 80 +/- 5 nM). The binding of two E-2 per EST subunit suggests that the partial inhibition occurs through binding at an allosteric site. In addition to providing the dissociation constants for the ligand-enzyme complexes, binding studies demonstrate that each substrate binds independently to the enzyme and that both the E.PAP.E2S and E.PAP.E-2 dead-end complexes form. These results strongly suggest a Random Ri Ri mechanism with two dead-end complexes.