Testing the Sulfotransferase Molecular Pore Hypothesis
Testing the Sulfotransferase Molecular Pore Hypothesis
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DOI：
10.1074/jbc.m112.445015
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发表时间：
2013-03-22
影响因子：
4.8
通讯作者：
Leyh, Thomas S.
中科院分区：
文献类型：
--
作者：
Cook, Ian;Wang, Ting;Leyh, Thomas S.
人的胞浆硫转移酶(Sults)通过将硫酰部分(-SO3)从活化的硫酸盐(3‘-磷酸腺苷-5’-磷酸硫酸盐)转移到异源和内生生物的羟基和伯胺上来调节数百种信号代谢物的活性。Sults如何从胞质环境中存在的大量竞争配体中选择底物是该领域的一个重要问题。选择性似乎是由一个分子孔空间控制的，分子孔的打开和关闭响应于核苷酸结合。这一观点是由显示核苷酸依赖的孔闭合的结构和核苷酸结合诱导异构化的事实所促进的，该异构化限制了对受体结合口袋的访问。分子动力学模型强调了孔异构化在选择性中的重要性，并预测特定的分子键稳定闭合的孔以响应核苷酸结合。为了验证孔模型，通过诱变破坏了SULT2A1中的这些连接，并确定了它们对选择性的影响。这些突变将核苷酸结合从选择性中分离出来，并产生了不再区分大小底物的酶。这些突变没有影响小底物的亲和力或周转率，但导致对大底物的催化效率提高了183倍。模型预测，当核苷酸结合时，覆盖在受体结合口袋上的11个残基的“瓣”可以打开并接纳大底物；一个突变结构证明了这一点。总而言之，该模型被证明是一个稳健、准确的结果结构和选择性的预测者，其一般特征可能适用于结果家族的其他成员。
Human cytosolic sulfotransferases (SULTs) regulate the activities of hundreds of signaling metabolites via transfer of the sulfuryl moiety (-SO3) from activated sulfate (3'-phosphoadenosine 5'-phosphosulfate) to the hydroxyls and primary amines of xeno- and endobiotics. How SULTs select substrates from the scores of competing ligands present in a cytosolic milieu is an important issue in the field. Selectivity appears to be sterically controlled by a molecular pore that opens and closes in response to nucleotide binding. This point of view is fostered by structures showing nucleotide-dependent pore closure and the fact that nucleotide binding induces an isomerization that restricts access to the acceptor-binding pocket. Molecular dynamics models underscore the importance of pore isomerization in selectivity and predict that specific molecular linkages stabilize the closed pore in response to nucleotide binding. To test the pore model, these linkages were disrupted in SULT2A1 via mutagenesis, and the effects on selectivity were determined. The mutations uncoupled nucleotide binding from selectivity and produced enzymes that no longer discriminated between large and small substrates. The mutations did not affect the affinity or turnover of small substrates but resulted in a 183-fold gain in catalytic efficiently toward large substrates. Models predict that an 11-residue "flap" covering the acceptor-binding pocket can open and admit large substrates when nucleotide is bound; a mutant structure demonstrated that this is so. In summary, the model was shown to be a robust, accurate predictor of SULT structure and selectivity whose general features will likely apply to other members of the SULT family.