Sulfatases, Trapping of the Sulfated Enzyme Intermediate by Substituting the Active Site Formylglycine *
Sulfatases, Trapping of the Sulfated Enzyme Intermediate by Substituting the Active Site Formylglycine *
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硫酸酯酶，通过取代活性位点甲酰甘氨酸捕获硫酸化酶中间体 *
DOI：
--
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发表时间：
1998
影响因子：
4.8
通讯作者：
K. von Figura
中科院分区：
文献类型：
--
作者：
Michael Recksiek;T. Selmer;T. Dierks;B. Schmidt;K. von Figura
磺化酶含有一个活性位点甲酰甘氨酸残基，是由翻译后修饰产生的。两种溶酶体硫酸酯酶的晶体结构与甲酰基甘氨酸位置上含有丝氨酸的碱性磷酸酶的催化位点有显著的相似性。为了阐明硫酸盐酯水解的催化机理，用丝氨酸取代了芳基磺化酶A和B的甲酰基甘氨酸。这些突变体与底物孵育后，在引入的丝氨酸上被共价硫酸化。该硫酸酸化酶中间体在pH为5时稳定。在碱性条件下缓慢水解。这些特性与碱性磷酸酶类似，碱性磷酸酶形成的磷酸丝氨酸中间体在pH值为5时是稳定的，但在碱性pH下会被水解。在野生型磺化酶中，形成硫酸化酶中间体所需的羟基是由甲酰甘氨酸的醛水合物提供的。第二，非酯化羟基的醛水合物是必不可少的快速脱硫酶在酸性pH值，这很可能发生通过消除。丝氨酸突变体中缺乏第二个羟基解释了硫酸化酶中间体的捕获。因此，在作为双酚时，甲酰甘氨酸残基允许在酸性环境中进行有效的酯水解。
Sulfatases contain an active site formylglycine residue that is generated by post-translational modification. Crystal structures of two lysosomal sulfatases revealed significant similarity to the catalytic site of alkaline phosphatase containing a serine at the position of formylglycine. To elucidate the catalytic mechanism of sulfate ester hydrolysis, the formylglycine of arylsulfatases A and B was substituted by serine. These mutants upon incubation with substrate were covalently sulfated at the introduced serine. This sulfated enzyme intermediate was stable at pH 5. At alkaline pH it was slowly hydrolyzed. These characteristics are analogous to that of alkaline phosphatase which forms a phosphoserine intermediate that is stable at pH 5, but is hydrolyzed at alkaline pH. In wild-type sulfatases the hydroxyl needed for formation of the sulfated enzyme intermediate is provided by the aldehyde hydrate of the formylglycine. The second, non-esterified hydroxyl of the aldehyde hydrate is essential for rapid desulfation of the enzyme at acidic pH, which most likely occurs by elimination. The lack of this second hydroxyl in the serine mutants explains the trapping of the sulfated enzyme intermediate. Thus, in acting as a geminal diol the formylglycine residue allows for efficient ester hydrolysis in an acidic milieu.
大肠杆菌碱性磷酸酶的精制结构，分辨率为 2.8 A。
影响因子：
5.6
作者：
Sowadski,JM;Handschumacher,MD;Murthy,HM;Foster,BA;Wyckoff,HW
通讯作者：
Wyckoff,HW
DOI：
--
发表时间：
1985
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Gettins,P;Metzler,M;Coleman,JE
通讯作者：
Coleman,JE