Recksiek M, Selmer T, Dierks T, Schmidt B, von Figura K
Institut für Biochemie und Molekulare Zellbiologie, Abt. Biochemie II, Universität Göttingen, Gosslerstr. 12d, 37073 Göttingen, Germany.
J Biol Chem. 1998 Mar 13;273(11):6096-103. doi: 10.1074/jbc.273.11.6096.
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.
硫酸酯酶含有一个通过翻译后修饰产生的活性位点甲酰甘氨酸残基。两种溶酶体硫酸酯酶的晶体结构显示，其与在甲酰甘氨酸位置含有丝氨酸的碱性磷酸酶的催化位点有显著相似性。为了阐明硫酸酯水解的催化机制，将芳基硫酸酯酶A和B的甲酰甘氨酸替换为丝氨酸。这些突变体与底物一起孵育时，在引入的丝氨酸处被共价硫酸化。这种硫酸化的酶中间体在pH 5时稳定。在碱性pH下，它会缓慢水解。这些特性与碱性磷酸酶类似，碱性磷酸酶形成在pH 5时稳定但在碱性pH下会水解的磷酸丝氨酸中间体。在野生型硫酸酯酶中，形成硫酸化酶中间体所需的羟基由甲酰甘氨酸的醛水合物提供。醛水合物的第二个未酯化羟基对于酶在酸性pH下的快速脱硫至关重要，这很可能是通过消除反应发生的。丝氨酸突变体中缺乏这个第二个羟基解释了硫酸化酶中间体的捕获。因此，甲酰甘氨酸残基作为偕二醇，在酸性环境中允许高效的酯水解。
