Knaust A, Schmidt B, Dierks T, von Bülow R, von Figura K
Institut für Biochemie und Molekulare Zellbiologie, Abteilung Biochemie II, Universität Göttingen, Germany.
Biochemistry. 1998 Oct 6;37(40):13941-6. doi: 10.1021/bi9810205.
Sulfatases contain a unique posttranslational modification in their active site, a formylglycine residue generated from a cysteine or a serine residue. The formylglycine residue is part of a sequence that is highly conserved among sulfatases, suggesting that it might direct the generation of this unique amino acid derivative. In the present study residues 68-86 flanking formylglycine 69 in arylsulfatase A were subjected to an alanine/glycine scanning mutagenesis. The mutants were analyzed for the conversion of cysteine 69 to formylglycine and their kinetic properties. Only cysteine 69 turned out to be essential for formation of the formylglycine residue, while substitution of leucine 68, proline 71, and alanine 74 within the heptapeptide LCTPSRA reduced the formylglycine formation to about 30-50%. Several residues that are part of or directly adjacent to an alpha-helix presenting the formylglycine 69 at the bottom of the active site pocket were found to be critical for catalysis. A surprising outcome of this study was that a number of residues fully or highly conserved between all known eukaryotic and prokaryotic sulfatases turned out to be essential neither for generation of formylglycine nor for catalysis.
硫酸酯酶在其活性位点含有一种独特的翻译后修饰，即由半胱氨酸或丝氨酸残基生成的甲酰甘氨酸残基。甲酰甘氨酸残基是硫酸酯酶中高度保守序列的一部分，这表明它可能指导这种独特氨基酸衍生物的生成。在本研究中，对芳基硫酸酯酶A中甲酰甘氨酸69两侧的68 - 86位残基进行了丙氨酸/甘氨酸扫描诱变。分析了突变体中半胱氨酸69向甲酰甘氨酸的转化及其动力学性质。结果发现只有半胱氨酸69对于甲酰甘氨酸残基的形成至关重要，而七肽LCTPSRA中的亮氨酸68、脯氨酸71和丙氨酸74被替换后，甲酰甘氨酸的形成减少到约30 - 50%。发现在活性位点口袋底部呈现甲酰甘氨酸69的α螺旋的一部分或直接相邻的几个残基对于催化作用至关重要。这项研究的一个惊人结果是，所有已知真核和原核硫酸酯酶之间完全或高度保守的一些残基，结果表明对于甲酰甘氨酸的生成和催化作用都不是必需的。
