Overexpression of inactive arylsulphatase mutants and in vitro activation by light-dependent oxidation with vanadate
Overexpression of inactive arylsulphatase mutants and in vitro activation by light-dependent oxidation with vanadate
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DOI：
10.1042/bj20040447
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发表时间：
2004-09-01
影响因子：
4.1
通讯作者：
Zankel, TC
中科院分区：
文献类型：
--
作者：
Christianson, TM;Starr, CM;Zankel, TC
芳基硫酸酯酶B（AS B）和A（阿萨）受到其功能所需的独特翻译后修饰。当这些酶过表达时，修饰反应，活性位点半胱氨酸转化为甲酰甘氨酸，变得饱和。我们已经通过表达ASB和阿萨的突变体消除了体内修饰的可能性，其中活性位点半胱氨酸被丝氨酸取代。当与相同条件下的天然酶相比时，这些突变体更有效地表达。纯化的ASB突变体，然后可以转化为催化活性ASB在体外使用钒酸盐和光。
Arylsulphatases B (ASB) and A (ASA) are subject to a unique post-translational modification that is required for their function. The modification reaction, conversion of an active-site cysteine into a formylglycine, becomes saturated when these enzymes are overexpressed. We have removed the possibility of in vivo modification by expressing mutants of ASB and ASA in which the active-site cysteine is substituted with a serine. These mutants are expressed much more efficiently when compared with the native enzymes under identical conditions. The purified ASB mutant can then be converted into catalytically active ASB in vitro using vanadate and light.