Expression, mutagenesis and kinetic analysis of recombinant K1E endosialidase to define the site of proteolytic processing and requirements for catalysis.
Expression, mutagenesis and kinetic analysis of recombinant K1E endosialidase to define the site of proteolytic processing and requirements for catalysis.
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DOI：
10.1046/j.1365-2958.2002.02908.x
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发表时间：
2002-05
影响因子：
3.6
通讯作者：
Luzio JP
中科院分区：
文献类型：
--
作者：
Leggate DR;Bryant JM;Redpath MB;Head D;Taylor PW;Luzio JP
从大肠杆菌中表达并纯化具有催化活性的噬菌体E内唾液酸酶的重组融合蛋白。具有添加到内唾液酸酶的氨基末端的不同融合配偶体的构建体具有酶活性。丝氨酸706和天冬氨酸707之间发生翻译后蛋白水解裂解，从90 kDa翻译产物产生76 kDa成熟酶。使用变性SDS-PAGE观察到在C-末端从天冬氨酸707截短的内唾液酸酶具有与野生型酶相同的76 kDa分子量，但在非变性PAGE条件下，未观察到形成约250 kDa三聚体野生型酶，这意味着酶的C-末端可能是活性三聚体正确组装所需的，而不是如先前所建议的作为活性部位的一部分。天冬氨酸138突变为丙氨酸大大降低了酶活性，而其他选择的天冬氨酸残基转化为丙氨酸的影响较小，与噬菌体E内切唾液酸酶和外切唾液酸酶的结构和催化机制之间的相似性一致。
Catalytically active, recombinant fusion proteins of bacteriophage E endosialidase were expressed and purified from Escherichia coli. Constructs with different fusion partners added to the amino terminus of the endosialidase were enzymatically active. A post-translational proteolytic cleavage was shown to occur between serine 706 and aspartate 707 to generate the 76 kDa mature enzyme from the 90 kDa translation product. Endosialidase truncated at the C-terminus from aspartate 707 was observed to have the same 76 kDa molecular weight as wild-type enzyme using denaturing SDS–PAGE but, under native PAGE conditions, was not observed to form the ≈250 kDa trimeric wild-type enzyme, implying that the C-terminus of the enzyme may be required for correct assembly of active trimer, rather than as part of the active site as has been previously suggested. Mutagenesis of aspartate 138 to alanine greatly reduced enzyme activity whereas conversion of other selected aspartate residues to alanine had less effect, consistent with similarities between the structure and catalytic mechanism of bacteriophage E endosialidase and those of exosialidases.
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影响因子：
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作者：
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通讯作者：
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