Mutation of amino acids in the alpha 1,3-fucosyltransferase motif affects enzyme activity and Km for donor and acceptor substrates
Mutation of amino acids in the alpha 1,3-fucosyltransferase motif affects enzyme activity and Km for donor and acceptor substrates
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DOI：
10.1093/glycob/cwh157
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发表时间：
2005-02-01
期刊：
影响因子：
4.3
通讯作者：
Macher, BA
中科院分区：
文献类型：
--
作者：
Jost, F;de Vries, T;Macher, BA
α 1，3-岩藻糖基转移酶（FucT）共有一个保守的氨基酸序列，称为α 1，3 FucT基序，该基序被认为对核苷酸糖结合很重要。为了评价该基序中氨基酸的重要性，在人FucT VI中用丙氨酸替换每个α 1，3 FucT基序氨基酸（丙氨酸扫描诱变），并分析所得突变蛋白的酶活性，并在突变蛋白具有足够活性的那些情况下进行动力学表征。其中两个突变蛋白是无活性的，六个突变蛋白的活性低于野生型的1%，四个突变蛋白的活性接近野生型的10 -50%。具有显著酶活性的突变体蛋白质中的三个对于GDP-岩藻糖具有比FucT VI野生型酶大得多的Km（5至15倍）。具有显著酶活性的第四突变蛋白（S249 A）对于受体底物具有比野生型FucT VI大至少10倍的K-m，对于GDP-岩藻糖仅具有稍大（2-3倍）的K-m。因此，α 1，3 FucT基序内的任何氨基酸突变为Ala影响α 1，3-FucT活性，并且Ala取代某些α 1，3 FucT基序氨基酸导致蛋白质对受体和供体底物的动力学常数改变。二级结构预测表明α 1，3 FucT基序的螺旋-环-螺旋折叠，这可用于合理化突变在3D结构方面的影响。
Alpha 1,3-fucosyltransferases (FucT) share a conserved amino acid sequence designated the alpha 1,3 FucT motif that has been proposed to be important for nucleotide sugar binding. To evaluate the importance of the amino acids in this motif, each of the alpha 1,3 FucT motif amino acids was replaced with alanine (alanine scanning mutagenesis) in human FucT VI, and the resulting mutant proteins were analyzed for enzyme activity and kinetically characterized in those cases in which the mutant protein had sufficient activity. Two of the mutant proteins were inactive, six had less than 1% of wild-type activity, and four had similar to10-50% of wild-type enzyme activity. Three of the mutant proteins with significant enzyme activity had substantially larger K-m (5 to 15 times) for GDP-fucose than FucT VI wild-type enzyme. The fourth mutant protein with significant enzyme activity (S249A) had a K-m at least 10 times larger than wild-type FucT VI for the acceptor substrate, with only a slightly larger (2-3 times) K-m for GDP-fucose. Thus mutation of any of the amino acids within the alpha 1,3 FucT motif to Ala affects alpha 1,3-FucT activity, and substitution of Ala for some of the alpha 1,3 FucT motif amino acids results in proteins with altered kinetic constants for both the acceptor and donor substrates. Secondary structure prediction suggests a helix-loop-helix fold for the alpha 1,3 FucT motif, which can be used to rationalize the effects of mutations in terms of 3D structure.