Substitution of the conserved tryptophan 31 in Escherichia coli thioredoxin by site-directed mutagenesis and structure-function analysis.
Substitution of the conserved tryptophan 31 in Escherichia coli thioredoxin by site-directed mutagenesis and structure-function analysis.
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通过定点诱变和结构功能分析取代大肠杆菌硫氧还蛋白中的保守色氨酸 31。
DOI：
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发表时间：
1991
影响因子：
4.8
通讯作者：
Arne Holmgren
中科院分区：
文献类型：
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作者：
Günter Krause;Arne Holmgren
所有原核和真核硫氧还蛋白含有保守的色氨酸残基，暴露在活性位点二硫化物/二硫醇。通过定点突变研究了该W31在大肠杆菌硫氧还蛋白（Trx）中的作用。四个突变体Trx与W31 Y，W31 F，W31 H，和W31 A的替代进行了表征。在所有突变体Trx中观察到剩余W28的色氨酸荧光发射非常低;减少导致荧光大幅但可变增加（高达11倍），达到高于天然或变性野生型Trx的水平，表明先前假设的变化涉及W28。所有W31突变体Trx都是E.大肠杆菌硫氧还蛋白还原酶。与野生型相比，W31 A、W31 H和W31 F Trx的表观Km值增加不到2倍，W31 Y Trx显示出甚至略高的催化效率（kcat/Km值）。功能减少Trx与核糖核苷酸还原酶和减少胰岛素二硫化物的W31置换，特别是在低pH值的A和H残基的影响更强烈。与W31 F或野生型蛋白相比，W31 Y、W31 A或W31 H的T7基因5蛋白和还原Trx产生的T7 DNA聚合酶活性降低了很大的因子。利用pUC 118-trxA在E. coli trxA-背景。具有W31 Y和W31 F取代的trxA基因完全和部分恢复了trxA-metE-测试菌株的甲硫氨酸亚砜利用; W31 A和W31 H突变导致无生长。M13的繁殖受到W31 Y和W31 F的中度阻碍，或受到W31 A和W31 H替代物的严重阻碍。噬菌体T3/7杂合体的生长仅在W31 Y和W31 F取代的情况下才可能，这反映了T7 DNA聚合酶的体外结果。
All prokaryotic and eukaryotic thioredoxins contain a conserved tryptophan residue, exposed at the active site disulfide/dithiol. The role of this W31 in Escherichia coli thioredoxin (Trx) was studied by site-directed mutagenesis. Four mutant Trx with W31Y, W31F, W31H, and W31A replacements were characterized. Very low tryptophan fluorescence emission from the remaining W28 was observed in all mutant Trx; reduction resulted in large, but variable increases (up to 11-fold) of fluorescence, to levels higher than in native or denatured wild-type Trx, demonstrating a previously postulated change involving W28. All W31 mutant Trx were good substrates for E. coli thioredoxin reductase. Compared with wild type, the apparent Km values were increased less than 2-fold for the W31A, W31H, and W31F Trx and the W31Y Trx showed even slightly higher catalytic efficiency (kcat/Km value). Functions of reduced Trx with ribonucleotide reductase and in reduction of insulin disulfides were more strongly influenced by the W31 replacements, in particular at low pH for A and H residues. T7 DNA polymerase activity generated by T7 gene 5 protein and reduced Trx was lowered by large factors for W31Y, W31A, or W31H compared with W31F or the wild-type protein. The in vivo function of Trx was studied by using pUC118-trxA expression in an E. coli trxA- background. The trxA genes with W31Y and W31F substitutions restored, fully and partly, the methionine sulfoxide utilization of a trxA- metE- test strain; W31A and W31H mutations resulted in no growth. Propagation of M13 was moderately impeded by W31Y and W31F or severely by W31A and W31H replacements. Growth of a phage T3/7 hybrid was possible only with the W31Y and W31F substitutions reflecting the in vitro results for T7 DNA polymerase.