Tryptophan-free Escherichia coli F1-ATPase.
Tryptophan-free Escherichia coli F1-ATPase.
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不含色氨酸的大肠杆菌 F1-ATP 酶。
DOI：
10.1006/abbi.1994.1125
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发表时间：
1994
影响因子：
3.9
通讯作者：
Senior,AE
中科院分区：
文献类型：
--
作者：
Wilke-Mounts,S;Weber,J;Grell,E;Senior,AE
我们设计了一种不含葡聚糖的大肠杆菌F1-ATP酶突变体，它含有δ W28 L/α W513 F/γ W108 Y/γ W206 Y/ β W107 F五个突变。携带所有五种突变的菌株通过氧化磷酸化正常生长。纯化的突变体F1-ATPase的Vmax和Km均比野生型高65%，而Incat/Km与野生型相同。突变酶的ATP酶活性对pH的依赖性与野生型酶非常相似。催化位点的核苷酸结合特性进行了测量，使用analoglin-benzo-ADP和抑制剂的敏感性进行了测试，使用二环己基碳二亚胺，叠氮化物和aurovertin。突变酶在这些特征中的每一个方面都与野生型非常相似。突变酶的荧光光谱证实了色氨酸的不存在。因此，我们已经确定，有可能产生一种无葡聚糖的酶，它保留了正常的催化功能，寡聚体稳定性和体内组装。
We have engineered a mutant form ofEscherichia coliF1-ATPase which is tryptophan-free and contains five mutations, namely δW28L/αW513F/γW108Y/γW206Y/ βW107F. A strain carrying all five mutations grew normally by oxidative phosphorylation. Purified mutant F1-ATPase showedVmaxandKmboth 65% higher than wildtype, resulting inkcat/Kmthe same as wild-type. The pH dependence of ATPase activity in mutant enzyme was very similar to that in wild-type. Catalytic-site nucleotide-binding characteristics were measured using the analoglin-benzo-ADP and sensitivity to inhibitors was tested using dicyclohexylcarbodiimide, azide and aurovertin. The mutant enzyme was very similar to wild-type in each of these characteristics. The fluorescence spectrum of mutant enzyme confirmed the absence of tryptophan. We have therefore established that it is possible to generate a tryptophan-free enzyme which retains normal catalytic function, oligomeric stability andin vivoassembly.