Tryptophan substitutions surrounding the nucleotide in catalytic sites of F1-ATPase.
Tryptophan substitutions surrounding the nucleotide in catalytic sites of F1-ATPase.
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F1-ATPase 催化位点核苷酸周围的色氨酸取代。
DOI：
10.1021/bi981089c
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Senior,AE
中科院分区：
文献类型：
--
作者：
Weber,J;Wilke-Mounts,S;Hammond,ST;Senior,AE
新的色氨酸取代，周围的核苷酸结合在催化位点，被引入到土方ichiacoliF 1-ATP酶。对突变体酶进行了纯化和荧光光谱研究。由β-Trp-404、β-Trp-410、β-Asp-158（排列在腺嘌呤结合口袋内）和β-Trp-153（靠近α/β-磷酸）组成的一簇Trp取代对MgADP、MgAMPPNP和MgATP显示出相同的荧光响应，并与MgADP和MgAMPPNP显示出相同的核苷酸结合模式，其中一个位点亲和力较高，两个位点亲和力较低。因此，在不存在催化转换（和γ-亚基旋转）的情况下，位点2和3的亲和力相似，并且这些Trp取代感测的催化位点区域不会随不同核苷酸改变构象。相比之下，α-Trp-291和β-Trp-297（均接近γ-磷酸）对MgADP和MgAMPPNP显示出非常不同的荧光响应，在这些情况下，响应完全或主要是由于第一个高亲和力催化位点处的核苷酸结合，从而允许特异性检测该位点。用MgATP滴定表明，在稳态条件下存在高亲和力位点，Vmax MgATP水解。
Novel tryptophan substitutions, surrounding the nucleotide bound in catalytic sites, were introduced intoEscherichiacoliF1-ATPase. The mutant enzymes were purified and studied by fluorescence spectroscopy. One cluster of Trp substitutions, consisting of β-Trp-404, β-Trp-410, β-Asp-158 (lining the adenine-binding pocket), and β-Trp-153 (close to the α/β-phosphates), showed the same fluorescence responses to MgADP, MgAMPPNP, and MgATP and the same nucleotide binding pattern with MgADP and MgAMPPNP, with one site of higher and two sites of lower affinity. Therefore, in absence of catalytic turnover (and of γ-subunit rotation), sites 2 and 3 appeared similar in affinity, and the region of the catalytic site sensed by these Trp substitutions did not change conformation with different nucleotides. In contrast, α-Trp-291 and β-Trp-297, both close to the γ-phosphate, showed very different fluorescence responses to MgADP versus MgAMPPNP, and in these cases the response was due exclusively or predominantly to nucleotide binding at the first, high-affinity catalytic site, thus allowing specific detection of this site. Titration with MgATP showed that the high-affinity site was present under conditions of steady-state,VmaxMgATP hydrolysis.