Uncoupling metallonuclease metal ion binding sites via nudge mutagenesis.
Uncoupling metallonuclease metal ion binding sites via nudge mutagenesis.
复制标题
通过微移诱变解偶联金属核酸酶金属离子结合位点。
DOI：
10.1007/s00775-007-0209-y
复制
发表时间：
2007
期刊：
影响因子：
--
通讯作者：
Dupureur,CynthiaM
中科院分区：
文献类型：
--
作者：
Papadakos,GrigoriosA;Nastri,Horacio;Riggs,Paul;Dupureur,CynthiaM
核酸酶水解磷酸二酯键对核酸加工至关重要。许多核酸酶利用金属离子辅因子，并且对于许多这些酶，已经检测到两个活性位点金属离子。测试提出的机制作用，为个别绑定金属离子的网站和合作行为之间的相似性受到阻碍。在homodimericPvuII限制性内切酶中，DNA结合的金属离子依赖性是S形的，并且与两类偶联金属离子结合位点一致。我们推断，保守的活性位点突变会干扰配体场，足以观察单个金属离子结合位点的滴定，而不会显著干扰酶功能。事实上，突变的酪氨酸残基5.5碱基从两种金属离子的酶底物晶体结构（Y 94 F）呈现的DNA结合双相的金属离子依赖性：两类金属离子结合位点在DNA的存在下变得不同。酶-Ca（II）和酶-Ca（II）-DNA复合物的~ 1H-~（15）N杂原子单量子相干谱支持了金属离子配位的微扰。游离Y 94 F与金属离子的结合基本上是不受干扰的：通过使用不同金属离子的多次实验，数据与低毫摩尔亲和力的每个亚基两个碱土金属离子结合位点一致，其行为与野生型非常相似。这里提出的结果表明，在DNA的存在下，在金属离子结合位点的偶联的Tyr 94的羟基基团的作用。它的去除导致两个金属离子结合位点的亲和力在底物存在下被解析。金属离子亲和力的这种调节对于确定单个结合金属离子对金属核酸酶功能的贡献的努力将是非常宝贵的。
The hydrolysis of phosphodiester bonds by nucleases is critical to nucleic acid processing. Many nucleases utilize metal ion cofactors, and for a number of these enzymes two active-site metal ions have been detected. Testing proposed mechanistic roles for individual bound metal ions has been hampered by the similarity between the sites and cooperative behavior. In the homodimericPvuII restriction endonuclease, the metal ion dependence of DNA binding is sigmoidal and consistent with two classes of coupled metal ion binding sites. We reasoned that a conservative active-site mutation would perturb the ligand field sufficiently to observe the titration of individual metal ion binding sites without significantly disturbing enzyme function. Indeed, mutation of a Tyr residue 5.5 Å from both metal ions in the enzyme–substrate crystal structure (Y94F) renders the metal ion dependence of DNA binding biphasic: two classes of metal ion binding sites become distinct in the presence of DNA. The perturbation in metal ion coordination is supported by1H–15N heteronuclear single quantum coherence spectra of enzyme–Ca(II) and enzyme–Ca(II)–DNA complexes. Metal ion binding by free Y94F is basically unperturbed: through multiple experiments with different metal ions, the data are consistent with two alkaline earth metal ion binding sites per subunit of low millimolar affinity, behavior which is very similar to that of the wild type. The results presented here indicate a role for the hydroxyl group of Tyr94 in the coupling of metal ion binding sites in the presence of DNA. Its removal causes the affinities for the two metal ion binding sites to be resolved in the presence of substrate. Such tuning of metal ion affinities will be invaluable to efforts to ascertain the contributions of individual bound metal ions to metallonuclease function.