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首页> 外文期刊>Journal of Molecular Biology >Conformational changes and cleavage by the homing endonuclease I-PpoI: a critical role for a leucine residue in the active site.
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Conformational changes and cleavage by the homing endonuclease I-PpoI: a critical role for a leucine residue in the active site.

机译：归巢内切核酸酶I-PpoI的构象变化和切割：活性位点中亮氨酸残基的关键作用。

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摘要

The homing endonuclease I-PpoI severely bends its DNA target, resulting in significant deformations of the minor and major groove near the scissile phosphate groups. To study the role of conformational changes within the protein catalyst and the DNA substrate, we have determined the structure of the enzyme in the absence of bound DNA, performed gel retardation analyses of DNA binding and bending, and have mutagenized a leucine residue that contacts an adenine nucleotide at the site of cleavage. The structure of the L116A/DNA complex has been determined and the effects of the mutation on affinity and catalysis have been measured. The wild-type protein displays a rigid-body rotation of its individual subunits upon DNA binding. Homing site DNA is not detectably bent in the absence of protein, but is sharply bent in both the wild-type and L116A complexes. These results indicate that binding involves a large distortion of the DNA and a smaller change in protein conformation. Leucine 116 is critical for binding and catalysis: it appears to be important for forming a well-ordered protein-DNA complex at the cleavage site, for maximal deformation of the DNA, and for desolvation of the nucleotide bases that are partially unstacked in the enzyme complex. Copyright 2000 Academic Press.
机译：归巢核酸内切酶I-PpoI严重弯曲了其DNA靶标，导致可裂解磷酸基团附近的小沟和大沟明显变形。为了研究蛋白质催化剂和DNA底物中构象变化的作用，我们确定了在不存在结合DNA的情况下酶的结构，对DNA结合和弯曲进行了凝胶阻滞分析，并诱变了与细胞接触的亮氨酸残基。裂解位点的腺嘌呤核苷酸。已经确定了L116A / DNA复合物的结构，并测量了该突变对亲和力和催化作用的影响。野生型蛋白质在DNA结合后显示出其各个亚基的刚性旋转。在没有蛋白质的情况下，归巢位点DNA不能被检测到弯曲，但是在野生型和L116A复合物中都被急剧弯曲。这些结果表明结合涉及DNA的大变形和蛋白质构象的较小变化。亮氨酸116对于结合和催化至关重要：它似乎对于在切割位点形成有序的蛋白质-DNA复合物，使DNA最大变形以及使部分未堆积在酶中的核苷酸碱基脱溶剂至关重要。复杂。版权所有2000学术出版社。

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