Fourmy D, Meinnel T, Mechulam Y, Blanquet S
Unité de Recherche Associée 240 du Centre National de la Recherche Scientifique Ecole Polytechnique, Palaiseau, France.
J Mol Biol. 1993 Jun 20;231(4):1068-77. doi: 10.1006/jmbi.1993.1352.
Cys/His motifs, found in several nucleic acid binding proteins, generally correspond to sites for the binding of metal atoms. Such a motif, comprising four Cys residues, occurs in the subunits of Escherichia coli methionyl-tRNA synthetase, a dimeric enzyme known to bind two zinc atoms. In this study, each of the four cysteines in the cysteine cluster (region 145 to 161) of E. coli methionyl-tRNA synthetase were successively changed into an alanine. Either substitution is sufficient to destabilize the tight binding of the zinc ion. Moreover, a peptide having a sequence corresponding to that of the 138 to 163 region of methionyl-tRNA synthetase has been prepared. It strongly binds one zinc atom, even in the presence of ethylene diamine tetraacetate. These data establish that, in methionyl-tRNA synthetase, the Cys motif of region 145 to 161 is actually the binding site for zinc. In addition, the mutation of each cysteine modifies the parameters of the methionine activation reaction, and appears to change the structure of the enzyme, as probed by an increased sensitivity of the mutant enzymes to trypsin attack. The possible role of the zinc atom and of its chelating residues in the folding of the active centre of methionyl-tRNA synthetase is discussed.
在几种核酸结合蛋白中发现的半胱氨酸/组氨酸基序通常对应于金属原子的结合位点。这种由四个半胱氨酸残基组成的基序存在于大肠杆菌甲硫氨酰 - tRNA合成酶的亚基中，该二聚体酶已知能结合两个锌原子。在本研究中，大肠杆菌甲硫氨酰 - tRNA合成酶半胱氨酸簇（区域145至161）中的四个半胱氨酸中的每一个都依次被替换为丙氨酸。任何一种替换都足以破坏锌离子的紧密结合。此外，已经制备了一种具有与甲硫氨酰 - tRNA合成酶138至163区域序列相对应的肽。即使在存在乙二胺四乙酸的情况下，它也能强烈结合一个锌原子。这些数据表明，在甲硫氨酰 - tRNA合成酶中，145至161区域的半胱氨酸基序实际上是锌的结合位点。此外，每个半胱氨酸的突变改变了甲硫氨酸活化反应的参数，并且似乎改变了酶的结构，这通过突变酶对胰蛋白酶攻击的敏感性增加来探测。讨论了锌原子及其螯合残基在甲硫氨酰 - tRNA合成酶活性中心折叠中的可能作用。
