Amino acid discrimination by a highly differentiated metal center of an aminoacyl-tRNA synthetase.
Amino acid discrimination by a highly differentiated metal center of an aminoacyl-tRNA synthetase.
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氨酰-tRNA 合成酶的高度分化金属中心可区分氨基酸。
DOI：
10.1021/bi034812u
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Hou,Ya-Ming
中科院分区：
文献类型：
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作者：
Zhang,Chun-Mei;Perona,JohnJ;Hou,Ya-Ming
大肠杆菌半胱氨酰-tRNA合成酶（CysRS）实现了高氨基酸特异性，而不需要编辑反应。晶体学和光谱学研究先前已经证明，特异性的主要决定因素是活性位点锌离子，其通过强烈的锌-硫醇盐相互作用识别底物半胱氨酸。CysRS的活性位点裂缝由高度或严格保守的氨基酸组成，包括四个内球锌配体，裂缝底部的五个组氨酸咪唑，以及在半胱氨酸结合时翻转以完成结合口袋形成的色氨酸。在这里，我们通过突变分析来确定活性位点裂缝的这些主要特征中的每一个的意义。取代通常导致对半胱氨酸、ATP和tRNA酶底物中的每一种的Kmandkcat参数的实质性有害影响。这些发现强调了活性位点高度分化性质的重要性，并为无编辑选择性的起源提供了新的见解。大多数突变体在tRNA氨酰化中的衰减比在腺苷酸合成中的衰减少，这表明tRNA结合驱动构象变化以帮助组装活性位点。
Escherichia colicysteinyl-tRNA synthetase (CysRS) achieves high amino acid specificity without the need for an editing reaction. Crystallographic and spectroscopic studies have previously demonstrated that a major determinant of the specificity is an active site zinc ion that recognizes the substrate cysteine through a strong zinc−thiolate interaction. The active site cleft of CysRS is composed of highly or strictly conserved amino acids, including four inner-sphere zinc ligands, five histidine imidazoles at the base of the cleft, and a tryptophan that flips down upon cysteine binding to complete formation of the binding pocket. Here we establish the significance of each of these major features of the active site cleft by mutational analysis. Substitutions generally lead to substantially deleterious effects onKmandkcatparameters with respect to each of the cysteine, ATP, and tRNACyssubstrates. These findings emphasize the importance of the highly differentiated nature of the active site and provide new insights into the origins of selectivity without editing. Most mutants are less attenuated in tRNA aminoacylation than in adenylate synthesis, suggesting that tRNA binding drives a conformational change to help assemble the active site.