Outersphere and innersphere coordinated metal ions in an aminoacyl-tRNA synthetase ribozyme.
Outersphere and innersphere coordinated metal ions in an aminoacyl-tRNA synthetase ribozyme.
复制标题
氨酰-tRNA 合成酶核酶中外球和内球协调金属离子。
DOI：
10.1093/nar/gkf641
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发表时间：
2002
影响因子：
14.9
通讯作者：
Suga,Hiroaki
中科院分区：
文献类型：
--
作者：
Saito,Hirohide;Suga,Hiroaki
金属离子是各种核酶的重要辅因子。在这里，我们剖析了金属离子在体外进化的氨酰-tRNA合成酶样核酶（ARS核酶）中的作用。这种核酶可以在tRNA切口上给苯丙氨酸充电，在那里它共价连接到tRNA的5′末端（即前体tRNA的一种形式），以及在那里它可以作为催化剂。镁离子的存在是该核酶显示完全催化活性所必需的。金属依赖性动力学，以及使用Tb 3+与Mg 2+或Co（NH3）63+竞争的结构映射，确定了嵌入tRNA结合位点附近的两个潜在金属结合位点。高亲和力金属结合位点可以用Mg 2+或Co（NH3）63+填充，因此活性依赖于与水或铵离子完全配位的金属离子。该位点也与氨基酸结合位点重叠，表明金属离子在构成催化核心中起作用。弱金属结合位点仅被可以与核酶中的配体形成内层接触的金属离子占据，因此，Mg 2+可以增强核酶活性，但Co（NH3）63+不能。在这项工作中描述的实验建立的金属离子，具有不同的协调性能的ARS核酶的作用。
Metal ions are essential cofactors for various ribozymes. Here we dissect the roles of metal ions in an aminoacyl‐tRNA synthetase‐like ribozyme (ARS ribozyme), which was evolvedin vitro. This ribozyme can charge phenylalanine on tRNA incis, where it is covalently attached to the 5′‐end of tRNA (i.e. a form of precursor tRNA), as well as intrans, where it can act as a catalyst. The presence of magnesium ion is essential for this ribozyme to exhibit full catalytic activity. Metal‐dependent kinetics, as well as structural mappings using Tb3+in competition with Mg2+or Co(NH3)63+, identified two potential metal‐binding sites which are embedded near the tRNA‐binding site. The high affinity metal‐binding site can be filled with either Mg2+or Co(NH3)63+and thus the activity relies on a metal ion that is fully coordinated with water or ammonium ions. This site also overlaps with the amino acid‐binding site, suggesting that the metal ion plays a role in constituting the catalytic core. The weak metal‐binding site is occupied only by a metal ion(s) that can form innersphere contacts with ligands in the ribozyme and, hence, Mg2+can enhance ribozyme activity, but Co(NH3)63+cannot. The experiments described in this work establish the roles of metal ions that have distinct coordination properties in the ARS ribozyme.