Activity of 3′-thioAMP derivatives as ribosomal P-site substrates
Activity of 3′-thioAMP derivatives as ribosomal P-site substrates
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DOI：
10.1093/nar/gki617
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发表时间：
2005-01-01
影响因子：
14.9
通讯作者：
Barta, A
中科院分区：
文献类型：
--
作者：
Dorner, S;Schmid, W;Barta, A
核糖体是一个大的RNP复合物，但其主要的酶活性，肽基转移酶，是一种核酶。由于许多RNA酶在催化中使用二价金属离子，因此提出的一个假设是金属离子可能有助于肽键的形成。为了能够测试金属离子与P位点底物的3 '-桥氧的可能配位，合成了3'-硫代AMP。其与N-乙酰基- L-亮氨酸的化学酰化产生单和二氨基酰化的3 '-硫代AMP。这些硫代底物进行了测试，在一个优化的片段反应中的肽键形成与它们的未修饰的对应物相比。由于该氨基酸主要与AcLeu-thioAMP中的非生产性2 '-OH连接（5），因此该底物几乎没有活性，不用于进一步分析。相比之下，二（AcLeu）-硫代AMP（4）比二（AcLeu）- AMP（2）更有活性，这与硫酯的较高能量一致。当在测定中使用含Mn 2+的缓冲液时，这两种活性都略有增强。这些数据表明，硫代化的P-位点底物在肽键形成中具有活性，并且原则上可以用于全翻译系统中的金属离子拯救实验。
The ribosome is a large RNP complex but its main enzymatic activity, the peptidyl transferase, is a ribozyme. As many RNA enzymes use divalent metal ions in catalysis, one of the hypotheses put forward proposed that metal ions might aid peptide bond formation. To be able to test a possible coordination of a metal ion to the 3'- bridging oxygen of P-site substrates, a 3'-thioAMP was synthesized. Its chemical acylation with N-acetyl- L- leucine yielded both mono and diaminoacylated 3'-thioAMP. These thioated substrates were tested for peptide bond formation in an optimized fragment reaction in comparison with their unmodified counterparts. As the amino acid was predominantly linked to the unproductive 2'-OH in AcLeu-thioAMP (5), this substrate was barely active and not used for further analysis. In contrast, Di(AcLeu)-thioAMP ( 4) was more active than Di( AcLeu)- AMP ( 2) which is in line with the higher energy of thioesters. Both activities were slightly enhanced when Mn2+ containing buffers were employed in the assay. These data show that thioated P-site substrates are active in peptide bond formation and can in principle be used for metalion-rescue experiments in a full translation system.