The G2447A mutation does not affect ionization of a ribosomal group taking part in peptide bond formation
The G2447A mutation does not affect ionization of a ribosomal group taking part in peptide bond formation
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DOI：
10.1261/rna.5600503
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发表时间：
2003-08-01
期刊：
影响因子：
4.5
通讯作者：
Rodnina, M
中科院分区：
文献类型：
--
作者：
Beringer, M;Adio, S;Rodnina, M
核糖体上肽键的形成是由RNA催化的。使用大肠杆菌核糖体的动力学研究表明，催化作用（>10（5）倍的总加速）很大程度上是由于底物定位。然而，pK（a）=7.5的核糖体基团质子化对肽键形成的抑制接近100倍，表明一般酸碱催化的贡献或活性位点内pH依赖性构象变化的抑制。一般认为23SrRNA的A2451具有一般碱基的功能，并认为A2451的pK（a）位移是由G2447参与的电荷传递系统引起的。使用快速动力学测定，我们发现G2447A突变，这对细胞生长基本上没有影响，降低了约10倍的肽键形成的速率，并不影响与pK（a）=7.5参与反应的核糖体基团的电离。这一结果不支持G2447参与的电荷传递机制和A2451作为一般碱在催化肽键形成中的作用。
Peptide bond formation on the ribosome is catalyzed by RNA. Kinetic studies using Escherichia coli ribosomes have shown that catalysis (>10(5)-fold overall acceleration) is due to a large part to substrate positioning. However, peptide bond formation is inhibited similar to100-fold by protonation of a ribosomal group with pK(a)=7.5, indicating either a contribution of general acid-base catalysis or inhibition by a pH-dependent conformational change within the active site. The function of a general base has been attributed to A2451 of 23S rRNA, and a charge relay system involving G2447 has been postulated to bring about the extensive pK(a) shift of A2451 implied in the model. Using a rapid kinetic assay, we found that the G2447A mutation, which has essentially no effect on cell growth, lowers the rate of peptide bond formation about 10-fold and does not affect the ionization of the ribosomal group with pK(a)=7.5 taking part in the reaction. This result does not support the proposed charge relay mechanism involving G2447 and the role of A2451 as general base in the catalysis of peptide bond formation.