Suwa Satoshi, Nagai Yasuhiro, Fujimoto Akihiro, Kikuchi Yo, Tanaka Terumichi
Division of Bioscience and Biotechnology, Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi, Aichi 441-8580, Japan.
J Biochem. 2009 Feb;145(2):151-60. doi: 10.1093/jb/mvn150. Epub 2008 Nov 13.
We prepared a series of shape variants of a pre-tRNA and examined substrate shape recognition by bacterial RNase P ribozyme and holoenzyme. Cleavage site analysis revealed two new subsites for accepting the T-arm and the bottom half of pre-tRNA in the substrate-binding site of the enzyme. These two subsites take part in cleavage site selection of substrate by the enzyme: the cleavage site is not always selected according to the relative position of the 3'-CCA sequence of the substrate. Kinetic studies indicated that the substrate shape is recognized mainly in the transition state of the reaction, and neither the shape nor position of either the T-arm or the bottom half of the substrate affected the Michaelis complex formation. These results strongly suggest that the 5' and 3' termini of a substrate are trapped by the enzyme first, then the position and the shape of the T-arm and the bottom half are examined by the cognate subsites. From these facts, we propose a new substrate recognition model that can explain many experimental facts that have been seen as enigmatic.
我们制备了一系列前体tRNA的形状变体，并研究了细菌RNase P核酶和全酶对底物形状的识别。切割位点分析揭示了该酶底物结合位点中用于接纳前体tRNA的T臂和下半部分的两个新亚位点。这两个亚位点参与了酶对底物切割位点的选择：切割位点并不总是根据底物3'-CCA序列的相对位置来选择。动力学研究表明，底物形状主要在反应的过渡态被识别，底物的T臂或下半部分的形状和位置均不影响米氏复合物的形成。这些结果有力地表明，底物的5'和3'末端首先被酶捕获，然后T臂和下半部分的位置和形状由相应的亚位点进行检查。基于这些事实，我们提出了一种新的底物识别模型，该模型可以解释许多曾被视为谜一样的实验事实。
