Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis
Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis
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DOI：
10.1261/rna.2905811
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发表时间：
2011-12-01
期刊：
影响因子：
4.5
通讯作者：
Kothe, Ute
中科院分区：
文献类型：
--
作者：
Wright, Jaden R.;Keffer-Wilkes, Laura C.;Kothe, Ute
假尿嘧啶合成酶通过位点特异性地将尿嘧啶异构化为假尿嘧啶，催化形成最丰富的功能性rna修饰。虽然这些酶的结构和底物特异性已被详细研究，但假尿嘧啶合成酶的动力学和催化机制仍不清楚。本文首次对三种大肠杆菌假尿嘧啶合成酶进行了稳态前动力学分析。一种新型的停流吸光度实验显示，tRNA与TruB的结合分两步进行，总速率为6秒（-1）。为了直接观察假尿嘧啶形成的催化作用，将传统的氚释放法应用于淬火流技术，首次允许观察单轮假尿嘧啶的形成。因此，TruB测定假尿嘧啶化单轮速率常数（k(Psi)）为0.5 sec（-1）。该速率常数与稳态实验中多次翻转条件下得到的k（cat）相似，表明催化是TruB的限速步骤。为了研究假尿嘧啶合成酶是否具有一般的慢催化特性，我们还对另外两种大肠杆菌酶RluA和TruA进行了快速动力学猝流分析，结果显示假尿嘧啶的生成速率常数分别为0.7和0.35秒（-1）。因此，均匀缓慢的催化可能是假尿嘧啶合酶的一个普遍特征，它们共享一个保守的催化结构域，并且可能使用相同的催化机制。
Pseudouridine synthases catalyze formation of the most abundant modification of functional RNAs by site-specifically isomerizing uridines to pseudouridines. While the structure and substrate specificity of these enzymes have been studied in detail, the kinetic and the catalytic mechanism of pseudouridine synthases remain unknown. Here, the first pre-steady-state kinetic analysis of three Escherichia coli pseudouridine synthases is presented. A novel stopped-flow absorbance assay revealed that substrate tRNA binding by TruB takes place in two steps with an overall rate of 6 sec(-1). In order to observe catalysis of pseudouridine formation directly, the traditional tritium release assay was adapted for the quench-flow technique, allowing, for the first time, observation of a single round of pseudouridine formation. Thereby, the single-round rate constant of pseudouridylation (k(Psi)) by TruB was determined to be 0.5 sec(-1). This rate constant is similar to the k(cat) obtained under multiple-turnover conditions in steady-state experiments, indicating that catalysis is the rate-limiting step for TruB. In order to investigate if pseudouridine synthases are characterized by slow catalysis in general, the rapid kinetic quench-flow analysis was also performed with two other E. coli enzymes, RluA and TruA, which displayed rate constants of pseudouridine formation of 0.7 and 0.35 sec(-1), respectively. Hence, uniformly slow catalysis might be a general feature of pseudouridine synthases that share a conserved catalytic domain and supposedly use the same catalytic mechanism.