Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada.
RNA. 2011 Dec;17(12):2074-84. doi: 10.1261/rna.2905811. Epub 2011 Oct 13.
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(Ψ)) 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.
假尿嘧啶核苷合成酶通过特异性地将尿苷异构化为假尿苷来催化功能性 RNA 中最丰富的修饰。尽管这些酶的结构和底物特异性已经得到了详细研究，但假尿嘧啶核苷合成酶的动力学和催化机制仍然未知。在这里，首次对三种大肠杆菌假尿嘧啶核苷合成酶进行了预稳态动力学分析。一种新的停流吸收测定法表明，TruB 结合 tRNA 分两步进行，总速率为 6 sec(-1)。为了直接观察假尿嘧啶形成的催化作用，传统的氚释放测定法被改编为猝灭流技术，首次允许观察单个假尿嘧啶形成循环。由此，TruB 的假尿嘧啶化的单轮速率常数 (k(Ψ)) 被确定为 0.5 sec(-1)。该速率常数与稳态实验中多次循环条件下获得的 k(cat)相似，表明催化是 TruB 的限速步骤。为了研究假尿嘧啶核苷合成酶是否普遍具有缓慢的催化作用，还对另外两种大肠杆菌酶 RluA 和 TruA 进行了快速动力学猝灭流分析，它们的假尿嘧啶形成速率常数分别为 0.7 和 0.35 sec(-1)。因此，均匀缓慢的催化可能是假尿嘧啶核苷合成酶的一般特征，它们具有保守的催化结构域，并且可能使用相同的催化机制。
