Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis
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Date
2011
Authors
Wright, Jaden R.
Keffer-Wilkes, Laura C.
Dobing, Selina R.
Wieden-Kothe, Ute
Journal Title
Journal ISSN
Volume Title
Publisher
Cold Springs Harbor Laboratory Press
Abstract
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 (kC) by TruB was determined to be 0.5 sec 1. This rate constant is similar to the kcat 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.
Description
Sherpa Romeo green journal. Open access article. Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0) applies
Keywords
Pseudouridine , Pseudouridine synthase , Kinetic mechanism , RNA modification , Pre-steady-state kinetic analysis
Citation
Wright, J. R., Keffer-Wilkes, L. C., Dobing, S. R., & Kothe, U. (2011). Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis. RNA, 17, 2074-2084. doi: 10.1261/rna.2905811