PMID:21998096
| Citation |
Wright, JR, Keffer-Wilkes, LC, Dobing, SR and Kothe, U (2011) Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis. RNA 17:2074-84 |
|---|---|
| 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 (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. |
| Links |
PubMed PMC3222121 Online version:10.1261/rna.2905811 |
| Keywords |
Catalysis; Escherichia coli/enzymology; Escherichia coli/genetics; Gene Expression Regulation, Bacterial; Intramolecular Transferases/genetics; Intramolecular Transferases/isolation & purification; Intramolecular Transferases/metabolism; Kinetics; Protein Binding; Pseudouridine/chemistry; Pseudouridine/metabolism; RNA, Transfer/metabolism |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<annotationlinks/>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
See also
References
See Help:References for how to manage references in EcoliWiki.