Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis

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dc.contributor.authorWright, Jaden R.
dc.contributor.authorKeffer-Wilkes, Laura C.
dc.contributor.authorDobing, Selina R.
dc.contributor.authorWieden-Kothe, Ute
dc.date.accessioned2019-06-02T21:42:32Z
dc.date.available2019-06-02T21:42:32Z
dc.date.issued2011
dc.descriptionSherpa Romeo green journal. Open access article. Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0) appliesen_US
dc.description.abstractPseudouridine 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.en_US
dc.description.peer-reviewYesen_US
dc.identifier.citationWright, 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.2905811en_US
dc.identifier.urihttps://hdl.handle.net/10133/5384
dc.language.isoen_USen_US
dc.publisherCold Springs Harbor Laboratory Pressen_US
dc.publisher.departmentDepartment of Chemistry and Biochemistryen_US
dc.publisher.facultyArts and Scienceen_US
dc.publisher.institutionUniversity of Lethbridgeen_US
dc.publisher.urlhttps://dx.doi.org/10.1021/bi7011492
dc.subjectPseudouridineen_US
dc.subjectPseudouridine synthaseen_US
dc.subjectKinetic mechanismen_US
dc.subjectRNA modificationen_US
dc.subjectPre-steady-state kinetic analysisen_US
dc.subject.lcshPseudouridine--Research
dc.subject.lcshRNA--Research
dc.titlePre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysisen_US
dc.typeArticleen_US
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