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dc.creatorSun, W
dc.creatorPertzev, A
dc.creatorNicholson, AW
dc.date.accessioned2021-02-01T22:23:24Z
dc.date.available2021-02-01T22:23:24Z
dc.date.issued2005-10-18
dc.identifier.issn0305-1048
dc.identifier.issn1362-4962
dc.identifier.doihttp://dx.doi.org/10.34944/dspace/5628
dc.identifier.other15699182 (pubmed)
dc.identifier.urihttp://hdl.handle.net/20.500.12613/5646
dc.description.abstractEscherichia coli ribonuclease III (RNase III; EC 3.1.24) is a double-stranded(ds)-RNA-specific endonuclease with key roles in diverse RNA maturation and decay pathways. E.coli RNase III is a member of a structurally distinct superfamily that includes Dicer, a central enzyme in the mechanism of RNA interference. E.coli RNase III requires a divalent metal ion for activity, with Mg2+ as the preferred species. However, neither the function(s) nor the number of metal ions involved in catalysis is known. To gain information on metal ion involvement in catalysis, the rate of cleavage of the model substrate R1.1 RNA was determined as a function of Mg2+ concentration. Single-turnover conditions were applied, wherein phosphodiester cleavage was the rate-limiting event. The measured Hill coefficient (nH) is 2.0 ± 0.1, indicative of the involvement of two Mg2+ ions in phosphodiester hydrolysis. It is also shown that 2-hydroxy-4H-isoquinoline-1,3-dione - an inhibitor of ribonucleases that employ two divalent metal ions in their catalytic sites-inhibits E.coli RNase III cleavage of R1.1 RNA. The IC50 for the compound is 14 μM for the Mg2+-supported reaction, and 8 μM for the Mn2+-supported reaction. The compound exhibits noncompetitive inhibitory kinetics, indicating that it does not perturb substrate binding. Neither the O-methylated version of the compound nor the unsubstituted imide inhibit substrate cleavage, which is consistent with a specific interaction of the N-hydroxyimide with two closely positioned divalent metal ions. A preliminary model is presented for functional roles of two divalent metal ions in the RNase III catalytic mechanism. © The Author 2005. Published by Oxford University Press. All rights reserved.
dc.format.extent807-815
dc.language.isoen
dc.relation.haspartNucleic Acids Research
dc.relation.isreferencedbyOxford University Press (OUP)
dc.subjectBase Sequence
dc.subjectCatalysis
dc.subjectCations, Divalent
dc.subjectEnzyme Inhibitors
dc.subjectEscherichia coli Proteins
dc.subjectHydrolysis
dc.subjectIsoquinolines
dc.subjectKinetics
dc.subjectMagnesium
dc.subjectManganese
dc.subjectModels, Chemical
dc.subjectMolecular Sequence Data
dc.subjectPhosphates
dc.subjectRNA
dc.subjectRibonuclease III
dc.titleCatalytic mechanism of Escherichia coli ribonuclease III: Kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysis
dc.typeArticle
dc.type.genreJournal Article
dc.relation.doi10.1093/nar/gki197
dc.ada.noteFor Americans with Disabilities Act (ADA) accommodation, including help with reading this content, please contact scholarshare@temple.edu
dc.date.updated2021-02-01T22:23:22Z
refterms.dateFOA2021-02-01T22:23:25Z


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