Kinetic Isotope Effect Analysis of RNA 2′-O-Transphosphorylation

Michael E. Harris, Darrin M. York, Joseph A. Piccirilli, Vernon E. Anderson

Research output: Chapter in Book/Report/Conference proceedingChapter

2 Scopus citations

Abstract

The breaking of RNA strands by 2′-O-transphosphorylation is a ubiquitous reaction in biology, and enzymes that catalyze this reaction play key roles in RNA metabolism. The mechanisms of 2′-O-transphosphorylation in solution are relatively well studied, but complex and can involve different transition states depending on how the reaction is catalyzed. Because of this complexity and the lack of experimental information on transition-state structure, pinning down the chemical details of enzyme-catalyzed RNA strand cleavage has been difficult. Kinetic isotope effects (KIEs) provide information about changes in bonding as a reaction proceeds from ground state to transition state, and therefore they provide a powerful tool for revealing mechanistic detail. Application of kinetic isotope analyses to RNA 2′-O-transphosphorylation faces three fundamental challenges: synthesis of RNA substrate isotopomers with 18O substitutions at the 2′-O, 5′-O and nonbridging phosphoryl oxygens; determination of the 18O/16O ratios in the residual unreacted substrate or product RNAs; and analyzing these data to allow calculation of the KIEs for use in evaluating different mechanistic scenarios. In this chapter, we outline methods for surmounting these challenges for solution RNA 2′-O-transphosphorylation reactions, and we describe their initial application to understand nonenzymatic solution reactions and reactions catalyzed by the enzyme ribonuclease A.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages433-457
Number of pages25
DOIs
StatePublished - 2017

Publication series

NameMethods in Enzymology
Volume596
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Keywords

  • Catalysis
  • Enzyme
  • Mass spectrometry
  • Metal ion
  • Phosphate
  • Phosphodiester
  • RNA
  • Ribonuclease
  • Ribozyme
  • Transphosphorylation

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