title: Reconsidering the energetics of ribonuclease catalysed RNA hydrolysis.
abstract: In principle, all biochemical reactions are reversible, though some are more reversible than others. The classical ribonuclease mechanism involves a reversible transphosphorylation step, followed by quasi irreversible hydrolysis of the cyclic intermediate. We performed isotope-exchange and intermediate-trapping experiments showing that the second hydrolysis step is readily reversible in the presence of RNase A or RNase T1. As a consequence, the equilibrium between a phosphodiester and a 2',3'-cyclophosphate accounts for all catalysed reactions, even if the leaving/attacking group is a water molecule. Therefore, ribonucleases are transferases rather than hydrolases. The equilibrium constant for the catalysed interconversion is close to 1 M. From this result, we estimate the effective concentration of the 2'-hydroxyl nucleophile in the cyclization step to be 10(7) M. The high effective concentration of the vicinal hydroxyl group balances the strain-associated and solvation-associated instability of the pentacyclic phosphodiester.
authors.affiliation: Dienst Ultrastructuur, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit, Brussels, Belgium.
authors.forename: S
authors.initials: S
authors.lastname: Loverix
authors.forename: G
authors.initials: G
authors.lastname: Laus
authors.forename: J C
authors.initials: JC
authors.lastname: Martins
authors.forename: L
authors.initials: L
authors.lastname: Wyns
authors.forename: J
authors.initials: J
authors.lastname: Steyaert
mesh_terms: D002384:Catalysis; D004734:Energy Metabolism; D006868:Hydrolysis; D007700:Kinetics; D012313:RNA / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism*
chemical_list: D012313:RNA; D012260:Ribonucleases