Reconsidering the energetics of ribonuclease catalysed RNA hydrolysis.

TitleReconsidering the energetics of ribonuclease catalysed RNA hydrolysis.
Publication TypeJournal Article
Year of Publication1998
AuthorsLoverix, S., G. Laus, J. C. Martins, L. Wyns, and J. Steyaert
JournalEur J Biochem
Volume257
Issue1
Pagination286-90
Date Published1998 Oct 1
ISSN0014-2956
KeywordsCatalysis, Energy Metabolism, Hydrolysis, Kinetics, Ribonucleases, RNA
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.

Alternate JournalEur. J. Biochem.
PubMed ID9799130