Reconsidering the energetics of ribonuclease catalysed RNA hydrolysis

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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

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DA - 19981110
IS - 0014-2956 (Print)
LA - eng
PT - Journal Article
PT - Research Support, Non-U.S. Gov't
RN - 63231-63-0 (RNA)
RN - EC 3.1.- (Ribonucleases)
SB - IM