Reconsidering the energetics of ribonuclease catalysed RNA hydrolysis.

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.