RECONSIDERING THE ENERGETICS OF RIBONUCLEASE CATALYZED RNA HYDROLYSIS

In principle, all biochemical reactions are reversible, though some ar e more reversible than others. The classical ribonuclease mechanism in volves a reversible transphosphorylation step, followed by quasi irrev ersible hydrolysis of the cyclic intermediate. We performed isotope-ex change and intermediate-trapping experiments showing that the second h ydrolysis step is readily reversible in the presence of RNase A or RNa se T1. As a consequence, the equilibrium between a phosphodiester and a 2',3'-cyclophosphate accounts for all catalysed reactions, even if t he leaving/attacking group is a water molecule. Therefore, ribonucleas es are transferases rather than hydrolases. The equilibrium constant f or the catalysed interconversion is close to 1 M. From this result, we estimate the effective concentration of the 2'-hydroxyl nucleophile i n the cyclization step to be 10(7) M. The high effective concentration of the vicinal hydroxyl group balances the strain-associated and solv ation-associated instability of the pentacyclic phosphodiester.