Experimental and computational evidence that ribonuclease A alters the transition state for RNA 2′‐O‐transphosphorylation

Abstract

Ribonuclease A (RNase A) is a widely used model system illustrating basic principles of enzyme catalysis. Yet, ambiguity regarding the transition state it stabilizes precludes a complete understanding of its mechanism. Here, we present kinetic isotope effect analyses that provide direct evidence for a late, anionic transition state for RNase A catalyzed RNA 2′‐O‐transphosphorylation similar to catalysis by hydroxide in solution, but with dramatically less charge accumulation on the 5′O. Molecular simulations indicate stabilization of negative charge on the NPOs by H‐bonding with a protonated His12 and Lys41. Quantum chemical calculations consistent with the experimental KIEs provide a unified mechanistic interpretation whereby P‐O5′ bond cleavage is concerted with proton transfer from a protonated His119. Thus, theory and experiment provide strong support that RNase A exploits both direct electrostatic stabilization of the anionic transition state and general acid catalysis, resulting in an altered transition state relative to that observed in solution. This work was supported by grants NIH GM096000 to M.E.H., NSF MCB0717850 to V.E.A., NIH AI081987 to J.A.P. and NIH GM064288 to D.M.Y. D.L.K was supported by NIH training grant T32‐GM008056.