PubMed2003
The second step in the enzyme-catalyzed hydrolysis of phosphate esters by ribonuclease A (RNase A) was studied using an ab initio quantum-based model of the active site including constrained parts of three critical residues, His-12, His-119, and Lys-41, and a small substrate. The competition between release of the cyclic phosphate intermediate and subsequent hydrolysis following transphosphorylation was explored to determine the electronic factors that contribute to preferential intermediate product release observed experimentally. The structural and energetic results obtained at both the RHF and MP2 levels reveal several contributing factors consistent with experimental observation. Although the intrinsic electronic effects tend to favor hydrolysis slightly with an overall activation free energy of approximately 70 kJ mol(-1), entropic and environmental effects favor release of the cyclic phosphate intermediate over hydrolysis. Exploration of the second, hydrolysis step also revealed interesting similarity with the transphosphorylation step, including the observation of autocatalysis by the substrate. Moreover, both steps of the overall RNase A reaction reveal multiple pathways involving proton transfers to sites of similar proton affinities. The anionic phosphate in both steps can act as a stable proton binding site as protons are moved around the active site throughout the progress of the reaction. These results suggest autocatalysis may be representative of more general behavior in enzymes containing highly charged substrates, especially phosphates.