Beyond the Plateau: pL Dependence of Proton Inventories as a Tool for Studying Ribozyme and Ribonuclease Catalysis.

Acid/base catalysis is an important catalytic strategy used by ribonucleases and ribozymes; however, understanding the number and identity of functional groups involved in proton transfer remains challenging. The proton inventory (PI) technique analyzes the dependence of the enzyme reaction rate on the ratio of D2O to H2O and can provide information about the number of exchangeable sites that produce isotope effects and their magnitude. The Gross-Butler (GB) equation is used to evaluate H/D fractionation factors from PI data typically collected under conditions (i.e., a "plateau" in the pH-rate profile) assuming minimal change in active site residue ionization. However, restricting PI analysis to these conditions is problematic for many ribonucleases, ribozymes, and their variants due to ambiguity in the roles of active site residues, the lack of a plateau within the accessible pL range, or cooperative interactions between active site functional groups undergoing ionization. Here, we extend the integration of species distributions for alternative enzyme states in noncooperative models of acid/base catalysis into the GB equation, first used by Bevilacqua and colleagues for the HDV ribozyme, to develop a general population-weighted GB equation that allows simulation and global fitting of the three-dimensional relationship of the D2O ratio (n) versus pL versus kn/k0. Simulations using the GPW-GB equation of PI results for RNase A, HDVrz, and VSrz illustrate that data obtained at multiple selected pL values across the pL-rate profile can assist in the planning and interpreting of solvent isotope effect experiments to distinguish alternative mechanistic models.