Acid/base catalysis is a ubiquitous catalytic strategy used by ribonucleases and ribozymes to stabilize transition states for RNA strand cleavage by 2’-O-transphosphorylation. However, it is a fundamental challenge in mechanistic enzymology to understand the position and timing of proton transfer. The proton inventory (PI) technique analyzes the dependence of enzyme reaction rate on the ratio of D2O to H2O and can infer for simple cases the number of exchangeable sites that produce isotope effects and their magnitude. The Gross-Butler equation in use since the 1930s estimates H/D fractionation factors in both the transition state (phi_TS) and equilibrium isotope effects in the ground state (phi_GS). Typically, experiments are conducted in the “plateau” regions of pL-rate profiles to minimize phi_GS contributions assuming minimal change in active site residue ionization. However, the pKa values and roles of active site groups in acid/base catalysis are often ambiguous, and many enzymes or their mutants lack a pL-independent plateau for PI analysis. Here, we describe a general population-weighted Gross-Butler equation (GPW-GB) that accounts for both equilibrium H/D effects on active site pKa values and kinetic effects on transition state proton transfer. Simulations of experimental results for alternative mechanisms with different transition state effects and active site acid and base pKa values reveal how the magnitude and curvature of PI results as a function of pL. Evaluation of PI results for ribozymes and RNase A from the literature suggests potential inaccuracies in the magnitude of reported effects, or alternative interpretations. The results show that in many cases this approach may aid in resolving kinetic ambiguity and suggest mechanistic details testable by computation or experiment. To establish the proton transfer model of pistol ribozyme, PIs and data fitting on GPW-GB are on the way.

Analysis of proton transfer mechanisms in ribonucleases and ribozymes: Derivation and application of a general population-weighted gross-butler (GPW-GB) equation

ACS Fall 2021

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