Chemical rescue of proton transfer in catalysis by carbonic anhydrases in the β- and γ-class

Catalysis of the dehydration of HCO₃^- by carbonic anhydrase requires proton transfer from solution to the zinc-bound hydroxide. Carbonic anhydrases in each of the α, β, and γ classes, examples of convergent evolution, appear to have a side chain extending into the active site cavity that acts as a proton shuttle to facilitate this proton transfer, with His 64 being the most prominent example in the α class. We have investigated chemical rescue of mutants in two of these classes in which a proton shuttle has been replaced with a residue that does not transfer protons: H216N carbonic anhydrase from Arabidopsis thaliana (β class) and E84A carbonic anhydrase from the archeon Methanosarcina thermophila (γ class). A series of structurally homologous imidazole and pyridine buffers were used as proton acceptors in the activation of CO₂ hydration at steady state and as proton donors of the exchange of ¹⁸O between CO₂ and water at chemical equilibrium. Free energy plots of the rate constants for this intermolecular proton transfer as a function of the difference in pK_a of donor and acceptor showed extensive curvature, indicating a small intrinsic kinetic barrier for the proton transfers. Application of Marcus rate theory allowed quantitative estimates of the intrinsic kinetic barrier which were near 0.3 kcal/mol with work functions in the range of 7-11 kcal/mol for mutants in the β and γ class, similar to results obtained for mutants of carbonic anhydrase in the α class. The low values of the intrinsic kinetic barrier for all three classes of carbonic anhydrase reflect proton transfer processes that are consistent with a model of very rapid proton transfer through a flexible matrix of hydrogen-bonded solvent structures sequestered within the active sites of the carbonic anhydrases.