Intramolecular proton transfer from multiple sites in catalysis by murine carbonic anhydrase V

The hydration of CO2 catalyzed by carbonic anhydrase requires proton transfer from the zinc-bound water at the active site to solution for each cycle of catalysis, In the most efficient of the mammalian carbonic anhydrases, isozyme II, this transfer is facilitated by a proton shuttle residue, His 64. Murine carbonic anhydrase V (mCA V) has a sterically constrained tyrosine at the analogous position; it is not an effective proton shuttle, yet catalysis by this isozyme still achieves a maximal turnover in CO2 hydration of 3 x 10(5) s(-1) at pH > 9. We have investigated the source of proton transfer in a truncated form of mCA V and identified several basic residues, including Lys 91 and Tyr 131, located near the mouth of the active-site cavity that contribute to proton transfer, Intramolecular proton-transfer rates between these shuttle groups and the zinc-bound water were estimated as the rate-determining step in k(cat) for hydration of CO2 measured by stopped-flow spectrophotometry and in the exchange of O-18 between CO2 and water measured by mass spectrometry. Comparison of k(cat) in catalysis by Lys 91 and Tyr 131 and the corresponding double mutant showed a strong antagonistic interaction between these sites, suggesting a cooperative behavior in facilitating the proton-transfer step of catalysis, Replacing four potential proton shuttle residues produced a multiple mutant that had 10% of the catalytic turnover k(cat) of the wild type, suggesting that the main proton shuttles have been accounted for in mCA V, These replacements caused relatively small changes in k(cat)/K-m for hydration, which measures the interconversion of CO2 and HCO3- in a stage of catalysis that is separate and distinct from the proton transfers; these measurements serve as a control indicating that the replacements of proton shuttles have not caused structural changes that affect reactivity at the zinc.