Intermolecular proton transfer in catalysis by carbonic anhydrase V

The dehydration of bicarbonate catalyzed by carbonic anhydrase is accompani ed by the transfer of a proton from solution to the zinc-bound hydroxide. W e have investigated the properties of proton transfer from donors in soluti on, mostly derivatives of imidazole and pyridine, to a truncated mutant of carbonic anhydrase V with replacements that render the active site cavity l ess sterically constrained, Tyr 64 --> Ala and Phe 65 --> Ala. Catalysis wa s measured by determining the rate of exchange of O-18 between the CO2-HCO3 - system and water, and rate constants for proton transfer were estimated a s the rate-limiting step in the release of (H2O)-O-18 from the enzyme to so lution. Each proton donor enhanced catalytic activity in a saturable manner . The resulting rate constants for proton transfer when compared with the v alues of pK(a) of the donor and acceptor gave a Bronsted plot of high curva ture. These data could also be described by Marcus theory which showed an i ntrinsic barrier for intermolecular proton transfer near 0.8 kcal/mol and a work term or thermodynamic contribution to the free energy of reaction nea r 10 kcal/mol. This low intrinsic kinetic barrier for proton transfer is ve ry similar to nonenzymic bimolecular proton transfer between nitrogen and o xygen acids and bases in solution. However, the significant thermodynamic c ontribution suggests appreciable involvement of solvent and active-site org anization prior to proton transfer. These Marcus parameters are very simila r to those describing intramolecular proton transfer from His 64 in carboni c anhydrase, suggesting similarities in the intra- and intermolecular proto n transfer processes.