Carbon dioxide hydration activity of carbonic anhydrase in mixtures of water and deuterium oxide.

We have measured the hydration of CO2 catalyzed by bovine red cell carbonic anhydrase in mixtures of H2O and D2O at pH(D) which is in the plateau region of the pH--activity profile. The steady-state parameters for hydration Vmax and Km both show an exponential dependence on the atom fraction of deuterium in solvent water. These observed solvent isotope effects cannot be fit to a model involving one hydrogen but must arise from the contributions of two or more hydrogens which change their fractionation factor in the transition state of the rate-limiting step of the catalysis. On the basis of previous evidence, this step is taken to be an intramolecular proton transfer which our results suggest involves water bridges. The dependence of the rate constant for the uncatalyzed reaction between CO2 and water has a nonlinear dependence on the atom fraction of deuterium in the solvent. We conclude that the observed isotope effect must arise from more than one hydrogen in the transition state.