Kinetics of carbonic anhydrase catalysis in solvents of increased viscosity: a partially diffusion-controlled reaction.

Steady-state kinetic studies of the bovine carbonic anhydrase B-catalyzed hydration of CO2, dehydration of HCO3-, and hydrolysis of p-nitrophenylacetate were made in glycerol/water solvents of increased viscosity in order that the effect of diffusion-control on the substrate association reactions could be determined. The minimum association rate constants (kmin = V/(Km[E0])) were obtained at low substrate concentrations. The esterase activity did not depend upon the solvent viscosity. However, both the CO2 hydration and HCO3- dehydration reactions depended upon the solvent viscosity consistent with partial diffusion control. Thus both chemical activation and diffusion control processes contribute to the observed kmin. In low-viscosity aqueous solutions both hydration and dehydration are largely controlled by chemical activation. However, at higher viscosities, equal to that found in the interior of the erythrocyte, both reactions are largely diffusion controlled. This result can be interpreted to mean that carbonic anhydrase is a highly evolved enzyme that has approached its maximum efficiency. The extent of diffusion control observed rules out H2CO3 as a significant reactant with the enzyme. Several models that yield minimum steric requirements for access of substrate to the active site are examined. Minimum steric constraints are less for the smaller CO2. The slower esterase reaction is not influenced by diffusion.