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.