Thermodynamics of Carbonic Anhydrase Catalysis

The COZ hydration and HC03dehydration activities of human red cell carbonic anhydrase isozymes B and C (HCAB and HCAC) have been studied as a function of temperature from 0” to 37°C. The Arrhenius plots of In kcat versus 1/T are linear for both isozymes in both hydration and dehydration reactions, indicating that the rate-determining steps remain unchanged over this temperature range. The 37°C hydration kc, , at pH 7.5, is 13 X 10‘ s-’ for isozyme C and 0.71 X lo6 s-’ for isozyme B. K,, for hydration, is 10 m~ for C and 5 m~ for B, and invariant with temperature. The uncatalyzed reactions are significantly affected by temperature, 30to 40-fold rate enhancements being observed from 0’ to 37°C. The enzyme-catalyzed processes are much less sensitive to temperature, the rate enhancements being 2to %fold for HCAB and 5to 6-fold for HCAC in this temperature range. These observations are consistent with a significant lowering of the free energy of activation by both isozymes. This effect is greater for C accounting for its higher catalytic power. The enthalpy of activation, at pH 7.5 and 8.2, in the rate-limiting step is considerably less for the B enzyme compared to C. This is, however, more than offset by a large negative entropy of activation in the case of HCAB. This observation indicates either a mechanistic difference in the rate-limiting events or a difference in the structural organizations of the active sites of the two isozymes, or both.