Abstract
The CO2 hydration and HCO3- dehydration activities of human red cell carbonic anhydrase isozymes B and C (HCAB and HCAC) have been studied as a function of temperature from 0 degrees to 37 degrees C. The Arrhenius plots of ln 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 degrees C hydration kcat, at pH 7.5, is 13 X 10(5) s-1 for isozyme C and 0.71 X 10(5) s-1 for isozyme B. Km, for hydration, is 10 mM for C and 5 mM for B, and invariant with temperature. The uncatalyzed reactions are significantly affected by temperature, 30- to 40-fold rate enhancements being observed from 0 degrees to 37 degrees C. The enzyme-catalyzed processes are much less sensitive to temperature, the rate enhancements being 2- to 3-fold for HCAB and 5- to 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.
Highlights
The COZhydration and HC03- dehydration activities HCOC dehydration of HCAB and HCAC isozymes and atof human red cell carbonic anhydrase isozymes B and tempt to provide a thermodynamic basis for the kinetic dif
The enzyme-catalyzedprocesses are muchless sensitive to temperature, the rate enhancements being 2- to %fold forHCAB and 5- to 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
Rates for both COZhydration from 0’ to 25°C and HC03- dehydration from 0” to 37°C were determined using the methods of Maren and Couto [12]
Summary
From the DeDartment of Pharmacolo-m_ andTheraDeutics, University of Florida College of Medicine, Gainesuille, Florida 32610. The enzyme-catalyzedprocesses are muchless sensitive to temperature, the rate enhancements being 2- to %fold forHCAB and 5- to 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. In dehydration is considerably less for the B enzyme compared to C. reaction, ionic strength varied with substrate (HCOa-)concentration This is, more than offset by a large negative entropy of activation in the case of HCAB. The CO, hydration kinetics at 37°C were studied in a Durrum-Gibson enzyme in human red cell occurs in two isozymes, designated B and C. Both isozymes are structurally well characterized, and their tertiary structuresshow considerable similarity [13]. E-AHt'RT.eASt'wRh,ere ka is Boltzmann's constant and h is Planck's constant
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