J. Biol. Chem., Vol. 262, Issue 34, 16417-16424, 12, 1987

A comparison of the mechanisms of CO2 hydration by native and Co2+- substituted carbonic anhydrase II

KA Kogut and RS Rowlett
Department of Chemistry, Colgate University, Hamilton, New York 13346.

We have measured the pH dependence of kcat and kcat/Km for CO2 hydration catalyzed by both native Zn2+-and metallo-substituted Co2+- bovine carbonic anhydrase II in the absence of inhibitory ions. For the Zn2+-enzyme, the pKa values controlling kcat and kcat/Km profiles are similar, but for the Co2+-enzyme the values are about 0.6 pH units apart. Computer simulations of a metal-hydroxide mechanism of carbonic anhydrase suggest that the data for both native and Co2+-carbonic anhydrase can be accounted for by the same mechanism of action, if we postulate that the substitution of Co2+ for Zn2+ in the active site causes a separation of about 0.6 pH units in the pKa values of His-64 and the metal-bound water molecule. We have also measured the activation parameters for kcat and kcat/Km for Co2+-substituted carbonic anhydrase II-catalyzed CO2 hydration and have compared these values to those obtained previously for the native Zn2+-enzyme. For kcat and kcat/Km we obtain an enthalpy of activation of 4.4 +/- 0.6 and approximately 0 kcal mol-1, respectively. The corresponding entropies of activation are -18 +/- 2 and -27 +/- 2 cal mol-1 K-1.
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