Mechanism of Action of Carbonic Anhydrase

Mn(II), Co(II), Ni(II), Cu(II), Zn(II), Cd(II), and Hg(II) form metallocarbonic anhydrases containing these metal ions at the same binding site. Of this series, only the Zn(II) and Co(II) enzymes catalyze the hydration of CO2 and the hydrolysis of p-nitrophenyl acetate. Likewise, only Zn(II) and Co(II) induce the metal ion-dependent binding of 3H-acetazolamide at the active center. Acetate, azide, cyanate, sulfide, and cyanide compete with 3H-acetazolamide for the binding site at the active center. Binding of 1 eq of sulfide and cyanide to the Zn(II) and Co(II) enzymes at pH values below the pKa values of H2S and HCN is accompanied by the release of H+ ions. Over the pH range in which the inhibitors are in the form CN- and HS-, binding is accompanied by the appearance of -OH ions. The experimental data fit the theoretical curves constructed by assuming a single additional H+ dissociation associated with the protein, coupled with the metal ion, and described by a pKa of 8.1. The latter coincides with the inflection point of the pH-rate profile for catalysis by human carbonic anhydrase B. Apo- and the inactive metallocarbonic anhydrases do not show the alterations in hydrogen ion equilibria accompanying the addition of CN-. Accompanying spectral data as a function of pH show that -OH, HCO3-, and CN- compete for the same binding site on the Co(II) enzyme. A mechanism based on a mixed enzyme-zinc-hydroxide as the active form of the enzyme is proposed for the hydration of CO2 and the hydrolysis of p-nitrophenyl acetate.