Inhibition by Cupric Ions of super(18)O Exchange Catalyzed by Human Carbonic Anhydrase II: Relation to the Interaction Between Carbonic Anhydrase and Hemoglobin

The inhibition of human carbonic anhydrase II by cupric ions has been investigated by determining the effect of these ions on two types of super(18)O exchange: the exchange of super(18)O between CO sub(2) and water; and the exchange of super(18)O between super(12)C-and super(13)C-containing species of CO sub(2). R sub(1) is the rate at chemical equilibrium of the interconversion of CO sub(2) and HCO sub(3) super(-). R sub(H2O) is the rate of the dissociation of water bearing substrate oxygen from the enzyme. Cupric ions inhibited R sub(H2O) with an IC sub(50) near 1 x 10 super(-7) M at pH 7.3 but did not inhibit R sub(1) at concentrations below 4 x 10 super(-6) M. Lowering the pH to 6 abolished this inhibition by Cu super(2+). Thus, Cu super(2+) binds to human carbonic anhydrase II at a site other than the active site and inhibits the exchange of water from the enzyme without affecting the equilibrium rate of hydration of CO sub(2). The data suggest that protonation of the copper-binding site as pH is lowered to 6 decreases the tightness of binding of Cu super(2+). In view of this inhibition the authors reinvestigated the report that micromolar concentrations of hemoglobin and histidine enhanced super(18)O exchange between CO sub(2) and H sub(2)O catalyzed by carbonic anhydrase II. This enhancement is due to removal of Cu super(2+) from its inhibitory binding site on the enzyme by hemoglobin and histidine, which themselves bind Cu super(2+) tightly.