Carbonic anhydrase catalysis: A volume profile analysis

The human carbonic anhydrase II catalyzed hydration of CO2 and dehydration of HCO3- at low and high buffer concentrations and at low and high substrate concentrations were studied using a pH-indicator method at pressures up to 130 MPa. Activation volumes, Delta V-double dagger, for K-cat(h), k(cat)(h)/K-m(h), k(cat)(d), and k(cat)(d)/K-m(d), are 0, -9 +/- 1, +9 +/- 1 (+10 +/- 3 in D2O), and +14.0 +/- 1.2 cm(3) mol(-1) in H2O solution, respectively; they were derived from rate constants obtained in the buffer concentration independent region. The value of Delta V-double dagger for both intra- and intermolecular proton-transfer steps is zero. The solvent kinetic isotope effect on k(cat)(d) is 3.6 +/- 0.4 (H2O/D2O) at pressures up to 100 MPa. Analysis of these results indicates that nucleophilic attack of the Zn2+ bound hydroxyl moiety on CO2 is associative, and that the CO2 release and the substitution of H2O by HCO3- on the zinc center are dissociative in character. The CO2 release and a proton transfer are concerted steps during the dehydration reaction. A reaction volume profile for the catalyzed hydration of CO2 and dehydration of HCO3- is constructed. When this has been analyzed in concert with that found for the uncatalyzed reactions and the kinetic results for the model complex catalyzed reactions, further mechanistic distinctions within the overall hydration/dehydration pathways of carbonic anhydrase catalysis were inferred. The nucleophilic attack of the Zn2+-bound hydroxyl moiety on CO2 takes place via an outer-sphere mechanism in which the oxygen from coordinated OH- directly attacks the carbon of CO2. The bicarbonate coordination with Zn2+ is most likely to be unidentate, and the proton of the bicarbonate species bridges two oxygen atoms, one of which coordinates with Zn2+ and the other binds to the carbon atom of the bicarbonate ion. Breakage and formation of the Zn-O bond are involved in the substitution reaction during which HCO3- is displaced by H2O in the hydration reaction, and H2O is displaced by HCO3- in the dehydration reaction.