Catalysis by cobalt(II)-substituted carbonic anhydrase II of the exchange of oxygen-18 between CO2 and H2O.
We have measured the catalysis by Co(II)-substituted bovine carbonic anhydrase II from red cells of the exchange of 18O between CO2 and H2O using membrane-inlet mass spectrometry. We chose Co(II)-substituted carbonic anhydrase II because the apparent equilibrium dissociation constant of HCO3- and enzyme at pH 7.4, KHCO3-eff approximately equal to 55 mM, was within a practicable range of substrate concentrations for the 18O method. For the native, zinc-containing enzyme KHCO3-eff is close to 500 mM at this pH. The rate constant for the release from the active site of water bearing substrate oxygen kH2O was dependent on the fraction of enzyme that was free, not bound by substrate HCO3- or anions. The pH dependence of kH2O in the pH range 6.0-9.0 can be explained entirely by a rate-limiting, intramolecular proton transfer between cobalt-bound hydroxide and a nearby group, probably His-64. The rate constant for this proton transfer was found to be 7 X 10(5) S-1 for the Co(II)-substituted enzyme and 2 X 10(6) S-1 for the native enzyme. These results are applied to models derived from proton-relaxation enhancement of water exchanging from the inner coordination shell of the cobalt in carbonic anhydrase. The anions iodide, cyanate, and thiocyanate inhibited catalysis of 18O exchange by Co(II)-substituted carbonic anhydrase II in a manner competitive with total substrate (CO2 and HCO3-) at chemical equilibrium and pH 7.4. These results are discussed in terms of observed steady-state inhibition patterns and suggest that there is no significant contribution of a ternary complex between substrate, inhibitor, and enzyme.
D000818:Animals; D002245:Carbon Dioxide; D002256:Carbonic Anhydrases / Q000097:blood*; D002417:Cattle; D003035:Cobalt / Q000494:pharmacology*; D004912:Erythrocytes / Q000201:enzymology; D007700:Kinetics; D010103:Oxygen Isotopes; D011485:Protein Binding; D014867:Water
D010103:Oxygen Isotopes; D014867:Water; D002245:Carbon Dioxide; D003035:Cobalt; D002256:Carbonic Anhydrases