Isotope effect evidence for the zinc hydroxide mechanism of carbonic anhydrase catalysis.
The carbon kinetic isotope effect on the enzymatic dehydration of HCO3- ion is k12/k13 = 1.011 and is independent, within experimental error, of the addition of sucrose, substitution of D2O for H2O, and substitution of enzyme-bound Zn2+ by Co2+. These results are consistent with a ping-pong mechanism in which proton transfer between enzyme and solvent is separated from HCO3- dehydration. For the dehydration half-reaction, diffusional processes are severalfold faster than dehydration, and the rate-determining step is the dehydration itself. The intrinsic isotope effect is approximately 1.011, indicating that hydration of CO2 occurs by reaction of zinc-bound OH-, rather than zinc-bound H2O.
D000818:Animals; D001639:Bicarbonates; D002256:Carbonic Anhydrases / Q000097:blood*; D002417:Cattle; D003035:Cobalt / Q000494:pharmacology; D003903:Deuterium; D017666:Deuterium Oxide; D004912:Erythrocytes / Q000201:enzymology*; D006878:Hydroxides; D007700:Kinetics; D010103:Oxygen Isotopes; D014867:Water; D015032:Zinc; D017967:Zinc Compounds
D001639:Bicarbonates; D006878:Hydroxides; D010103:Oxygen Isotopes; D017967:Zinc Compounds; D014867:Water; D003035:Cobalt; D003903:Deuterium; D002256:Carbonic Anhydrases; C018021:cobaltous chloride; D015032:Zinc; D017666:Deuterium Oxide; C052745:zinc hydroxide