The Catalytic Mechanism of Carbonic Anhydrase. Hydrogen-Isotope Effects on the Kinetic Parameters of the Human C Isoenzyme

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Abstract
1 The steady-state kinetics of the interconversion of CO2 and HCO−3 catalyzed by human car-bonic anhydrase C was studied using 1H2O and 2H2O as solvents. The pH-independent parts of the parameters Kcat and Km are 3–4 times larger in 1H2O than in 2H2O for both directions of the reaction, while the ratios kcat/Km show much smaller isotope effects. With either CO2 or HCO−3 as substrate the major pH dependence is observed in kcat, while Km appears independent of pH. The pKa value characterizing the pH-rate profiles is approximately 0.5 unit larger in 2H2O than in 1H2O. 2 The hydrolysis of p-nitrophenyl acetate catalyzed by human carbonic anhydrase C is approxi-mately 35 % faster in 2H2O than in 1H2O. In both solvents the pKa values of the pH-rate profiles are similar to those observed for the CO2-HCO−3 interconversion. 3 It is tentatively proposed that the rate-limiting step at saturating concentrations of CO2 or HCO−3 is an intramolecular proton transfer between two ionizing groups in the active site. It cannot be decided whether the transformation between enzyme-bound CO2 and HCO−3 involves a proton transfer or not.