The pH Dependence of the Hydration of COz Catalyzed by Carbonic Anhydrase I11 from Skeletal Muscle of the Cat

We have measured the pH dependence of the kinetics of C 0 2 hydration catalyzed by carbonic anhydrase I11 from the skeletal muscle of the cat. Two methods were used: an initial velocity study in which the change in absorbance of a pH indicator was measured in a stopped flow spectrophotometer, and an equilibrium study in which the rate of exchange of "0 between C 0 2 and H 2 0 was measured with a mass spectrometer. We have found that the steady state constants k22 and Kcmoo' are independent of pH within experimental error in the range of pH 5.0 to 8.5; the rate of release from the enzyme of the oxygen abstracted from substrate HCO, in the dehydration is also independent of pH in this range. This behavior is very different from that observed for carbonic anhydrase I1 for which k22 and the rate of release of substrate oxygen are very pHdependent. The rate of interconversion of C 0 2 and HC03 at equilibrium catalyzed by carbonic anhydrase I11 is not altered when the solvent is changed from HzO to 98% D20 and 2% H20. Thus, the interconversion probably proceeds without proton transfer in its ratelimiting steps, similar to isozymes I and 11.