Kinetics of Enzymes with Isomechanisms: Britton Induced Transport Catalyzed by Bovine Carbonic Anhydrase II, Measured by Rapid-Flow Mass Spectrometry

Induced transport of13CO2to H13CO−3by bovine carbonic anhydrase II in the presence of excess H12CO−3at pH 6.35 causes a temporary decrease in the concentration of13CO2from 0.169 to 0.092 ± 0.003 mM, measured in less than half a second with a new rapid-flow, membrane-inlet mass spectrometer. From this perturbation, a value of 83 ± 0.3 M−1is calculated for the α term in Eq. [26] of H. G. Britton (Biochem. J.133, 255–261, 1973). Combining α with theKmfor bicarbonate (32 ± 1 mM) and Eqs. [7] and [21] of K. L. Rebholz and D. B. Northrop (Methods Enzymol.249, 211–240, 1995) yields a ratio of less than 0.57 ± 0.04 for the apparent rate constants representing the isomerization segment and chemical conversion segment, respectively, of the enzyme-catalyzed dehydration of bicarbonate. These results provide proof positive for the previously inferred (but unproven despite universal acceptance) isomechanism for the carbonic anhydrases. Moreover, the data and new equations quantify the proposed internal proton transfer to be 64 ± 4% rate-limiting for the bovine type II isoenzyme, a value similar to but more precise than estimates based upon solvent isotope effects and product inhibition kinetics.