Kinetics of enzymes with isomechanisms: britton induced transport catalyzed by bovine carbonic anhydrase II, measured by rapid-flow mass spectrometry.
Induced transport of 13CO2 to H13CO3- by bovine carbonic anhydrase II in the presence of excess H12CO3- at pH 6.35 causes a temporary decrease in the concentration of 13CO2 from 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-1 is calculated for the alpha term in Eq. [26] of H. G. Britton (Biochem. J. 133, 255-261, 1973). Combining alpha with the K(m) for 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.
Northrop
D B
DB
Division of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin, Madison 53706, USA.
Simpson
F B
FB
D000818:Animals; D001639:Bicarbonates / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism; D002247:Carbon Isotopes; D002256:Carbonic Anhydrases / Q000378:metabolism*; D002417:Cattle; D004798:Enzymes / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods
D001639:Bicarbonates; D002247:Carbon Isotopes; D004798:Enzymes; D002245:Carbon Dioxide; D002256:Carbonic Anhydrases