Comparison of intra- and intermolecular proton transfer in human carbonic anhydrase II.
The catalysis of the hydration of CO2 by human carbonic anhydrase II (HCA II) includes the transfer of a proton from zinc-bound water to histidine 64 utilizing a network of intervening hydrogen-bonded water molecules, then the proton is transferred to buffer in solution. We used stopped-flow spectrophotometry and 18O exchange between CO2 and water measured by mass spectrometry to compare catalytic constants dependent on proton transfer in HCA II and in the mutant H64A HCA II containing the replacement His64-->Ala. Maximal velocities and oxygen-18 exchange catalyzed by H64A HCA II showed that nearly all of the proton transfer with this mutant proceeded through the imidazole buffer. The following parameters were very similar or identical in catalysis by H64A HCA II compared with catalysis by wild-type HCA II both in the presence of large concentrations of imidazole (100 mM): the maximal rate of initial velocity and of exchange of 18O between CO2 and water, solvent hydrogen isotope effects on the maximal velocity, and the dependence of these isotope effects on the atom fraction of deuterium in solvent water. These results indicate that the proton transfer involving the zinc-bound water in catalysis is not significantly affected by the difference between the mobility of the free imidazole buffer and the side chain of His 64. Moreover, data for both the wild-type and mutant enzymes are consistent with proton transfer through intervening hydrogen-bonded water bridges in the active sites. These features of the proton transfer are discussed in terms of a model in which the first proton transfer from the zinc-bound water to an adjacent water is rate limiting.
Taoka
S
S
Department of Pharmacology and Therapeutics, University of Florida, Gainesville 32610.
Tu
C
C
Kistler
K A
KA
Silverman
D N
DN
D002256:Carbonic Anhydrases / Q000737:chemistry*; D002384:Catalysis; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D011522:Protons
D011522:Protons; D002256:Carbonic Anhydrases