Carbonic anhydrase VII (CA VII) appears to be the most highly conserved of the active mammalian
carbonic anhydrases. We have characterized the catalytic activity and inhibition properties of a recombinant murine CA VII. CA VII has steady-state constants similar to two of the most active
isozymes of
carbonic anhydrase, CA II and IV; also, it is very strongly inhibited by the
sulfonamides ethoxzolamide and
acetazolamide, yielding the lowest Ki values measured by the exchange of 18O between CO2 and water for any of the mammalian
isozymes of
carbonic anhydrase. The catalytic measurements of the hydration of CO2 and the
dehydration of HCO3- were made by stopped-flow spectrophotometry and the exchange of 18O using mass spectrometry. Unlike the other
isozymes of this class of CA, for which Kcat/K(m) is described by the single ionization of
zinc-bound water, CA VII exhibits a pH profile for Kcat/K(m) for CO2 hydration described by two ionizations at pKa 6.2 and 7.5, with a maximum approaching 8 x 10(7) M-1 s-1. The pH dependence of kcat/K(m) for the hydrolysis of
4-nitrophenyl acetate could also be described by these two ionizations, yielding a maximum of 71 M-1 s-1 at pH > 9. Using a novel method that compares rates of 18O exchange and
dehydration of HCO3-, we assigned values for the apparent pKa at 6.2 to the
zinc-bound water and the pKa of 7.5 to His 64. The magnitude of Kcat, its pH profile, 18O-exchange data for both wild-type and a H64A mutant, and inhibition by CuSO4 and
acrolein suggest that the
histidine at position 64 is functioning as a
proton-transfer group and is responsible for one of the observed ionizations. A truncation mutant of CA VII, in which 23 residues from the amino-terminal end were deleted, has its rate constant for intramolecular
proton transfer decreased by an order of magnitude with no change in Kcat/K(m). This suggests a role for the amino-terminal end in enhancing
proton transfer in catalysis by
carbonic anhydrase.