The catalytic properties of murine carbonic anhydrase VII

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 O-18 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 O-18 using mass spectrometry, Unlike the other isozymes of this class of CA, for which k(cat)/K-m is described by the single ionization of zinc-bound water, CA VII exhibits a pH profile for k(cat)/K-m for CO2 hydration described by two ionizations at pK(a) 6.2 and 7.5, with a maximum approaching 8 x 10(7) M-1 s(-1). The pH dependence of k(cat)/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 O-18 exchange and dehydration of HCO3-, we assigned values for the apparent pK(a) at 6.2 to the zinc-bound water and the pK(a) of 7.5 to His 64, The magnitude of k(cat), its pH profile, O-18-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 k(cat)/K-m. This suggests a role for the amino-terminal end in enhancing proton transfer in catalysis by carbonic anhydrase.