Catalytic properties of murine carbonic anhydrase IV

A cDNA encoding the murine carbonic anhydrase IV (mCA IV) gene, modified to resemble a form of mature human carbonic anhydrase IV (Okuyama, T,, Waheed, A., Kusumoto, W., Zhu, X. L., and Sly, W. S. (1995) Arch. Biochem. Biophys, 320, 315-322), was expressed in Escherichia coli. Inactive inclusion bodies were collected and refolded, and active enzyme was purified; the resulting mCA IV was used to characterize the catalysis of CO2 hydration using stopped flow spectrophotometry and O-18 exchange between CO2 and water, Unlike previously studied isozymes in this class of carbonic anhydrase, the pH profile for k(cat) for hydration of CO2 catalyzed by mCA TV could not be described by a single ionization, suggesting multiple proton transfer pathways between the zinc bound water molecule and solution, A role for His(64) in transferring protons between the zinc-bound water and solution was confirmed by the 100 fold lower activity of the mutant of mCA IV containing the replacement His(64) --> Ala, The remaining activity in this mutant at pH levels near 9 suggested a second proton shuttle mechanism, The maximal turnover number k(cat) for hydration of CO2 catalyzed by mCA IV was 1.1 x 10(6) s(-1) at pH > 9. A pK(a) of 6.6 was estimated for the zinc-bound water molecule in mCA IV.