Abstract
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 18O exchange between CO2 and water. Unlike previously studied isozymes in this class of carbonic anhydrase, the pH profile for kcat for hydration of CO2 catalyzed by mCA IV could not be described by a single ionization, suggesting multiple proton transfer pathways between the zinc-bound water molecule and solution. A role for His64 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 His64 --> Ala. The remaining activity in this mutant at pH levels near 9 suggested a second proton shuttle mechanism. The maximal turnover number kcat for hydration of CO2 catalyzed by mCA IV was 1.1 x 10(6) s-1 at pH > 9. A pKa of 6.6 was estimated for the zinc-bound water molecule in mCA IV.
Highlights
The mammalian carbonic anhydrases (CAs1) constitute a gene family of at least seven distinct isozymes [1] that catalyze the hydration of CO2 to form bicarbonate and a proton: CO2 ϩ H2O u HCO3Ϫ ϩ Hϩ
Murine CA IV—A murine CA IV cDNA coding sequence closely corresponding in structure to human CA II was inserted into a pET31 vector and protein expressed in E. coli BL21(DE3)pLysS [18]
The murine CA IV coding sequence was truncated to mimic that of the endogenous rat protein, removing the first 17 amino acids and converting the residue into a start methionine
Summary
The mammalian carbonic anhydrases (CAs1) constitute a gene family of at least seven distinct isozymes [1] that catalyze the hydration of CO2 to form bicarbonate and a proton: CO2 ϩ H2O u HCO3Ϫ ϩ Hϩ These isozymes are characterized by a high degree of amino acid identity (28 –59%) [2], they are quite diverse in their cellular distribution, catalytic activity, and physiological function (reviewed in Ref. 3). Among these isozymes, CA IV is the only known membrane-associated form. Sulfonamides inhibit CA IV, but the average inhibition constant is 17-fold less than for CA II [12]
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