GLY-63-]GLN SUBSTITUTION ADJACENT TO HIS-64 IN RODENT CARBONIC-ANHYDRASE IVS LARGELY EXPLAINS THEIR REDUCED ACTIVITY

Carbonic anhydrase (CA) IV is a glycosyl-phosphatidylinositol-anchored isozyme expressed on plasma membranes of capillary endothelial cells and certain epithelial cells of the nephron, the colon, and the genito urinary tract. CA IVs purified from bovine and rabbit lungs are high-a ctivity enzymes, like human CA IV, while CA IV from mouse and rat lung s had only 10-20% as much catalytic activity. To explain the molecular basis for these differences in activity, we isolated and characterize d the full-length cDNAs for bovine and rabbit CA IVs and compared thei r sequences to those we previously reported for human, murine, and rat CA IVs. These comparisons led us to postulate that a Gly-63 --> Gin s ubstitution adjacent to His-64 in the rodent enzymes accounts for thei r lower activity. To test this hypothesis, we made the Gly-63 -->, Gin mutants of bovine and rabbit CA IVs and the Gln-63 --> Gly mutant of murine CA IV by site-directed mutagenesis, and compared the activities of mutant and wild-type CA IVs expressed in COS-7 cells. In addition, we produced recombinant cDNAs expressing secretory forms of the Gly-6 3 and Gln-63 forms of each of the three enzymes and compared the activ ities of the enzymes purified from transfected COS-7 cell secretions w ith the activities of CA IVs purified from lungs. These studies demons trated that Gly-63 is important for the high activity of bovine and ra bbit CA IVs, and they showed that the low activity of murine CA IV cou ld be improved by the Gln-63 --> Gly substitution, We suggest that the lower activity of the rodent CA IVs can be largely explained by the G ln-63 substitution which reduces the efficiency of proton transfer by the adjacent His-64.