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 genitourinary tract. CA IVs purified from bovine and rabbit lungs are high-activity enzymes, like human CA IV, while CA IV from mouse and rat lungs had only 10-20% as much catalytic activity. To explain the molecular basis for these differences in activity, we isolated and characterized the full-length cDNAs for bovine and rabbit CA IVs and compared their sequences to those we previously reported for human, murine, and rat CA IVs. These comparisons led us to postulate that a Gly-63-->Gln substitution adjacent to His-64 in the rodent enzymes accounts for their lower activity. To test this hypothesis, we made the Gly-63-->Gln 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-63 and Gln-63 forms of each of the three enzymes and compared the activities of the enzymes purified from transfected COS-7 cell secretions with the activities of CA IVs purified from lungs. These studies demonstrated that Gly-63 is important for the high activity of bovine and rabbit CA IVs, and they showed that the low activity of murine CA IV could 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 Gln-63 substitution which reduces the efficiency of proton transfer by the adjacent His-64.
Tamai
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Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Health Sciences Center, MO 63104, USA.
Waheed
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Cody
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Sly
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D015723:Gene Library; D005973:Glutamine; D005998:Glycine; D006639:Histidine; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D007527:Isoenzymes; D011994:Recombinant Proteins; D005973:Glutamine; D006639:Histidine; D002256:Carbonic Anhydrases; D005998:Glycine