Gly-63 → Gln substitution adjacent to His-64 in rodent carbonic anhydrase IVs largely explains their reduced activity

Abstract

Carbonic anhydrase (CA) IV is a glycosylphosphatidylinositol-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 3 Gln substitution adjacent to His-64 in the rodent enzymes accounts for their lower activity. To test this hypothesis, we made the Gly-63 3 Gln mutants of bovine and rabbit CA IVs and the Gln-63 3 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 3 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.

A transcriptomic approach of salinity response in the euryhaline teleost, Dicentrarchus labrax

“…These motifs are known to be correlated with transcript stability (Shaw and Kamen, 1986), and have been found in other cDNAs (Fucci et al, 1995;Tanguy et al, 2005). We also observed seven putative N-glycosylation motifs Asn-Xaa-Thr/Ser (Marshall, 1974) in the coding region of D. labrax CA gene, as described in the mouse CA gene (Tamai et al, 1996).…”

Catalytic Properties of Murine Carbonic Anhydrase IV

“…It is noteworthy that murine CA IV has glutamine at residue 63, whereas human CA II has glycine at this position; hence, in this position also murine CA IV has a bulkier side chain that could hinder the role of His 64 as a proton shuttle. Tamai et al (41) have reported evidence consistent with the influence of residue 63 in the overall catalysis by CA IV; the replacement Gly 63 3 Gln in bovine CA IV decreases catalytic activity measured in a colorimetric assay near neutral pH, and the replacement Gln 63 3 Gly in murine CA IV increases activity. This role of residue 63 can also explain the differences in the pH profile for k cat obtained for murine CA IV in this work and that of Baird et al (42) for human CA IV.…”

Selection of Carbonic Anhydrase Variants Displayed on Phage

“…However, the effect on k cat does not parallel changes in the pK a of the zinc-solvent ligand, as observed for transfer of a proton between an acid to a base in model reactions and between His 64 and bases (55)(56)(57). Therefore, the decreases in k cat are likely caused by small changes in the positioning of the active site solvent/His 64 proton transfer pathway, as has been observed for amino acid substitutions at positions near His 64 (58,59). Interestingly, the variants with the largest increase in the zinc dissociation rate constant (Ͼ300-fold relative to wild-type CAII) also display the biggest decreases in CO 2 , k cat /K M is decreased 9 -74-fold relative to wild-type CAII.…”