The Catalytic Properties of Murine Carbonic Anhydrase VII

Abstract

Carbonic anhydrase VII (CA VII) appears to be the most highly conserved of the active mammalian carbonic anhydrases. We have characterized the catalytic activity and inhibition properties of a recombinant murine CA VII. CA VII has steady-state constants similar to two of the most active isozymes of carbonic anhydrase, CA II and IV; also, it is very strongly inhibited by the sulfonamides ethoxzolamide and acetazolamide, yielding the lowest Ki values measured by the exchange of 18O between CO2 and water for any of the mammalian isozymes of carbonic anhydrase. The catalytic measurements of the hydration of CO2 and the dehydration of HCO3- were made by stopped-flow spectrophotometry and the exchange of 18O using mass spectrometry. Unlike the other isozymes of this class of CA, for which Kcat/K(m) is described by the single ionization of zinc-bound water, CA VII exhibits a pH profile for Kcat/K(m) for CO2 hydration described by two ionizations at pKa 6.2 and 7.5, with a maximum approaching 8 x 10(7) M-1 s-1. The pH dependence of kcat/K(m) for the hydrolysis of 4-nitrophenyl acetate could also be described by these two ionizations, yielding a maximum of 71 M-1 s-1 at pH > 9. Using a novel method that compares rates of 18O exchange and dehydration of HCO3-, we assigned values for the apparent pKa at 6.2 to the zinc-bound water and the pKa of 7.5 to His 64. The magnitude of Kcat, its pH profile, 18O-exchange data for both wild-type and a H64A mutant, and inhibition by CuSO4 and acrolein suggest that the histidine at position 64 is functioning as a proton-transfer group and is responsible for one of the observed ionizations. A truncation mutant of CA VII, in which 23 residues from the amino-terminal end were deleted, has its rate constant for intramolecular proton transfer decreased by an order of magnitude with no change in Kcat/K(m). This suggests a role for the amino-terminal end in enhancing proton transfer in catalysis by carbonic anhydrase.

Tribute to R. G. Boutilier: Evidence of a high activity carbonic anhydrase isozyme in the red blood cells of an ancient vertebrate, the sea lamprey Petromyzon marinus

“…Unlike carp CA, which grouped within the previously described fish cytoplasmic CA clade (Lund et al, 2002;Tufts et al, 2003;Esbaugh et al, 2004;Esbaugh et al, 2005), lamprey CA grouped closely with vertebrate CA VII. Interestingly, vertebrate CA VII is thought to be ancestral to both mammalian CA I, II and III, as well as the fish cytoplasmic CAs (Lund et al, 2002;Esbaugh et al, 2004;Esbaugh et al, 2005), and is not known to have any physiological function (Lakkis et al, 1996;Lakkis et al, 1997;Earnhardt et al, 1998). It is unclear whether lamprey CA is actually a CA VII-like isozyme due to the discrepancies between the NJ and MP methods; however, it is certain that the lamprey isozyme is ancestral to cytoplasmic isozymes found in more derived vertebrates.…”

“…This enzyme has been found in virtually all living organisms, but only members of the ␣-CA gene family are found in vertebrates (Tashian, 1992;Hewett-Emmett and Tashian, 1996;Earnhardt et al, 1998). To date, 15 different ␣-CA isozymes have been characterized in mammals by means of their kinetic properties, subcellular location and/or molecular structure (Chegwidden and Carter, 2000;HewettEmmett, 2000).…”

Tautomerism of Histidine 64 Associated with Proton Transfer in Catalysis of Carbonic Anhydrase

“…It is widely accepted that the hydration of CO 2 catalyzed by hCAII proceeds through several chemical steps as shown in Scheme 1 (1,4,5): the direct nucleophilic attack of the zinc-bound hydroxide ion on the carbonyl carbon of substrate CO 2 (structures 1-2), the formation of a zinc-bound bicarbonate intermediate (structures [2][3], the isomerization of the bicarbonate ion (structures 3-4), the exchange of the product bicarbonate ion with a H 2 O (structures 4 -5), and the regeneration of the zinc-bound hydroxide ion by the transfer of a proton to bulk solvent (structures [1][2][3][4][5]. The proton transfer step (structures 1-5) consists of two substeps: 1) an intra-molecular transfer of protons to another residue in the enzyme and 2) a release of protons to the outside of the enzyme with the aid of a base.…”

Expression of hypoxia‐inducible, membrane‐bound carbonic anhydrase isozyme XII in mouse tissues

“…Four of these are cytoplasmic (CA I, CA II, CA III, and CA VII) (Sly and Hu, 1995;Earnhardt et al, 1998), two are mitochondrial (CA VA and CA VB) (Fujikawa-Adachi et al, 1999), one is secreted (CA VI) (Murakami and Sly, 1987), and four are membrane associated (CA IV, CA IX, CA XII, and CA XIV) (Zhu and Sly, 1990;Pastorek et al, 1994;Ivanov et al, 1998;Tü reci et al, 1998;Mori et al, 1999). Some isozymes such as CA II are expressed in a number of different tissues, whereas others (e.g., CA VI, IX, and XIV) show a more limited distribution.…”