Carbonic Anhydrase

 The enzyme carbonic anhydrase (CA) catalyses the reversible hydration of carbon dioxide to bicarbonate and a proton, using a catalytic zinc ion bound at the active site (1, 2). Carbonic anhydrases are of interest because of their varied metabolic roles, efficient catalytic mechanisms, and high metal ion specificity. The enzyme is found in animals, plants, and bacteria, where it plays roles in respiration, photosynthesis, and CO₂ fixation. Three genetically distinct families of CA proteins have been identified: aCA isozymes, found in all animals; bCA, commonly found in plants and bacteria; and gCA, found in a variety of bacteria (3). Of these classes of CA proteins, the a-CA family is the best characterized. This article provides a brief overview of the metabolic roles and genetic structure of the a-CA human isozymes and describes the catalytic reaction mechanism and metal-binding properties of the high activity, well-characterized human a-CA isozyme II (CAII). Recent work on the b- and g-CA enzymes, which catalyze CO₂ hydration using radically different protein structures, is summarized.

1. Metabolic Function and Genetic Structure of Human a-CA Isozymes

 Carbonic anhydrase II (CAII) was first discovered in 1933 in human erythrocytes (4), where it facilitates CO₂ transport in respiration by converting CO₂, released as a metabolic by-product from tissues, to bicarbonate. Bicarbonate is carried by the bloodstream to pulmonary capillaries, where its conversion back to CO₂ is catalyzed by CA for release by the lung. CAII is also expressed in many other tissues, including ocular epithelium, where it plays a role in maintaining intraocular pressure (5) . A human genetic disease in which CAII is nonfunctional indicates that this isozyme is also crucial for bone resorption and kidney function (6). Furthermore, the inhibition of CAII by aromatic sulfonamides (RSO₂NH₂) is used clinically to treat glaucoma and altitude sickness (5). Additionally, at least six more CA human isozymes with varied activities and locations have been discovered (5). The cytosolic isozymes CAI, CAIII, and CAVI are expressed in high concentrations in red blood cells, muscle cells, and salivary glands, respectively. CAIV is a membrane-bound isozyme important for regulating bicarbonate levels in the kidney, whereas CAV is a mitochondrial isozyme. CAVI is secreted by the salivary glands. The exact physiological role of several of these isozymes is still under investigation.

 The genomic structure of the human CA isozymes lends insight into their evolution and regulation of their transcription. CAI, CAII, and CAIII have been mapped to chromosome 8q22, CAV and VII to chromosome 16, CAIV to chromosome 17q, and CAVI to chromosome 1 (3). This chromosomal mapping, along with comparisons of DNA sequence homologies, indicates that gene duplication events over 450 million years ago led to distribution of the CA isozymes on four different chromosomes; further gene duplications occurred later, resulting in closely linked isozymes CAI, CAII, and CAIII on one chromosome, and CAIV and VI on another (3). The structures of the various human CA genes currently are being studied to determine the mechanism of their tissue-specific expression. CAI has an unusually long 5′-untranslated region containing two promoters that direct expression of CAI in erythroid cells and in the colon (7). Other CA genes are also likely to have complex structures to direct their expression in different locations and developmental stages.

2. Catalytic Mechanism and Zinc-Binding Properties of Human CAII

Carbonic anhydrase II is the most thoroughly characterized and most highly active of the CA isozymes, catalyzing the hydration of CO₂ with a rate constant of ~ 10⁶ s¹ (1). The crystal structure of CAII reveals a tightly bound zinc ion coordinated by the nitrogen atoms of three protein histidine residues, His94, His96, and His119, located at the bottom of a deep active site cleft (Fig. 1) (8). At physiological pH, a hydroxide ion is also bound by zinc, completing the tetrahedral geometry of the metal site. Residue Thr199 forms a hydrogen bond with the zinc hydroxide, orienting it for efficient catalysis. Catalysis occurs in two main steps (1), initiated by nucleophilic attack of the hydroxide ion on the carbonyl carbon of CO₂ to form bicarbonate.

Water displaces bicarbonate, and the product is released. In the second, rate-limiting step in catalysis (Eq. (2)), the catalytically active enzyme species is regenerated by transfer of the product proton from the zinc-water to protein residue His64. This residue is exposed to solvent and transfers the proton to buffer B:

This proton shuttle is essential for rapid catalysis, as transfer of the proton to buffer is much faster than transfer to water. Carbonic anhydrase is one of the few biological systems in which proton transfer can be studied (9).

Figure 1. The crystal structure of human CAII determined by X-ray crystallography (8). It reveals a twisted b-sheet structure in which three histidine residues coordinate the catalytic zinc ion. Figure generated using MOLSCRIPT (13).

Just as the active site of CA has evolved for rapid catalysis, the protein structure is optimized for  tight, specific binding of the zinc ion, with a Kd of ~ 2 pM (2, 10). CAII binds only Cu²⁺, and Hg²⁺ with comparable affinity, and neither of these metals confers catalytic activity to the enzyme (10). CAII has a much lower affinity, with a Kd of nanomolar, for metals such as Co²⁺, Mg²⁺, Cd²⁺, and Ni²⁺, and only the Co²⁺-substituted enzyme is active. As the metal binding properties of this protein have been studied extensively, CAII is often used as a model for creating novel metal sites in proteins.

 Studies in which site-directed mutagenesis has been used to alter the nature of conserved residues in the zinc site (2) reveal the importance of several factors for high metal affinity. First, in almost all CAII variants studied, the zinc ion retains tetrahedral geometry even if the surrounding protein structure must be rearranged to accommodate new side-chain positions. Second, the distance between the metal and ligand is crucial. Third, conserved residues that form hydrogen bonds with the histidine zinc ligands each contribute modestly to zinc affinity but play a large role in controlling the rates of metal equilibration (2, 11). The histidine ligands, the residues that form hydrogen bonds to these ligands, and the surrounding protein structure contribute to the reactivity and binding properties of the zinc ion.

3.Genetically Distinct CA Families Arose Through Functional Convergence

Examples of the b-CA and g-CA carbonic anhydrase families are only now being discovered and characterized. In an amazing example of evolutionary functional convergence, these protein families are completely unrelated genetically to one another and to the a-CA family, yet these enzymes catalyze the same reaction using a catalytic zinc ion (3). The structure determined by X-ray crystallography of a g-CA isolated from archaebacteria (12) reveals a trimeric protein containing three active sites; in each active site, a zinc ion is bound at the monomer interface by three histidine residues from different polypeptide chains. As in a-CA proteins, the zinc is bound in tetrahedral geometry, the fourth ligand being a solvent molecule. In this case, a glutamic acid residue located near the zinc ion in g-CA may be the functional analog of Thr199 in human CAII. No structure of b-CA has been solved yet, but phylogenetic and spectroscopic data suggest that the zinc ion may be coordinated by glutamate or cysteine residues rather than histidine (3).

4. Summary

In recent years, the techniques of site-directed mutagenesis and X-ray crystallography have greatly expanded our knowledge of the relationships between protein structure and function in human CAII (2). The structure of CAII appears to have evolved for maximum catalytic activity and metal affinity. Applying these methods to the carbonic anhydrase enzymes that have evolved independently to catalyze the same reaction will give further insight into how protein structure can dictate the reactivity and affinity of metals.

References

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4. W. C. Stadie and H. O''Brien (1933) J. Biochem. 103, 521–529. 

5. S. J. Dodgson, R. E. Tashian, G. Gros, and N. D. Carter (1991) The Carbonic Anhydrases: Cellular Physiology and Molecular Genetics, Plenum Press, New York. 

6. W. S. Sly et al. (1983) Proc. Natl. Acad. Sci. USA 80, 2752–2756. 

7. H. J. M. Brady et al. (1991) Biochem. J. 277, 903–905. 

8. K. Hakansson, M. Carlsson, L. A. Svensson, and A. Liljas (1992) J. Mol. Biol. 227, 1192–1204.

9. D. N. Silverman (1995) Meth. Enzymol. 249, 479–503.

10. S. Lindskog and P. O. Nyman (1964) Biochim. Biophys. Acta 85, 462–474. 

11.  C.-C. Huang et al. (1996) Biochemistry 35, 3439–3446. 

12. C. Kisker et al. (1996) EMBO J. 15, 2323–2330. 

13. P. Kraulis (1991) J. Appl. Crystallogr. 24, 946–950.