Carbonic Anhydrase

Carbonic anhydrase (CA) is a zinc-containing metalloenzyme. Up to now, at least eight isoenzymes have been found in mammals. Their structure, distribution and properties are different, and are related to the secretion of H- and bicarbonate from various epithelial cells. Carbonic anhydrase catalyzes the CO₂ hydration reaction and some lipid and aldehyde hydration reactions, and participates in various ion exchanges to maintain the homeostasis of the organism.

Distribution

CA is widely distributed. CAI and CAII were isolated from red blood cells for the first time. CAIII was first discovered in skeletal muscle cytoplasm, and all of them were 29kD in humans. Membrane-associated CAIV was purified from calf lung, human kidney, and rat lung. CAIV (29kD) was found in mitochondria; CAVI (42kD) purified from the salivary glands is a secreted enzyme.

The new CA-associated gene CAVIII recently discovered in salivary glands and cerebellar Purkinje cells is also an intracytoplasmic enzyme. Gastrointestinal tract, renal distal tubules and collecting ducts, adrenal bulb cells, stenosis cells at the beginning of the epididymis, distal cephalic and proximal tail epithelial cells, fast contracting skeletal muscle cells, cerebral choroid plexus epithelial cells, and pith Phospholipid-producing cells and ocular ciliary processes, corneal and retinal cells all have CAII. CAIV is also distributed in the gastrointestinal tract, renal distal tubules, and ascending branch of the ascending medullary epithelial cells on the top and lateral plasma membrane surfaces, vas deferens and its ampulla epithelial microvilli, the top plasma membrane, subcutaneous smooth muscle layer, brain capillary epithelial cell surface, myocardium, eye capillary bed, choroid vascular layer and skeletal muscle, liver, lacrimal gland, etc.

Molecular Structure

There is a zinc atom in the active centers of CAI, II, and III, but CAI, II exist in monomeric form, while CAIII exists in the form of a disulfide-linked dimer. The three-dimensional structures of human CAI and CAII are almost identical using x-ray crystallography. The amino acid sequences of the two CAIs and CAII are about 60% homologous. CAIV has 260 amino acids and is anchored to the plasma membrane through phosphatidylinositol glycerol bonds. It is resistant to SDS dissociation and shares 30% to 36% homology with cytosolic CA. Its structure varies with different species. Human lung CAIV (36 kD) lacks N-linked oligosaccharide chains, whereas murine lung CAIV (39 kD) and other mammals mostly have the chain. CAVI has two oligosaccharide chains that are present in the recombination.

Biological Functions

Participate in the hydration process of CO₂: CAII reversibly catalyzes the hydration reaction of CO₂. The specific process is CO₂ + H₂O → H₂CO₃ → HCO₃^- + H⁺. The structural accuracy of CAII in human blood has been able to be determined to 1.54 x 10^-10 m, which can show that the catalytically active structural Zn is located in the fissure of its bottom 1.5 x 10^-10 m depth. Zn forms a polyhedron containing a tetravalent metal together with the 3 nitrogen atoms on the imidazole side chain in the histidine residue, and the oxygen atoms in the CA catalytically active nucleophilic group OH^-. The active region has an important polar residue, T199, which can accept a hydrogen bond from a Zn-bound hydroxide, and can also provide a hydrogen bond to E106. The role of H64 is to provide a proton “shuttle” that converts water bound to Zn to hydroxide. In the region adjacent to Zn bound OH^-, there is a hydrophobic cavity that is presumed to be the pre-catalyzed linking region of the CO₂ substrate, where the only competitive inhibitor of CO₂ hydration reaction can also be linked here.

Phosphatase activity: CAIII has phosphatase activity when measured with a nitrophenyl group. In mammals, CAIII can be glutathiolation, ie, the two cysteines of glutathione are connected by disulfide bonds to form disulfides, and the process is reversible. When Cys186 is involved in this process, CAIII has phosphatase activity. When Cys181 is involved in this process, CAIII loses phosphatase activity. The glutathionylation process activates the phosphatase activity of CAIII. In oxidative stress, glutathionylation is enhanced, CA III phosphatase activity is increased, and plays a role in cell signal transduction.

Activity Regulation

Figure 1. Catalytic, inhibition, and activation mechanisms of CAI. (Supuran C.T. 2016)

The main inhibitors of CA are sulfonamides. The effect of surfactants such as DDT in inhibiting CA may be related to the dissociation facilitation of the group. Different CAs have different susceptibility to sulfonamide inhibitors. The binding ability of CAII 198 site variants and inhibitors was investigated. It was found that the charge, hydrophobicity, and drug affinity of the 198 residue side chain were related to the phenylalanine side chain at CAII I198. The phenyl groups on the plug pack hydrophobic "bags", resulting in low catalysis and low sensitivity. In addition, the surfactant can inhibit the formation of polymers at high concentrations of CA, thus reducing the polymer's competitive inhibition of the formation of CA refolding protein, and promoting the initiation phase of protein refolding, and ultimately expanding the scope of the CA activity concentration.

It was showed that estradiol E2 has a certain effect on CA, and the effect varies from tissue to tissue: CA on the duodenal mucosa of rats can decrease activity under the influence of E2, while rat kidney CA, although there was a downward trend under E2, there was no significant difference. It was also found that the level of CAII protein and mRNA in the para-prostate of castrated rats was decreased, and the dorsal side was reversed. This change was reversible after treatment with testosterone, reversed with the changes of the lateral side after treatment, and had no effect on the dorsal side. The treatment of non-castrated rats showed that the level of dorsal CAI increased and that of the lateral CAII did not change. The basic structure, general distribution, and basic physiological functions of carbonic anhydrase have been recognized as early as the 1960s. Along with molecular biology techniques, the extensive application of recent research focused on the exploration of the relationship between CA's structure and function. And the understanding of the structure, function and distribution of CA will be further concretized.

Clinical Application

CA catalyzes the production of HCO₃ by CO₂ and H₂O in the ciliary body epithelial cells and is secreted into the aqueous humor through the luminal membrane. Since the fluid in the aqueous humor is kept electrically neutral, the secretion of Na⁺ to the aqueous humor is increased and the Cl^- is moved to the aqueous humor, so that the formation of high aqueous osmotic pressure, then promote the flow of H₂O to the aqueous humor. Glaucoma patients, due to poor return of aqueous humor, cause increased intraocular pressure. CA inhibitors (CAIs) inhibit the activity of CA, reduce the production of HCO₃ and lower the intraocular pressure, and are used clinically to treat glaucoma and reduce intraocular pressure. CAIs as a drug for the treatment of glaucoma are divided into three generations according to their development process and pharmacological effects: the first generation of oral CAIs, the second generation of topical CAIs, and the third generation of long-term non-stimulating topical CAIs.

Reference

1. Supuran C.T. Structure and function of carbonic anhydrases. Biochem J. 2016, 473(14):2023-32.