Breathing is a fundamental function in life - ever wondered what really happens when we breathe? The air we breathe in has precious oxygen that fuels the breakdown of sugars and fat in our cells. In our lungs, oxygen diffuses into the blood, binds to hemoglobin and is transported to all the cells of our body (see the Molecule of the Month feature on hemoglobin). Carbon dioxide is a byproduct of sugar and fat breakdown in cells and needs to be removed from our body. Again, blood acts as a transport medium. Carbon dioxide diffuses out of cells and is transported in blood in a few different ways: less than 10% dissolves in the blood plasma, about 20% binds to hemoglobin, while the majority of it (70%) is converted to carbonic acid to be carried to the lungs. An enzyme present in red blood cells, carbonic anhydrase, aids in the conversion of carbon dioxide to carbonic acid and bicarbonate ions. When red blood cells reach the lungs, the same enzyme helps to convert the bicarbonate ions back to carbon dioxide, which we breathe out. Although these reactions can occur even without the enzyme, carbonic anhydrase can increase the rate of these conversions up to a million fold.
Green Plants and Corals
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Plants also use oxygen for producing energy, and also release carbon dioxide. Green plants can convert water and carbon dioxide into sugars in the presence of sunlight. This process, photosynthesis, uses carbon dioxide from the atmosphere. Gaseous carbon dioxide is stored in plants as bicarbonate ions. In both land and water plants, carbonic anhydrase plays a role in converting bicarbonate ions back to carbon dioxide for photosynthesis. Another interesting biological phenomenon where this enzyme plays a role is the calcification of corals. Seawater calcium reacts with the bicarbonate produced by carbonic anhydrase from the coral polyps, forming calcium carbonate. This is deposited as the hard exterior of corals.
Carbonic anhydrase is an enzyme that assists rapid inter-conversion of carbon dioxide and water into carbonic acid, protons and bicarbonate ions. This enzyme was first identified in 1933, in red blood cells of cows. Since then, it has been found to be abundant in all mammalian tissues, plants, algae and bacteria. This ancient enzyme has three distinct classes (called alpha, beta and gamma carbonic anhydrase). Members of these different classes share very little sequence or structural similarity, yet they all perform the same function and require a zinc ion at the active site. Carbonic anhydrase from mammals belong to the alpha class, the plant enzymes belong to the beta class, while the enzyme from methane-producing bacteria that grow in hot springs forms the gamma class. Thus it is apparent that these enzyme classes have evolved independently to create a similar enzyme active site. PDB entries 1ca2, 1ddz and 1thj, shown here from top to bottom, are examples of the alpha, beta and gamma carbonic anhydrase enzymes, respectively. The zinc ions in the active site are colored blue in these figures. Note that the alpha enzyme is a monomer, while the gamma enzyme is trimeric. Although the beta enzyme shown here is a dimer, there are four zinc ions bound to the structure indicating four possible enzyme active sites. Other members of this class form tetramers, hexamers or octamers, suggesting that dimer is probably a building block for this class.
Mammalian carbonic anhydrases occur in about 10 slightly different forms depending upon the tissue or cellular compartment they are located in. These isozymes have some sequence variations leading to specific differences in their activity. Thus isozymes found in some muscle fibers have low enzyme activity compared to that secreted by salivary glands. While most carbonic anhydrase isozymes are soluble and secreted, some are bound to the membranes of specific epithelial cells. For a deeper look at carbonic anhydrase from a genomic perspective, please visit the Protein of the Month feature at the European Bioinformatics Institute
Carbonic Anhydrase in Health and Disease
Since this enzyme produces and uses protons and bicarbonate ions, carbonic anhydrase plays a key role in the regulation of pH and fluid balance in different parts of our body. In our stomach lining it plays a role in secreting acid, while the same enzyme helps to make pancreatic juices alkaline and our saliva neutral. The transport of the protons and bicarbonate ions produced in our kidney and eyes influence the water content of the cells at these locations. Thus carbonic anhydrase isozymes perform different functions at their specific locations, and their absence or malfunction can lead to diseased states, ranging from the loss of acid production in the stomach to kidney failure.
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When there is a build up of fluid that maintains the shape of our eyes, the fluid often presses on the optic nerve in the eye and may damage it. This condition is called glaucoma. In recent years, inhibitors of carbonic anhydrase are being used to treat glaucoma. Blocking this enzyme shifts the fluid balance in the eyes of the patient to reduce fluid build up thereby relieving pressure. The structure of PDB entry 1cnw shows how one such inhibitor (a sulfonamide), colored green in the figure, is bound to human carbonic anhydrase (isozyme II). Note that this inhibitor binds near the active site and disrupts the interactions of the water bound to the zinc ion, blocking the enzyme action. Unfortunately, prolonged use of this drug can affect the same enzyme present in other tissues and lead to side effects like kidney and liver damage.
Exploring the Structure
The alpha carbonic anhydrase enzymes have been well studied, leading to an understanding of how the enzyme works. The left hand figure shows the structure of carbonic anhydrase II from PDB entry 1ca2. Note the large beta sheet in the center of the structure colored in yellow. The active site lies at the bottom of a deep cleft in the enzyme where a zinc atom is bound, shown with a gray sphere. Nitrogen atoms of three histidines--numbered 94, 96 and 119 (colored in yellow)--directly coordinate the zinc. These amino acids are highly conserved in all isozymes. Atoms from threonine 199 and glutamate 106 (colored magenta) interact indirectly through the bound water, shown with a red sphere. Note that these residues in addition to the histidine 64 (also colored in magenta) help to charge the zinc with a hydroxyl ion. Some of the isozymes have differences in these and other residues, which may explain their difference in enzyme activity.
Zinc is the key to this enzyme reaction. The water bound to the zinc ion is actually broken down to a proton and hydroxyl ion. Since zinc is a positively charged ion, it stabilizes the negatively charged hydroxyl ion so that it is ready to attack the carbon dioxide. A close up of the amino acid side chains in the active site and the zinc ion is shown in the two right hand figures. The top figure shows a hydroxyl ion, shown with a red sphere, bound to the zinc ion in PDB entry 1ca2. Zinc directs the transfer of this bound hydroxyl to carbon dioxide, forming a bicarbonate ion. The bottom figure shows an intermediate structure where the bicarbonate ion, shown with red and white spheres, has just formed and is still bound to the enzyme (PDB entry 1cam). Note that the side chain for amino acid 199 is modeled as an alanine in this structure. Histidine 64 swings towards and away from the zinc ion in each cycle of enzyme action while helping the zinc to recharge with a new hydroxyl ion. The two positions of this residue, shown in the bottom right figure, represent its movement during enzyme action. As soon as the zinc is reloaded with a new water molecule and the bicarbonate ion has been released, the enzyme will be ready for action on another carbon dioxide molecule.
Carbonic anhydrases are enzymes that catalyze the hydration of carbon dioxide and the dehydration of bicarbonate:
CO2 + H2O <-----> HCO3- + H+
These carbonic anhydrase-driven reactions are of great importance in a number of tissues. Examples include:
Parietal cells in the stomach secrete massive amounts of acid (i.e. hydrogen ions or protons) into the lumen and a corresponding amount of bicarbonate ion into blood.
Pancreatic duct cells do essentially the opposite, with bicarbonate as their main secretory product.
Secretion of hydrogen ions by the renal tubules is a critical mechanism for maintaining acid-base and fluid balance.
Carbon dioxide generated by metabolism in all cells is removed from the body byred blood cells that convert most of it to bicarbonate for transport, then back to carbon dioxide to be exhaled from the lungs.
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Carbonic anhydrase isozymes are metalloenzymes consisting of a single polypeptide chain (Mr ~ 29,000) complexed to an atom of zinc. They are incredibly active catalysts, with a turnover rate (kcat) of about 106 reactions per second! Catalytic activity depends on ionization of a group of pKa 7 and, as you might expect from thinking about when and where the above reactions take place, the hydration reaction depends on the ionized group being in the basic form, and for dehydration in the acidic form.
Carbonic anhydrase inhibitors have been used theraputically. The prototype of such drugs is acetazolamide, which is still sometimes used as a diuretic to treat certain edematous conditions and for therapy of some types of glaucoma. The discovery of this drug is actually an interesting story. It is a member of the sulfonamides, a group of antibacterial agents which, when intially investigated, were shown to induce a metabolic acidosis because they inhibited excretion of hydrogen ion from the kidney.
By Jennifer McDowall
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Carbon dioxide (CO2) is a key metabolite in all living organisms. Carbon dioxide exists in equilibrium with bicarbonate (HCO3-), which is poorly soluble in lipid membranes compared to carbon dioxide; carbon dioxide can freely diffuse in and out of the cell, while bicarbonate must be transported. The conversion of bicarbonate to carbon dioxide facilitates its transport into the cell, while the conversion of carbon dioxide to bicarbonate helps trap the carbon dioxide in the cell. The interconversion of carbon dioxide and bicarbonate proceeds slowly at physiological pH, so organisms produce enzymes to speed up the process. Carbonic anhydrases are zinc-containing enzymes that catalyse the reversible reaction between carbon dioxide hydration and bicarbonate dehydration. Carbonic anhydrases have been found in all kingdoms of life. They have essential roles in facilitating the transport of carbon dioxide and protons in the intracellular space, across biological membranes and in the layers of the extracellular space; they are also involved in many other processes, from respiration and photosynthesis in eukaryotes to cyanate degradation in prokaryotes.
Mechanism of action
Carbonic anhydrase catalyses the following reaction:
H2O + CO2 ïƒŸïƒ H+ + HCO3-
This reaction is ubiquitous in nature, involving the interchange of gaseous and ionic species crucial to a wide range of physiological and biochemical processes. The mechanism of action of the mammalian carbonic anhydrase has been studied in depth. The enzyme employs a two-step mechanism: in the first step, there is a nucleophilic attack of a zinc-bound hydroxide ion on carbon dioxide; in the second step, the active site is regenerated by the ionisation of the zinc-bound water molecule and the removal of a proton from the active site. The active site can exist in two forms: a high pH form that is active in the hydration of carbon dioxide and a low pH form that is active in the dehydration of bicarbonate.
A multitude of isozymes
Species can produce many different carbonic anhydrase isozymes, some of which act in the cytosol, while others are membrane-bound. For instance, in humans there are three cytosolic isozymes (I, II and III), five membrane-bound isozymes (IV, VII, IX, XII and XIV), a mitochondrial isozyme (V), and a secreted salivary isozyme (VI), as well as several related proteins that lack catalytic activity. Carbonic anhydrases are often arranged in clusters along membranes or localised in extracellular spaces, which may contribute to the ability of carbonic anhydrase to facilitate the intracellular diffusion of carbon dioxide and protons (H+). By increasing the movement of protons, carbonic anhydrase can dissipate intracellular pH gradients, thereby helping the cell to maintain a uniform cellular pH. The removal of protons is essential for several reactions within the cell, such as for the functioning of ATPases, which are inhibited by a build-up of protons; as a result, the inhibition of carbonic anhydrase could reduce muscle contractility and calcium handling. Carbonic anhydrases can also create localised gradients, which may aid in processes such as facilitated diffusion across a membrane.