Carbonic anhydrase is an enzyme that assists the rapid conversion of carbon dioxide into bicarbonates and ions. The enzyme was first identified in 1933, when it was first found to be abundant in the cow's red blood cell. Since then it was found to be abundant in all tissues of mammals, plants algae and bacteria. Carbonic anhydrase is classified into 3 distinct classes, called alpha beta and gamma CA. These three 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 found in mammals are classified as alpha CA, plants have beta CA and the methane producing bacteria that grow in hot places are the gamma CA. The active sites of all carbonic anhydrases contain a zinc ion, as seen in the figure the zinc ions are colored blue. The figures show that the alpha enzyme is a monomer, the beta enzyme is a dimer and the gamma enzyme is trimetric though the beta enzyme shown here is a dimer, there are four zinc ion bonds in the structure indicating the four possible active sites which can turn it into other forms such as tetramers, hexamers or octamers.(1)
Figure 3. Beta Carbonic anhydrase (1)
Figure 1. Alpha Carbonic Anhydrase (1)
Figure 3. Gamma Carbonic Anhydrase (1)
Structure of Carbonic anhydrase
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Carbonic anhydrase is a type of enzyme that rapidly catalyzes the conversion of carbon dioxide into a bicarbonate and proton ion (HCO3-). This reaction is slow in the absence of the anhydrase catalyst, as the reaction with the enzyme takes place typically ten thousand to one million times per second. The active site to which the enzyme binds contains a zinc ion (Zn2+), in which the pka is lowered and allows for a nucleophilic attack on the carbon dioxide group. In humans, this reaction mechanism is vital in maintaining the pH balance and transporting carbon dioxide out of the tissues and into the lungs. CO2 hydration needs a buffer because a buffer can work either as an acid or as a base, and in this case the buffer helps enzyme to reach its highest catalytic rate. In some cases, the active site of the carbonic anhydrase is inaccessible to larger buffers, interfering with efficient proton transfer. In response to this CA II developed a proton shuttle made up of histidine residues that removes an H+ from the bound water molecule, activating its nucleophilicity and then transfers the proton to the edge of a the protein allowing for an easy removal by the buffer. Therefore the reaction uses both acid base catalysis and metal ion catalysis. (6)
Figure 4. Carbonic anhydrase structure (1)
Alpha carbonic anhydrase enzymes have since been carefully studied which led to an understanding of how the enzyme works. The left hand part of figure 4 shows the structure of carbonic anhydrase II from PDB entry 1ca2. Take not of the large beta sheet in the center which is colored yellow. The active site of the enzyme lies at to bottom deep cleft in the enzyme where a zinc atom is bound (shown in a gray sphere). The nitrogen atoms of three histidines (numbers 94, 96 and 119) directly coordinate the zinc. These amino acids are always conserved in all isozymes. Atoms from threonine 199 and glutamate 106 (color violet) indirectly interacts through the bound water. The residues plus the addition of histidine 64 helps to charge the zinc with a hydroxyl ion. Some of the isoenzymes have a difference in these and other residues which could explain the difference in their enzyme activity. (6)
Zinc is the key to this enzyme reaction. The water molecule bound to the zinc ion is broken down into proton and hydroxyl ions. Since zinc is positively charged, it would stabilize the negatively charged hydroxyl ion so that it is ready to attack the carbon dioxide. A close up image of the amino acid chains in the active site and the zinc ion is shown in the two right hand figures. The upper right figure shows the hydroxyl ion (red sphere) bound to the zinc ion in PDB entry 1ca2. Zinc would direct the transfer of this bound hydroxyl to CO2 forming a bicarbonate ion. While the bottom right figure shows an intermediate structure where the bicarbonate ion (red and white spheres) has formed and it is still bound the enzyme PDB entry 1cam. The side chains for the amino acid 199 are modeled as an alanine in this structure. Histidine 64 swings towards and away from the zinc ion in each enzyme action cycle while in the process it helps to recharge zinc with a new hydroxyl ion. This would also show the movement of the enzyme. As soon as zinc gains a new water molecule and the bicarbonate ion has been released, the enzyme will be ready for action on another carbon dioxide molecule.(6)
Mechanism of Carbonic anhydrase
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Zinc's role in carbonic anhydrase is to facilitate the creation of H+ proton and a nucleophilic hydroxide ion. The nucleophilic water molecules will attack the carbonyl group of CO2 to convert it into a bicarbonate. This is cause by the +2 charge that zinc possess which attracts the oxygen of water and deprotonates it converting it into a nucleophile so that the newly converted hydroxyl ion can attack the carbon dioxide.(2)
Water naturally deprotonates itself, but is it's a rather slow process and not in large quantities. Zinc deprotonates water by providing a positive charge for the hydroxide ion. Zinc alone cannot deprotonate water fast enough to reach the 106 per second rate that it has been measured, however, the proton is donated temporarily to the surrounding amino acid residues, which will later be given to the environment, while allowing the reaction to continue and not slowing down the process. Metal ions are good because it increases the reactivity of the chemicals and can create strong bonds. Zinc is able to help the deprotonation of water by lowering the pka of water. Binding of water to zinc lowers the pka of water from about 15.7 to 7. This means more water molecules are now able to deprotonate at a lower pH than normal, and this makes it easier for water to turn into a hydroxide ion which is a better nucleophile (2)
Figure 5. CA mechanism (2)
pH affects carbonic anhydrase in a sigmoidal fashion. The higher the pH, the more active the enzyme is (since it is in the optimal conditions for deprotonation).
1) The binding of zinc lowers the pKa of water from 15.7 to 7, generating a hydroxide ion (OH-) to attack carbon dioxide. Zinc releases a proton from a water molecule to generate this hydroxide ion. pH decreases as a result from the decrease in the pKa. According to Le Chatelier's principle, this drives the reaction towards deprotonation.
2) The carbon dioxide substrate binds to the enzymes active site and is positioned for optimal interaction.
3) The hydroxide ion (being a great nucleophile) attacks the carbonyl of carbon dioxide, converting it to bicarbonate ion through the neuclophilic attack. Oxygen on the carbon dioxide molecule forms an intermediate bond with the Zn metal during the conversion process. (5)
4) The enzyme is regenerated and the bicarbonate ion is released. The enzyme is ready for another reaction to occur. This regenerative ability of this enzyme allows for this reaction to be highly efficient and kinetically fast to constantly process carbon dioxide within the blood cells.
Associated Diseases and Importance of Carbonic anhydrase
For land animals, breathing is a fundamental function of life. The air we intake has oxygen in it which helps fuel the breakdown of sugars and fats found in our cells. In our lungs, oxygen diffuses into the blood then binds to the hemoglobin which would then be transported to all the cells of our body. Carbon dioxide is a byproduct of sugar and fat breakdown in cells. It needs to be removed from our body. CO2 diffuses out of the cell and is transported in different ways, less than 10% are dissolved in the blood plasma, 20% ends up binding into the hemoglobin and the majority is converted to carbonic acid which is carried back to the lungs. An enzyme which is present in red blood cells, carbonic anhydrase, aids in the conversion of CO2 into carbonic acid and bicarbonate ions. When the red blood cells reach the lungs, carbonic anhydrase again converts the bicarbonate ions back into carbon dioxide, which is the air we exhale. Although these reactions can occur without carbonic anhydrase, the enzyme actually helps speed up the process a million fold!!
Carbonic anhydrase inhibitors are pharmaceutical substances that repress the action of carbonic anhydrase - an enzyme that plays a major role in regulating pH and fluid levels in the human body. These drugs are often utilized to control glaucoma, epilepsy, and mountain sickness. They can also be used as diuretics, in the treatment of certain kinds of gastric ulcers, some neurological disorders, and osteoporosis.
To understand the role of carbonic anhydrase inhibitors in treating various diseases, it can be helpful to understand how carbonic anhydrase functions in the human body. It is largely responsible for converting carbon dioxide to carbonic acid and bicarbonate ions. Some of the tasks associated with this action are the regulation of acid levels in the stomach, and the water content in kidney and eye cells, as well as other bodily tissues. It also helps with ridding excess carbon dioxide from the body and ensuring proper pancreatic function.
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When carbonic anhydrase inhibitors are used, they typically work by reducing the body's uptake of bicarbonate ions. They also decrease salt absorption. This has the effect of lowering fluid levels in the body, hence their use as diuretic agents.
Anti-glaucoma pharmaceuticals that are carbonic anhydrase inhibitors include acetazolamide, dichlorphrenomide, and methazolamid, among others. These medications typically work by reducing the amount of fluid - known as aqueous humor, which is usually regulated by bicarbonate ions - that the eye produces. The most common method of administering these drugs is via eye drops. This alleviates pressure on the eye caused by glaucoma and helps to preserve vision. (100
Risks associated with prolonged use of carbonic anhydrase inhibitors include kidney failure and liver disease. These drugs also tend to raise blood and urine sugar levels in diabetics. Further, these medicines can increase shortness of breath in patients suffering from emphysema.
Some of the most common side effects patients may experience when taking carbonic anhydrase inhibitors are fatigue, weakness, diarrhea, nausea, and numbness in the extremities. Some less common side effects include blood in or difficulty with urination, lower back pain, and depression. More rare side effects are hives, convulsions, and unusual bruising or bleeding, among others. (10)
Medical research on carbonic anhydrase inhibitors suggests that they may play a role in helping to prevent kidney cells from being attacked by certain kinds of renal cancers. This seems to be a result of these pharmaceuticals' ability to affect pH levels. It is possible that they would be a good complementary treatment to other kinds of chemotherapy used to treat kidney cancer. (10)
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