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)
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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
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)
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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 substances that suppress the action of carbonic anhydrase, which is an enzyme that has a major role in regulating pH and fluid levels in the human body. These drugs are used to control glaucoma epilepsy and mountain sickness; them may also be used as diuretics for treatment of some neurological disorders and osteoporosis.
To further understand the role of CA inhibitors in treating various diseases, it is helpful to understand how carbonic anhydrase functions in the human body. Carbonic anhydrase is largely responsible for the conversion of carbon dioxide to carbonic acid and bicarbonate ions, some tasks associated with this action is the regulation of acid levels in the stomach, and the water content in eye and kidney cells. Also as with other body tissues it also helps excrete excess carbon dioxide from the body ensuring a proper pancreatic function. When carbonic anhydrase inhibitors are used, they act as a reducing agent to help the body lessen its it's uptake of bicarbonate ions and salt absorption. This has the effect of lowering the fluid levels in our bodies, hence their use as diuretic agents.
Anti-glaucoma medicines that are CA inhibitors include but not limited to be acetazolamide, dichlorphrenomide and methazolamide. These medications work by reducing the amount of aqueous humor, which is a fluid regulated by bicarbonate ions that are being produced by the eye. The most common way of administering these drugs is via eye drops, this method alleviates the pressure on the eye cause by glaucoma and helps preserve vision. Though it has wonderful effects it may also cause some side effects, the more common ones being fatigue, weakness, diarrhea and numbness in the extremities, while some not so common side effects are difficulty in urination, lower back pain and depression. And in rare cases patients experienced hives, convulsions and unusual bruising and bleedings.
A medical study done on CA inhibitors suggests that they might play a role on helping prevent kidney cells from attacks caused by some types of renal cancers. This effect is believed to be caused by the medicines ability to affect pH levels. It is possible that they would be a good complimentary treatment to other kinds of chemotherapy currently used to treat kidney cancers. (9)
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