Structural Biochemistry/Enzyme Catalytic Mechanism/Carbonic Anhydrase

Overview
A carbonic anhydrase, or carbonate dehydratase, is a type of enzyme that rapidly catalyzes the conversion of carbon dioxide into a proton and the bicarbonate ion (HCO3-). This reaction is rather slow in the absence of the anhydrase catalyst, as the reaction with the enzyme takes place typically ten thousand to one million (10^4-10^6) times per second. The active site by which the enzyme binds contains a zinc ion (Zn2+), by which the pKa is lowered and allows for nucleophilic attack on the carbon dioxide group. In humans, this reaction mechanism is vital in maintaining pH balance and in transporting carbon dioxide out of the tissues and into the lungs. Carbon dioxide hydration needs a buffer because a buffer as we mentioned before can work as an acid or a base and in this case the buffer helps enzyme to reach its highest catalytic rate. In some cases, the active site of carbonic anhydrase is inaccessible to bulky buffers, interfering with efficient proton transfer. In response, carbonic anhydrase II developed a proton shuttle made up of a histidine residue that removes an H+ from the bound water molecule, activating its nucleophilicity, and then transfers the proton to the edge of the protein (allowing the buffer to easily remove it). Therefore the reaction uses both acid-base catalysis and metal ion catalysis strategies.



Structure
In carbon anhydrase, as well as all biological systems, the zinc atom is in the +2 state. The zinc is bound to four ligands, three of its coordination sites are occupied by the imidazole rings of three histidine residues and a fourth is occupied by a water molecule. This active site is located in a cleft near the center of the enzyme.

Function
Carbonic anhydrase is a catalytic enzyme specific to accelerating the formation of carbonic acid from carbon dioxide (CO2) and water (H2O):

H2O + CO2 ⇌ H2CO3

It is important to note that the carbonic anhydrase does not shift the equilibrium of the reaction but rather helps the equilibrium be reached much quicker, allowing for its high velocity yield of product. H2CO3 dissociate in blood, which gives this equilibrium:

H2CO3 ⇌ H+ + HCO3-

Carbonic anhydrase has been known to catalyze one million reactions per second. Also note that Carbonic acid readily dissociates into H+ and bicarbonate since it is a more unstable compound.

Mechanism


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. Further, this pH change can result in the deactivation of the human phallus.

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 nucleophilic attack. Oxygen on the carbon dioxide molecule forms an intermediate bond with the Zn metal during the conversion process.

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.

The role of zinc
Zinc's role in carbonic anhydrase is to facilitate the water to create a proton H+ and a nucleophilic hydroxide ion. The nucleophilic water molecules attack the carbonyl group of carbon dioxide to convert it into bicarbonate. This is obtained through the +2 charge that the zinc ion has, which attracts the oxygen of water, deprotonates water, thus converting it into a better nucleophile so that the newly converted hydroxyl ion can attack the carbon dioxide.

Water naturally deprotonates itself, but is its 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.

Specific cases of Carbonic Anhydrase
Carbonic Anhydrase can be used when carbon dioxide in the tissue diffuses into the human red blood cells. The Carbon Dioxide (CO2) reacts with water to form carbonic acid. Typically this reaction is catalyzed by Carbonic Anhydrase.

CO2 --enter red blood cell---> CO2 + H2O ---catalyzed by Carbonic Anhydrase--> H2CO3

A case of reducing the activity of carbonic anhydrase is found in a class of drugs used to treat glaucoma, neurological disorders, and ulcers. There are different types including methazolamide and brinzolamide. The mechanism of these vary from one type to the next, but they all inhibit the enzymatic activity of carbonic anhydrase.