Carbonic Anhydrase: Structure, Mechanisms and Functions

INTRODUCTION

Carbonic anhydrase, abbreviated as CA, is the first identified zinc containing enzyme, (CA; carbonate hydro-lyase, EC 4.2.1.1) It is an enzyme that catalyzes the reversible hydration and dehydration of carbon dioxide to form carbonic acid, bicarbonate ions and protons. Being one of the fastest enzyme known, it is believed that one molecule of CA can process one million molecules of carbon dioxide per second. The basic molecular structure of CA includes specific amino acid threonine 199, glutamate 106, histidine 64 and histidine residues namely His 93, His 95, and His 118. The mode of regulation of CA is being inhibited by various medically prescribed substances that act as non competitive inhibitors, an example is Acetazolamide. CA plays a major key role in the fluid balance and regulatory of pH in different parts of the body thus, Mutation of this enzyme may lead to several diseases.(1)

CARBONIC ANYHYDRASE

THE START: Breathing, a fundamental function in life

The air that we breathe in has some valuable oxygen, an important molecule wherein it helps the breakdown of fats and sugars in our cells. From the blood, oxygen diffuses then binds with the hemoglobin to be transported in the cells of our body. A by product of sugar and fat breakdown in cells is called Carbon dioxide CO2). It is a key metabolite in all living organism and it needs to be removed from our body.

Carbon dioxide is diffuse out of the cells and transported in the blood in different ways to get to the lungs. CA is transported in numerous forms, mainly as bicarbonate, HCO3-. Bicarbonate is a CO2- with an attached OH group. When the HCO3- reaches the lungs, it is transformed back to a CO2, so it can be exhaled from the body. The conversion of bicarbonate to carbon dioxide facilitates its transport into the cell; while the conversion of carbon dioxide to bicarbonate assists trap the carbon dioxide in the cell. This interconversion of carbon dioxide and bicarbonate develop at a slow physiological pH hence organism tend to produces an enzyme to hasten the process. This enzyme responsible for the speed up interconversion, which can be found in the red blood cells, is called carbonic anhydrase. Although the interconversion of bicarbonate to carbon dioxide can happen without the enzyme, CA can great increase the rate of the conversions up to a millions of fold. (2)

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STRUCTURE

The CA molecule in general has ellipsoidal shape with the estimated dimension 4.1 x 4.1 x 4.7 nm. The active site is situated in a cavity having an approximately conical shape. The cavity is assessed 1.5 m wide at the way in and about 1.6 nm deep attaining almost the center of the molecule. The zinc ion is next to the peak of the cone and liganded into 3 imidazole groups.

Taken as a whole, the CA is composed of 10-stranded anti-parallel beta-sheet enclosed with various elements of other secondary structure. The 6 alpha-helices and 10-beta sheets make up the secondary structure of carbonic anhydrase. The basic function of CA is basically to regulate the oxygen and carbon dioxide content of the blood that is needed in a human body. As the function suggests, the chemical structure of CA extremely lies with the presence of zinc that lies deep within its active site. Its common amino acid composition includes threonine, glutamate and histidine. The specificity of these 3 amino acids (threonine 199, glutamate 106, and histidine 64) plays a critical role in relation to the presence of zinc by charging it with a hydroxyl ion. The zinc cation is associated with three histidine residue protein backbone namely: His93, His95, and His118. As stated, zinc plays a major role in the reaction of CA. The zinc present in the active side of CA is being bound to water to be able to dissociate it into a proton and hydroxyl ion. The hydroxyl ion is being stabilized by the positively charged zinc, in this way; the hydroxyl ion is being prepared to attack the carbon dioxide inside the RBC.

A closer look with CA can be seen in the figure below where the amino acid chains in the active site together with the zinc are evident. The role of the zinc basically includes the command of directional transfer of the bound hydroxyl to the carbon dioxide to be able to form bicarbonate ion. From the figure, it shows that the intermediate structure where the bicarbonate ion is still attached to the enzyme. The alanine replicated the side chain for amino acid 199 in this arrangement. Histidine 64 swings in the direction of and away from the zinc ion in every cycle of enzyme action although it is helping the zinc to recharge with a novel hydroxyl ion. The two locations of this residue, revealed in the bottom right figure, symbolize its movement throughout the action of enzyme. Almost immediately as the zinc is reloaded with an original water molecule together with the release of bicarbonate ion, the enzyme is set for another action on some new carbon dioxide molecule. (3)

MECHANISM OF CATALYSIS

The rate of catalysis of the CA is exceedingly pH dependent. It means that, the higher the pH, the catalysis is faster and as the pH reduces, the speed of the reaction falls down. The mean pH of this transition is near pH 7. (5)

Figure 2.0 shows the mechanism of CA catalysis. A zinc atom which is generally bound to four or more ligands differs in CA. In CA, three locations are engaged by the imidazole rings of three histidine residues and an additional site is occupied by a water molecule. Thus the geometry form of the active site is tetrahedral. The zinc atom plays an important role in the mechanism of CA catalysis because it is responsible for the release of a proton H+ from a water molecule, which then generates a nucleophilic hydroxide ion. Then carbon dioxide substrate attaches to the enzyme’s active site and is situated to react with the hydroxide ion. The zinc-bound OH- attacks the carbon of CO2 therefore converting it into a bicarbonate ion. This occurs since the zinc ion has the +2 charge, which attracts the oxygen of water. It then deprotonates the water, thus, converting it into a better nucleophile so that the newly converted hydroxyl ion can attack the carbon dioxide. After the nucleophilic attack of zinc bound OH-, addition of water molecule displaces the bicarbonate ion from the metal ion. The CA is then ready for another cycle of catalysis. (7)

KINETICS OF REACTIONS

CA inhibitors are class of pharmaceuticals that control the activity of carbonic anhydrase. It is inhibited by two classes of compounds, a metal complex forming anions and others are isosteres and sulfonamides. Inhibitors ionize upon binding with the enzyme to give way an NH- group that relocates the zinc hydroxide ions and shares a hydrogen bond. There are roughly 25 clinically used CA inhibitors as a drugs. It is mainly established as antiglaucoma drugs, diuretics, hypotensive agents, anticonvulsants, anticancer agents, antiepileptics, with additional use in the management of duodenal and gastric ulcers, osteoporosis and neurological disorder. (8)

• Acetazolamide
• Methazolamide
• Dorzolamide
• Topiramate

Figure 3. Illustrations of some CA inhibitors (9)

Figure 3.0 shows some CA inhibitors like Acetozolamide which acts as a mild diuretic. It cures glaucoma, altitude sickness, and some benign intracranial hypertension. Methazolamide treats glaucoma present in dogs which is called Open-angle glaucoma. While Topiramate which is a weak inhibitor, alleviate epilepsy, lennox gastuat syndrome and migraine headaches. And another CA inhibitor is the, Dorzolamide or sulphonamide which treat ocular hypertension or open-angele glaucoma. (10)

CA activator regulates the proton transfer processes between the active site and the solvent system. It also binds at the entrance of the enzyme of the active site. One of the strong activator of CA is Histidine. Some amines and amino acids like l-Trp (tryptophan), l-Phe (Phenylalanine), d-DOPA (D- 3,4-dihydroxyphenylalanine), l-Tyr (Tyrosine), 4-amino-l-Phe also works as activators of CA. These CA activators are potentially target for drug development that can be useful as a derivative for the enhancement of synaptic efficacy which can be able to treat various conditions like, depression, alzheimer’s disease, ageing, spatial learning and memory therapy enhancer. (11)

MODE OF REGULATION: Acetazolamide Inhibitor

In case of excessive contents of CA in blood and peripheral areas of the lungs, proper regulation and inhibition is needed. Acetazolamide is a non competitive inhibitor that is effective in giving control with the catalytic reaction of the enzyme. This chemical complex substance is medically used o treat different conditions of moderate up to severe metabolic or respiratory alkalosis. Alkalosis may happen if excess CA is being reacted with the bicarbonate and carbon dioxide ions in the RBC, causing extreme absorption of bicarbonate thus giving the erythrocyte more basicity rather than having enough and sufficient pH level. Acetazolamide action is explained by interfering with bicarbonate (HCO3-) reabsorption in the kidneys, thereby giving enough acidity in the RBC, and further results to alkalinizing the urine. The action of inhibition results further to decreased synthesis of aqueous humor of the eye and causes the lowering of intraocular pressure.

The interaction of Acetazolamide with CA does not occur with the active site, only close or remote to the active site. The net effect of this inhibitor basically changes the shape of CA that obviously leads to the inability of the substrate to bind properly, results to no catalytic reaction. (12)

CARBONIC ANHYDRASE IN HEALTH AND DISEASE:

Carbonic Anhydrase is found in numerous places in the body, including in the cerebro-spinal fluid, cytosol of some cells and mainly in the red blood cells. Since CA generates and utilizes protons and bicarbonate ions, it plays a major key role in the fluid balance and regulatory of pH in different parts of the body. Absence or mutation of the CA enzyme may lead to several diseases. Also, CA inhibitor contributes to several treatments of diseases.

One of the linked diseases of CA is the Osteopetrosis with cerebral calcification and renal acidosis. It is a syndrome deficient with CA in the body commonly called as Marble brain disease. This happens because sulfonamide inhibitor of CA can produce metabolic acidosis and have shown that CA inhibitors blocks the parathyroid hormone-induced the release of calcium bone which causes bone resorption. And since CA is present in the brain and CA inhibitors inhibits the production of cerebral spinal fluid, mutation of CA lead to cerebral calcification.

Other disease associated with the deficiency of specific type of CAIII is the Myastenia gravis. It is an autoimmune neuromuscular disorder that results to a weak muscle of a person. Defects in CA IV can cause retinitis pigmentosa, a degeneration of retinal photoreceptor, which a patient experiences night vision blindness and loss of midperipheral visual. (13)

Glaucoma, a condition wherein a build up of fluid in the eyes occurs and this presses the optic nerve that caused damage, is treated with the use of CA inhibitors like acetazolamide, brinzolamide, dorzolamide, and methazolamide. These inhibitors lessen the amount of fluid in the eye rapidly by 40% to 60% thus lowering the pressure inside the eye of a person with glaucoma. It now lessens the risk of optic nerve damage which promote vision loss. But prolong use of this drug affects the same enzyme in the tissue and may lead to kidney and liver damage

The CA also plays an important role in the secretion of acid through the catalyzed hydration of excreted CO2 in the stomach lining which is mainly responsible in digestion of food. It helps to make pancreatic juice alkaline and our saliva neutral. In summary, CA performs different role and functions at their specific locations. (14)