Carbonic anhydrase (CA) is a zinc containing metalloenzyme. Up to now, at least 11 isoenzymes have been found in mammals. Their structures, distributions and properties are different from those of variousepithelial cellsSecretion of H - is related to bicarbonate, which catalyzes COtwoHydration reaction and hydration reaction of some lipids and aldehydes, participate in multiple ion exchange, and maintain the bodyInternal environment steady state。
The first and most important zinc enzyme discovered in 1940.More than 80 kinds of zinc enzymes have been reported, ranking first among all kinds of metals.Distributed in the human bodyRenal tubular epithelial cells, gastric mucosa, pancreas, red blood cells, central nerve cells and ciliary body epithelial cells.
In human and animal blood, carbonic anhydrase is one of the main protein components in red blood cells, and its importance and content are second only tohemoglobin。[1]
CA is widely distributed.CA Ⅰ and Ⅱ were isolated from red blood cells for the first time.CA Ⅲ was first found in the cytosol of skeletal muscle, and all three are 29 kD intracellular enzymes in humans;The membrane associated enzyme CA Ⅳ has been purified from calf lung, human kidney and rat lung;CA Ⅳ (29kD) was found in mitochondria;CA Ⅵ (42kD) purified from salivary glands by Murakami in 1987 is a secretory enzyme;Recently in salivary glands and cerebellumPurkinje cellCA Ⅷ, a new CA related gene, is also an intracellular enzyme.CA Ⅱ was found in the gastrointestinal tract, the distal convoluted tubules and collecting ducts of the kidney, the cells of the adrenal glomerular zone, the narrow cells of the initial segment of the epididymis, the epithelial cells of the distal head and body and the proximal tail, the fast contracting skeletal muscle cells, the epithelial cells of the choroid plexus of the brain, myelin forming cells, and the cells of the ciliary process, cornea, and retina of the eye;CA Ⅳ is also distributed on the surface of the apical plasma membrane and the basal plasma membrane of the epithelial cells in the gastrointestinal tract, the distal convoluted tubules of the kidney and the thick segment of the ascending branch of the medullary loopthe epididymis, microvilli, parietal plasma membrane, subcutaneous smooth muscle layer, epithelial cell lumen of cerebral capillaries, myocardium, capillary bed of eye, choroidal vascular layer, skeletal muscle, liver, lacrimal gland, etc.
molecular structure
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Structure diagram of CAII in human body
CA Ⅰ and Ⅱ have a zinc containing monomer in structure, but CA Ⅰ and Ⅱ exist in the form of monomer, while CA Ⅱ exists in the form of dimer connected by disulfide bond. The three-dimensional structure of human CA Ⅰ and CA Ⅱ is almost the same as measured by X-ray crystal diffraction pattern.Their amino acid sequences are about 60% homologous.CA Ⅳ has 260 amino acids, anchored to the plasma membrane by phosphoinositide glycerol bond;It can resist the dissociation of SDS and has 30-36% homology with CA in cytoplasm. Its structure varies with different species. CA Ⅳ (36kD) in human lung lacks N-linked oligosaccharide chain, while CA Ⅳ (39kD) in mouse lung and other mammals mostly have this chain.CA Ⅵ has two oligosaccharide chains that exist repeatedly.
Important position
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Carbonic anhydrase is one of the main protein components of red blood cells, ranking second only tohemoglobin。It contains a curly protein chain and a zinc (Ⅱ) ion.The molecular weight is about 30000.Zinc ion is in the coordination environment of deformed tetrahedron.The most important reaction of catalysis iscarbon dioxideThe reversible hydration of (carbonic anhydride) enables it to proceed quickly under physiological pH conditions (pH=7).For catalytic COtwo(g) + HtwoO → HtwoCOthreeThe factor of enzyme accelerating the hydration of carbon dioxide is about 10 ^ 7.These reactions are very important for respiration.
Carbonic anhydrase is a zinc protein (zoogenic), which exists in red blood cells of vertebrates, various tissues of many animals and leaves of plants.It can rapidly convert carbonic acid and bicarbonate ions in red blood cells.It plays a role in the secretion of hydrochloric acid in the stomach. Generally speaking, it has the function of regulating the pH of body fluids.It is also believed to be related to the photosynthesis of plants.
major function
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1. Maintain acid-base balance in blood and other tissues.
2. Help the body tissues eliminate carbon dioxide.
3. Ensure that COtwoAnd HCOthree-To maintain an appropriate substrate concentration for the enzyme that catalyzes the substrate.
4. In plants, CA can help increase CO in chloroplaststwoTo increase ribulose diphosphateCarboxylaseCarboxylation rate of.
fiveMethanogenCA is involved in the catabolism of acetate.[1]
Activity regulation
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The main inhibitors of CA are sulfonamides,surface active agent For example, the inhibitory effect of DDT on CA may be related to the facilitation of group dissociation.Different CAs have different sensitivities to sulfonamide inhibitors. Studying the binding power of CA Ⅱ 198 variant with inhibitors, it is found that the charge, hydrophobicity and drug affinity of the 198 residue side chain are related to thePhenylalanineThe phenyl on the side chain fills the hydrophobic "bag", resulting in low catalysis and low sensitivity.In addition, surfactants can inhibit CA from forming polymers at high concentrations, thus reducing the competitive inhibition of the polymer on the formation of CA refolding proteins, promoting the start-up phase of protein refolding, and ultimately expanding the active concentration range of CA.
Chen et al. (1991) showed that estradiol E2 has a certain effect on CA, and the effect varies with different tissues: CA on the duodenal mucosa of rats can reduce the activity under the influence of E, while CA in the kidney of rats has a downward trend under the effect of E2, but there is no significant difference.In the same year, Pirkko et al. found that the level of CA Ⅱ protein and mRNA in the side of prostate of castrated rats decreased, while that in the dorsal side was the opposite;This change could be reversed after testosterone treatment;After treatment, the lateral changes were reversed, but the dorsal changes were not affected;The basic structure, general distribution, and basic physiological function of carbonic anhydrase were initially recognized by humans in the 1960s when it was found that the level of dorsal CA Ⅰ was increased while the level of lateral CA Ⅱ was unchanged in the treatment of non castrated ratsMolecular Biology TechnologyThe recent research focuses on the exploration of the relationship between CA structure and function, and the understanding of CA structure, function and distribution will be further concretized.
clinical
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CA in ciliary bodyepithelial cellsIt catalyzes CO2 and H2O to generate HCO3, which is secreted in aqueous humor through the cavity membrane. Because the aqueous humor must remain electrically neutral, the secretion of Na+to aqueous humor increases, and at the same time, it drives Cl - to move to aqueous humor, so that aqueous humor forms hyperosmotic pressure, thus promoting the flow of H2O to aqueous humor;Maintain aqueous balance and normal pH value.In glaucoma patients, the intraocular pressure is increased due to the obstruction of aqueous humor return.CA inhibitors (CAIs) can inhibit the activity of CA, reduce the production of HCO3 and reduce intraocular pressure. They are mainly used to treat glaucoma and reduce intraocular pressure clinically.
As a drug to treat glaucoma, CAIs can be divided into three generations according to their development process and pharmacological effects: the first generation of oral CAIs, the second generation of local CAIs, and the third generation of long-term non irritating local CAIs.
The first generation of oral CAIs acetazolamide is the first oral treatment drug for glaucoma. Because of its low fat solubility and less intraocular distribution, it needs a larger dose (1000-2000 mg) to be effective, so the white drug concentration is high;Long term drug use is prone to hypokalemia and metabolic acidosis (hyperchloremic acidosis), so clinical application is decreasing.The inhibition of oral CAIs on CA in non ocular tissues will cause serious systemic adverse reactions.Polyuria, gastrointestinal discomfort and fatigue occurred at the initial stage of drug use, some patients developed kidney stones, and very few cases occurredAgranulocytosis。
The second generation of local CAIs synthesized dolzolamide and brinzolamide in the early 1990s.It has strong selective inhibition of CA activity and high water solubility and fat solubility, which is convenient for preparing eye drops and can penetrate the cornea to make the drug reach effective drug concentration in the ciliary body.In 1995, dolzolamide eye drops were listed in Germany, without systemic adverse reactions.However, because the pH value of the aqueous solution is 5.5, it is slightly irritating to the conjunctiva.Brinzolamide was listed in 1998, and its water solubility is poor, so it is prepared into suspension.Its inhibitory effect on CA is stronger than dolzolamide, and its eye irritation is less.And the action time is short, it can only last for 2-3 hours, and it needs several times every day to maintain the effective drug concentration
The third generation of long-acting and non irritating topical application CAIs researchers have modified the structure of CAIs compounds and obtained two CAIs with strong effect and long action time (5-6 hours). However, the pH value of the prepared drops is about 5.5, which is still irritating to the eyes.In order to solve the pH problem, the researchers changed the dicarboxylic group at the end of the compound to amino acid, so that the pH value of the drip solution was between 6.5-7, and the eye irritation was basically eliminated.So far, the embryonic form of the third generation of CAIs has basically taken shape.
