Russian name
Trypsin + ChymotrypsinLatin name of the substances Trypsin + Chymotrypsin
Trypsinum + Chymotrypsinum ( genus. Trypsini + Chymotrypsini)Pharmacological group of substances Trypsin + Chymotrypsin
Typical clinical and pharmacological article 1
Pharmaceutical action. Proteolytic combined agent of protein nature. Obtained from the pancreas of a large cattle. When applied locally and externally, it breaks down necrotic tissue and fibrinous formations; dilutes viscous secretions, exudate, blood clots. Hydrolyzes proteins and peptones with the formation of low molecular weight peptides, breaks down bonds formed by aromatic amino acid residues (tyrosine, troptophan, phenylalanine, methionine).
Indications. Suppurative bronchopulmonary diseases (bronchiectasis, lung abscess, atelectasis, exudative pleurisy). Diseases of the ENT organs: purulent sinusitis, purulent otitis (acute, subacute, chronic), eustacheitis with viscous exudate, after tracheostomy to facilitate the removal of thick viscous exudate, chronic rhinitis. Burns, bedsores, trophic ulcers, purulent wounds. In ophthalmology: diseases of the cornea (ulcer, herpetic keratitis); burn of the mucous membrane of the eye, obstruction of the tear ducts, sluggish healing of eyelid skin wounds.
Contraindications. Hypersensitivity, malignant neoplasms, destructive forms pulmonary tuberculosis, stage II-III CHF, pulmonary emphysema with respiratory failure. Do not inject into bleeding cavities or apply to ulcerated surfaces of malignant tumors.
Carefully. Pleural empyema of tuberculous etiology (resorption of exudate can contribute to the development of bronchopleural fistula).
Dosing. Locally, externally.
For treatment purulent wounds and bedsores, sterile napkins moistened with a solution: 25-50 mg of the drug in 10-50 ml of 0.25% procaine solution, applied for a period of 2 to 24 hours (depending on the thickness of the purulent-necrotic masses) on the wound surface. The dressings are changed every 3-5 days.
At thermal burns III Art. a thin layer of the drug is applied to the scab at the rate of 1 g per 100 sq. cm of the wound surface, covered with a bandage moistened with a 0.9% NaCl solution or a 0.25% procaine solution; apply a waterproof bandage. The dressing is changed every other day. During each dressing change, easily removable areas of necrotic tissue are removed. For wounds covered with a thick scab, it is cut before applying a bandage.
For suppurative bronchopulmonary diseases use inhalation of 25-30 mg of the drug, diluted in 5 ml of distilled water, 1-3 times a day. The solution can also be administered through a bronchoscope, endotracheal tube, or tracheostomy. Antibiotics that bronchodilate drugs can be added to the solution.
For the treatment of ulcers, corneal burns and keratitis, it is used in the form of eye baths in a solution of 1:500 daily, for 2-3 days, or 2 drops of a 0.25% solution are instilled, 4 times a day, for 1-2 days. Obstruction of the tear ducts and wounds of the skin of the eyelids: wash the tear ducts or irrigate the wounds with a 1% solution. Solutions are prepared ex tempore.
At chronic rhinitis instill or irrigate the nasal cavity with a solution (5 mg in 5 ml of 0.9% NaCl solution) 2-3 times a day.
For chronic purulent otitis complicated by cholesteatoma, after washing the cavity with a 0.9% NaCl solution, instill a 0.5% solution of the drug (diluted by 0.9% NaCl solution), 2-3 times a day for 20-30 minutes.
Side effect. Allergic reactions, increased body temperature to subfebrile, tachycardia.
After inhalation administration: irritation of the mucous membrane of the upper respiratory tract, hoarseness of voice.
In ophthalmology: irritation and swelling of the conjunctiva (in this case it is recommended to reduce the concentration of the solution used).
Special instructions. In the next few hours after inhalation, the patient should carefully cough up sputum or remove it with suction.
om is involved in the proteolysis of food proteins in small intestine Chymotrypsin preferentially cleaves bonds formed by COOH groups of amino acids having hydrophobic side chains, and is characterized by a broader specificity of action than. Unlike trypsin, X. curdles milk.
Chymotrypsin is produced by exocrine cells of the pancreas (Pancreas) in the form of an inactive proenzyme - chymotrypsinogen, which is secreted in duodenum, where under the influence of trypsin it turns into chymotrypsin. In humans and most mammals, two types of chymotrypsinogen are found - A and B, differing in their physical and chemical properties. During the activation of chymotrypsinogen A, several active forms of chymotrypsin are formed, differing in solubility, crystal shape and the magnitude of enzymatic activity. The main form of chymotrypsin is chymotrypsin A - a protein with a molecular weight of about 25,000. It is most stable in a slightly acidic environment; in neutral and slightly alkaline environments it undergoes autolysis. The optimum action of chromium is at pH 7.5-8.2.
To determine the activity of chemicals, a number of methods based on the breakdown of proteins and synthetic substrates have been proposed. Spectrophotometrically controlled esters of M-substituted tyrosine are widely used.
X.'s activity is inhibited by a number of synthetic and natural inhibitors. Protein inhibitors of chromium are present in the blood plasma and tissues, protecting against destruction by this enzyme. Many of them are polyvalent and also inhibit other proteases (for example, α 2 -macroglobulin, some trypsin inhibitors); α 1 -antichymotrypsin is a specific inhibitor of Ch. in blood plasma.
Chymotrypsin is used as medicine, capable of selectively breaking down proteins of necrotic tissues, fibrous formations, liquefying viscous exudates, purulent masses. It has an anti-inflammatory, anti-edematous effect and promotes wound healing. Medications Chemotrypsinogen A is obtained from chymotrypsinogen A, which is isolated from the pancreas of cattle and, after recrystallization, is activated with trypsin. IN medical practice use chymotrypsin A, produced under the name “crystalline chymotrypsin”. This white, highly soluble in water and isotonic sodium chloride solution.
Indications for use, methods of use, doses, and possible complications the same as for crystalline trypsin. In addition, X. is used for intracapsular cataract extraction. Together with trypsin, X. is part of chymopsin, pancreatin, and some other drugs used for pancreatic insufficiency.
Release form: hermetically sealed bottles or ampoules containing 0.005 G (5 mg) and 0.01 G (10 mg) crystalline chymotrypsin. Storage in a place protected from light at a temperature not exceeding +10°.
II Chymotrypsinan enzyme of pancreatic secretion, belonging to the class of hydrolases (EC 3.4.21.1), catalyzing the hydrolytic breakdown of proteins, peptides, amides and amino acid esters during the digestion of food in the intestine.
1. Small medical encyclopedia. - M.: Medical encyclopedia. 1991-96 2. First health care. - M.: Great Russian Encyclopedia. 1994 3. encyclopedic Dictionary medical terms. - M.: Soviet Encyclopedia. - 1982-1984.
Synonyms:See what "Chymotrypsin" is in other dictionaries:
Chymotrypsin... Spelling dictionary-reference book
CHYMOTRYPSIN, a substance produced in the body that helps digest food. Chymotrypsin is an enzyme that breaks down proteins. It is produced in the small intestine through complex chemical reactions from chymotrypsinogen... Scientific and technical encyclopedic dictionary
Digestive enzyme of pancreatic juice; participates in the breakdown of proteins in the intestines. Produced as inactive chymotrypsinogen... Big Encyclopedic Dictionary
Proteolytic an enzyme of most vertebrates that participates, together with trypsin and other peptidases, in the breakdown of proteins into small intestine; synthesized by pancreatic cells in the form of an inactive precursor of chymotrypsinogen and... ... Biological encyclopedic dictionary
Noun, number of synonyms: 1 enzyme (253) ASIS Dictionary of Synonyms. V.N. Trishin. 2013… Synonym dictionary
chymotrypsin- Proteolytic enzyme of vertebrates, synthesized in the pancreas in the form of a precursor of chymotrypsinogen. [Arefyev V.A., Lisovenko L.A. English Russian Dictionary genetic terms 1995 407 pp.] Topics genetics EN chymotripsin ... Technical Translator's Guide
Digestive enzyme of pancreatic juice; participates in the breakdown of proteins in the intestines. Produced as inactive chymotrypsinogen. * * * CHYMOTRYPSIN CHYMOTRYPSIN, digestive enzyme of pancreatic juice; participates in… … encyclopedic Dictionary
- (gr.chymoscok + trypsin) an enzyme that breaks down food proteins; produced by pancreatic cells. New dictionary foreign words. by EdwART, 2009. chymotrypsin a, pl. No m. (… Dictionary of foreign words of the Russian language
Chymotrypsin chymotrypsin [EC 3.4.21.1 2]. A proteolytic enzyme of vertebrates, synthesized in the pancreas in the form of a precursor of chymotrypsinogen. (
Chymotrypsin- proteolytic enzyme; catalyzes the breakdown of proteins and peptides. Refers to peptide hydrolases. Together with trypsin participates in the proteolysis of food proteins in the small intestine Chymotrypsin preferentially cleaves bonds formed by COOH groups of amino acids having hydrophobic side chains, and is characterized by a broader specificity of action than trypsin. Unlike trypsin, X. curdles milk.
Chymotrypsin is produced by exocrine cells pancreas in the form of an inactive proenzyme - chymotrypsinogen, which is secreted into the duodenum, where, under the influence of trypsin, it is converted into chymotrypsin. In humans and most mammals, two types of chymotrypsinogen are found - A and B, which differ in their physicochemical properties. During the activation of chymotrypsinogen A, several active forms of chymotrypsin are formed, differing in solubility, crystal shape and the magnitude of enzymatic activity. The main form of chymotrypsin is chymotrypsin A - a protein with a molecular weight of about 25,000. The enzyme is most stable in a slightly acidic environment; in neutral and slightly alkaline environments it undergoes autolysis. The optimum action of chromium is at pH 7.5-8.2.
To determine the activity of chromium, a number of methods have been proposed,
based on the breakdown of proteins and synthetic substrates. Spectrophotometrically controlled hydrolysis of M-substituted tyrosine esters is widely used.X.'s activity is inhibited by a number of synthetic and natural inhibitors. Protein inhibitors of chromium are present in the blood plasma and tissues, protecting proteins from destruction by this enzyme. Many of them are polyvalent and also inhibit the activity of other proteases (for example, a 2 -macroglobulin, some trypsin inhibitors); a specific inhibitor of Ch. in blood plasma is a 1 -antichymotrypsin.
Chymotrypsin is used as a drug that can selectively break down the proteins of necrotic tissues and formations, and dilute viscous exudates and purulent masses. It has an anti-inflammatory, anti-edematous effect and promotes wound healing. Chromium medicinal preparations are obtained from chymotrypsinogen A, which is isolated from the pancreas of cattle and, after recrystallization, is activated with trypsin. In medical practice, chymotrypsin A is used, produced under the name “crystalline chymotrypsin”. It is a white powder, highly soluble in water and isotonic sodium chloride solution.
Indications for use,
Methods of use, doses, contraindications and possible complications are the same as for crystalline trypsin. In addition, X. is used for intracapsular cataract extraction. Together with trypsin, X. is part of chymopsin, pancreatin, and some other drugs used for pancreatic insufficiency.Release form: hermetically sealed bottles or ampoules containing 0.005 G (5 mg) and 0.01 G (10 mg) crystalline chymotrypsin. Storage in a place protected from light at a temperature not exceeding +10°.
Chymotrypsin enzyme
Chymotrypsin
Pharmachologic effect:
When applied topically, it breaks down necrotic (dead) tissue and fibrinous formations (blood clots); dilutes viscous secretions (secretion of special glands, for example, sputum), exudates (protein-rich liquid secreted from small vessels of the tissue), blood clots. Compound:
Chymotrypsin is a proteolytic enzyme produced in the pancreas of mammals. For medical use it is obtained from the pancreas of cattle. Pancreatic juice contains an inactive state in the form of chymotrypsinogen (chymotrypsinogen A and B), which is activated under the influence of trypsin, and a number of forms are formed from chymotrypsinogen A: a, b, g, s and p - chymotrypsins, and from chymotrypsinogen B - chymotrypsin B. All forms of chymotrypsin are similar in enzymatic properties, but differ in activity. A-chymotrypsin, which is marketed under the name “crystalline chymotrypsin,” is currently of practical importance as a medicine.a- Chymotrypsin is a protein with a relative molecular weight of 21,600 - 27,000. Belongs to the group of proteolytic enzymes. Like trypsin, it hydrolyzes proteins and peptones to form relatively low molecular weight peptides. It differs from trypsin in that it primarily cleaves bonds formed by aromatic amino acid residues (tyrosine, tryptophan, phenylalanine, methionine). In some cases, chymotrypsin produces deeper protein hydrolysis than trypsin. It also differs from trypsin in that it causes milk to clot. More stable than trypsin and inactivated more slowly.
Chymotrypsin crystalline is shiny flakes or white powder. Soluble in water and isotonic sodium chloride solution; pH of 0.2% aqueous solution 4.5 - 6.5. In dry form the racks; aqueous solutions are quickly inactivated, especially at high temperatures.
Indications for use:
Thrombophlebitis (inflammation of the vein wall with blockage), inflammatory-dystrophic forms of periodontal disease (dental diseases), osteomyelitis (inflammation of the bone marrow and adjacent bone tissue), sinusitis (inflammation of the maxillary sinus), otitis (inflammation of the ear cavity), iritis (inflammation of the iris), iridocyclitis (eye disease); tracheitis (inflammation of the trachea), bronchitis (inflammation of the bronchi), cataract extraction (removal of the clouded lens of the eye).Mode of application:
Intramuscularly for adults, 0.0025 g 1 time per day. For injection, dissolve immediately before use 0.005 g of crystalline chymotrypsin in 1-2 ml isotonic solution sodium chloride or 0.5-2% novocaine solution. The solution is injected deep into the upper outer quadrant of the gluteal muscle. Course - 6-15 injections.Side effects:
Allergic reactions, burning at the site of application. Bleeding from granulating (healing) areas. When introduced into cavities, substances with a histamine-like effect may be released.Contraindications:
Bleeding wounds and decaying malignant tumors. Individual intolerance. The drug is not suitable for intravenous administration.Release form:
Powder in hermetically sealed bottles of 0.005 g and 0.01 g.Attention!
Before using Chymotrypsin, you should consult your doctor. These instructions for use are provided in free translation and are intended for informational purposes only. For more complete information, please refer to the manufacturer's instructions.
Serine proteinases.
The mechanism of action of chymotrypsin and carboxypeptidase.
It was previously mentioned that the pancreas is responsible for the production of a whole group of proteolytic enzymes that hydrolyze peptide bonds formed by different amino acids. Enzymes involved in protein digestion and their specificity for peptide bonds formed by different amino acids are given in Table. 3.1. The most important and fully studied representative of intestinal proteinases belonging to the family serine proteinases, is chymotrypsin. Chymotrypsin is a digestive enzyme synthesized as a zymogen by pancreatic acinar cells.
Table 3.1
Substrate specificity of gastrointestinal proteinases
Active enzyme |
Zymogen |
Activator |
fissile |
|
peptide bond |
||||
Carboxyproteinases |
Auto activation, |
|||
Pepsin A |
Pepsinogen A |
active pepsin |
||
Serine |
||||
proteinases |
||||
Trypsin |
Trypsinogen |
Enterokinase, |
||
Trypsin |
||||
Chymotrypsin |
Chymotrypsinogen |
Trypsin |
Tyr, Phe, Trp, Met |
|
Elastase |
Proelastase |
Trypsin |
||
peptidases |
||||
Carboxypeptidase A |
Procarboxypepti- |
Val, Leu, Ile, Ala with |
||
Carboxypeptidase B |
Procarboxypepti- |
|||
Aminopeptidase |
Proaminopepti- |
Elimination of N- |
||
end |
||||
balances (for |
||||
except Pro) |
||||
Inside acinar cells, newly synthesized protein molecules are transported from the endoplasmic reticulum to the Golgi apparatus, where they are surrounded by a protein-lipid membrane - this is how zymogen granules are formed, which look like very dense bodies in an electron microscope (Fig. 3.3).
Rice. 3.3 Electron micrograph of zymogen granules in pancreatic acinar cells (after drawing by G. Palade from Strayer L., Biochemistry
"Mir", M., vol. 2, 1985).
Zymogen granules containing chymotrypsinogen accumulate at the tip of the acinar cells and are then released into the duct leading to the duodenum under the influence of a hormonal or neuronal signal.
Chymotrypsinogen is represented by one polypeptide chain of 245 amino acids. The chain is stabilized by five disulfide bridges. Activation of chymotrypsinogen is carried out under the action of trypsin, which cleaves the peptide bond between arginine-15 and isoleucine-16.
The active π-chymotrypsin formed in this process acts on other chymotrypsinogen molecules (Fig. 3.4).
Rice. 3.4 Scheme illustrating the sequence of stages of chymotrypsin activation
Next, π-chymotrypsin undergoes the additional proteolytic action of chymotrypsin with the cleavage of two dipeptides Ser14-Arg15 and Thr147-Asn148 and the formation of a stable form of the enzyme - α-chymotrypsin.
Since chymotrypsin is one of the most fully studied enzymes, it is advisable to consider its structure and mechanism of action in more detail. The α-chymotrypsin molecule consists of three polypeptide chains connected by two interchain disulfide bonds (Fig. 3.5).
Rice. 3.5 The α-chymotrypsin molecule has two interchain disulfide bridges and three intrachain disulfide bonds.
The molecular weight of α-chymotrypsin is about 25,000 Da. The molecule has a compact ellipsoidal shape measuring 51 × 40 × 40 Å. All charged groups, with the exception of the three required for catalysis, are on the surface of the molecule.
The equilibrium of the hydrolysis reaction is shifted almost completely towards the cleavage of peptide bonds. However, chymotrypsin is not capable of hydrolyzing every peptide bond at a high rate. He acts selectively on connections formed carboxyl groups of aromatic acids and amino acids with hydrophobic radicals big size, for example methionine.
A characteristic feature of chymotrypsin is its ability to hydrolyze ester bonds. Essentially, much of the information about the mechanism of action of chymotrypsin was obtained by studying the hydrolysis
esters.
Chymotrypsin has been shown to catalyze the hydrolysis of peptide and ester bonds in two distinct steps. This was first discovered when studying the kinetics of hydrolysis of the ester bond of n-nitrophenylacetate.
Analysis of the reaction showed that the release of one of the products, n-nitrophenol, during hydrolysis, occurs in two stages: first, n-nitrophenol is released explosively, and then it is formed with
lower stationary speed.
IN general outline the process of hydrolysis of n-nitrophenylacetate is reduced to the formation of an enzyme-substrate complex, then the ester bond in the substrate is cleaved and n-nitrophenol is released, while the acetyl group of the substrate remains covalently bound in the active center of the enzyme. Next, water attacks the acetyl-enzyme complex to form acetate ion and regenerated enzyme (Fig. 3.6)
Rice. 3.6 Acylation: formation of an acetyl-enzyme complex as an intermediate. Deacylation: hydrolysis of the acetyl-enzyme complex intermediate.
The rapid initial phase of n-nitrophenol release is associated with the formation of the acyl-enzyme complex shown in Fig. 3.6 icon
(*). This step is called acylation. The second stage, called deacylation, corresponds to the stationary stage of the reaction, which is also limiting. The acyl-enzyme complex turned out to be so stable that under certain conditions it can be isolated. After being isolated at pH 3 pure form this intermediate acyl-enzyme complex, it was possible to characterize the site at which the acyl group is added to the enzyme. It turned out that the acyl group is connected to the oxygen atom of a specific serine residue
– serine-195 (Ser-195). It is for this reason that chymotrypsin is classified as serine proteinases. This serine residue exhibits unusually high reactivity. It can be specifically marked
organic fluorophosphate – diisopropyl fluorophosphate (Fig. 3.7).
Rice. 3.7 Diisopropylphosphate (DPPP) inactivates chymotrypsin by forming the diisopropylphosphoryl derivative of serine-195.
The increased reactivity of serine-195 is evidenced by the fact that the remaining 27 serine residues in chymotrypsin do not react at all with diisopropyl fluorophosphate (DPFP). By the way, chymotrypsin is not the only enzyme that is inhibited by DPPP. Many other proteolytic enzymes such as trypsin, elastase,
thrombin, bacterial subtilisin also react specifically with DPPP, completely losing activity. As in the case of chymotrypsin, these proteinases interact with DPPP through a single serine residue. Thus, it is appropriate to talk about the existence of a whole family serine proteinases.
However, not only the Ser-195 side radical takes part in the catalytic acts of chymotrypsin. It was shown that the histidine residue His-57 also plays an important role in the active site of the enzyme. It was possible to prove its participation in catalysis using the so-called affinity tag, which binds to chymotrypsin like a substrate on the one hand and forms a covalent bond with a certain group in the active center, on the other hand.
Such a label for chymotrypsin is the compound tosyl-L- phenylalanine chloromethyl ketone, the structure of which is shown in Fig. 3.8.
Rice. 3.8 Structure of tosyl-L-phenylalanine chloromethyl ketone used as an affinity tag for chymotrypsin (R – tosyl group).
The presence of a side chain tag represented by phenylalanine in the molecule ensures its specific interaction with the enzyme. The reactive group of the label, chloromethyl ketone, interacts only with the His-57 residue and alkylates one of the nitrogen atoms of the histidine ring. The interaction of such an affinity tag with chymotrypsin completely deprives it of enzymatic activity. There is several evidence for the high specificity of the interaction of the tag with chymotrypsin:
− firstly, the affinity tag is highly stereospecific. The D-isomer of the label does not interact with chymotrypsin;
− secondly, a competitive inhibitor of chemtrypsin, β-phenylpropionate, inhibits the process of interaction of the label with the protein;
- thirdly, the rate of inactivation of chymotrypsin upon addition of a label is in the same dependence on pH as the rate of catalysis.
The interaction scheme of tosyl-L-phenylalanine chloromethyl ketone with His-57 is shown in Fig. 3.9.
Rice. 3.9 Alkylation of histidine-57 in chymotrypsin upon interaction with tosyl-L-phenylalanine chloromethyl ketone.
Thus, for the manifestation of the catalytic activity of chymotrypsin, the presence of residues Ser-195 and His-57 in the active center of the enzyme is fundamental. As was shown later, next to these residues in the chymotrypsin molecule there is also an aspartic acid residue - Asp-102. X-ray diffraction analysis showed that all three residues are located next to each other and create the so-called charge transfer system due to the fact that Asp-102 forms a hydrogen bond with His-57, which in turn is hydrogen bonded with Ser195 (Fig. 3.10).
Rice. 3.10 Charge transfer system in the active site of chymotrypsin in the absence of substrate (Blow D.M., Steitz T.A., X-ray diffraction studies of enzymes, Ann. Rev. Biochem., 1976, 39, 86-95).
The carboxylate ion Asp-102 polarizes the imidazole group of His-57, which increases its ability to perform proton shuttle binding, and when the substrate interacts with the chymotrypsin molecule, Asp-102 and His-57 accept the proton of the hydroxyl group of Ser-195.
Rice. 3.11 Charge transfer system in the active center of chymotrypsin in the presence of a substrate. When a substrate is added, intermediate proton binding occurs with aspartate-102 and histidine-57 (Blow D.M., Steitz T.A., X-ray diffraction studies of enzymes, Ann. Rev. Biochem., 1976, 39, 86-95).
The localization of specific substrate binding sites and the probable orientation of the hydrolyzed peptide bond were established as a result of X-ray diffraction analysis of chymotrypsin complexes with substrate analogues. In particular, it was shown by the example of studying complexes of chymotrypsin with a non-hydrolyzable analogue of the substrate - formyl-L-tryptophan, the existence in the enzyme of a deep hydrophobic
pocket near serine-195, which corresponds in size to the side radicals of aromatic amino acids (Fig. 3. 12).
Rice. 3.12 Schematic representation of the binding of formyl-L-tryptophan by chymotrypsin.
It is the presence of this deep pocket that explains the specificity of chymotrypsin for amino acids with an aromatic or other large hydrophobic side chain. X-ray diffraction analysis of complexes of chymotrypsin with analogues of polypeptide substrates revealed big number hydrogen bonds between the main chains of the enzyme and the substrate, which are located in the same way as in antiparallel β-sheet layers.
The mechanism of the catalytic action of chymotrypsin
Intensive and comprehensive X-ray diffraction and biochemical research chymotrypsin allowed us to come to a certain conclusion regarding the mechanism of its catalytic action. As already mentioned, His-57 and Ser-195 are directly involved in the cleavage of the substrate peptide bond. Hydrolysis of this bond begins when the oxygen atom of the OH group of Ser-195 attacks the carbon atom of the carbonyl group
hydrolyzed peptide bond of the substrate. As a result, the bond between the C and O atoms in the carbonyl group becomes single and the O atom acquires negative charge. In this case, four different atoms associated with the carbon of the carbonyl group are arranged in the form of a tetrahedron. Education of such tetrahedral intermediate turns out to be possible due to the formation of hydrogen bonds between a negatively charged oxygen atom (called an oxyanion) and two NH groups of the enzyme's main chain (Fig. 3.13).
Rice. 3.13 Tetrahedral intermediate in the acylation and deacylation reactions of chymotrypsin. The stability of the intermediate is ensured by hydrogen bonds formed by the NH groups of the enzyme's main chain.
This region of chymotrypsin is called oxyanion cavity. In the mechanism of formation of the mentioned intermediate, the most important role is played by the proton transfer from Ser-195 to His-57.
Rice. 3.14 The first stage of peptide hydrolysis with chymotrypsin is acylation. A tetrahedral intermediate is formed. The amine component of the substrate is then quickly separated from the enzyme, and the enzyme itself is converted into an intermediate product, the acyl enzyme.
Proton transfer is greatly facilitated by the presence of a charge transfer system. The Asp-102 residue strictly orients the position of the histidine-57 imidazole and partially neutralizes the charge appearing on this ring. The proton bound by the His-Asp pair then goes to the nitrogen atom of the hydrolyzed peptide bond, which is broken as a result. At this stage, the amine component of the substrate is hydrogen bonded to His-57, and the acid component is linked by an ester (covalent) bond to Ser-195, thereby completing the acylation step. Thus, upon acylation of chymotrypsin, an intermediate tetrahedral compound (1) is formed and, subsequently, the amine component of the substrate (2) is quickly separated from the enzyme. The enzyme itself is converted into an intermediate product of catalysis - acyl enzyme (3) (Fig. 3.14).
Rice. 3.15 The second stage of peptide hydrolysis with chymotrypsin is deacylation. The acyl enzyme is hydrolyzed by water. Deacylation is essentially the reverse reaction of acylation, but water takes the place of the amine component of the substrate.
At the next stage, deacylation, the amine component diffuses from the enzyme, and water takes its place in the active center.
In fact, deacylation is the reverse process of acylation. First, the charge transfer system abstracts a proton from the water.
