Mechanism of action of enzymes

The mechanism of action of enzymes can be considered from two positions: from the point of view of changes in the energy of chemical reactions and from the point of view of events in the active center.

A. Energy changes during chemical reactions

Any chemical reactions proceed in accordance with two basic laws of thermodynamics: the law of conservation of energy and the law of entropy. According to these laws, the total energy of a chemical system and its environment remains constant, while the chemical system tends to decrease order (increase entropy). To understand the energy of a chemical reaction, it is not enough to know the energy balance of the reagents entering and exiting the reaction; it is necessary to take into account energy changes during the process of a given chemical reaction and the role of enzymes in the dynamics of this process. Consider the decomposition reaction of carbonic acid:

H 2 CO 3 > H 2 0 + C0 2.

Carbonic acid is weak; the reaction of its decomposition will proceed under normal conditions if the molecules of carbonic acid have an energy exceeding a certain level, called the activation energy E a (Fig. 2-10).

Activation energy is the additional amount of kinetic energy required for the molecules of a substance to react.

When this energy barrier is reached, changes occur in the molecule, causing a redistribution of chemical bonds and the formation of new compounds. Molecules possessing E a are said to be in a transition state. The energy difference between the initial reagent H 2 CO 3 and the final compounds H 2 O and CO 2 is called the change in free energy

Rice. 2-10. Change in free energy during the decomposition of carbonic acid.

Reaction energies DG. Molecules H 2 O and CO 2 are more stable substances than H 2 CO 3, i.e. have less energy and practically do not react under normal conditions. The energy released as a result of this reaction is dissipated in the form of heat into the environment.

The more molecules have energy exceeding the level of E a, the higher the rate of the chemical reaction. You can increase the rate of a chemical reaction by heating. This increases the energy of the reacting molecules. However, high temperatures are destructive for living organisms, so enzymes are used in cells to speed up chemical reactions. Enzymes provide a high rate of reactions under optimal conditions existing in the cell by lowering the level of E a. Thus, enzymes reduce the height of the energy barrier, as a result the number of reactive molecules increases, and therefore the reaction rate increases.

In the mechanism of enzymatic catalysis, the formation of unstable intermediate compounds is of decisive importance - the enzyme-substrate complex ES, which undergoes transformation into an unstable transition complex EP, which almost instantly disintegrates into a free enzyme and the reaction product.

Thus, biological catalysts (enzymes) do not change free energy

substrates and products and therefore do not change the equilibrium of the reaction (Fig. 2-11).

An enzyme, performing the function of a catalyst for a chemical reaction, obeys the general laws of catalysis and has all the properties characteristic of non-biological catalysts, but also has distinctive properties associated with the structural features of enzymes.

The similarity between enzymes and non-biological catalysts is that:

• Enzymes catalyze energetically possible reactions;
• · the energy of a chemical system remains constant;
• · during catalysis the direction of the reaction does not change;
• Enzymes are not consumed during the reaction.

The differences between enzymes and non-biological catalysts are that:

• · the rate of enzymatic reactions is higher than reactions catalyzed by non-protein catalysts;
• Enzymes are highly specific;
• · the enzymatic reaction takes place in the cell, i.e. at a temperature of 37 °C, constant atmospheric pressure and physiological pH;
• · The speed of the enzymatic reaction can be adjusted.

1. Formation of the enzyme-substrate complex

The fact that enzymes have high specificity allowed us to put forward a hypothesis in 1890, according to which the active center of the enzyme is complementary to the substrate, i.e. corresponds to it like a “key to a lock”. After the interaction of the substrate (“key”) with the active center (“lock”), chemical transformations of the substrate into the product occur. The active center was considered as a stable, strictly determined structure.

In 1959, another version of the “lock and key” hypothesis was proposed to explain events in the active site of the enzyme. According to this hypothesis, the active center is a flexible structure

Rice. 2-11. Change in free energy during a chemical reaction, uncatalyzed and catalyzed by enzymes.

The enzyme reduces the activation energy E a, i.e. reduces the height of the energy barrier, as a result, the proportion of reactive molecules increases, therefore, the reaction rate increases with respect to the substrate. The substrate, interacting with the active center of the enzyme, causes a change in its conformation, leading to the formation of an enzyme-substrate complex, favorable for chemical modifications of the substrate. At the same time, the substrate molecule also changes its conformation, which ensures higher efficiency of the enzymatic reaction. This “induced correspondence hypothesis” was subsequently confirmed experimentally.

2. Sequence of events during enzymatic catalysis

The process of enzymatic catalysis can be divided into the following stages (Fig. 2-12). substrate catalysis chemical reaction

The first, second and fourth stages of catalysis are short and depend on the concentration of the substrate (for the first stage) and the binding constants of ligands in the active site of the enzyme (for the first and third stages). Changes in the energy of the chemical reaction at these stages are insignificant.

The third stage is the slowest; its duration depends on the activation energy of the chemical reaction. At this stage, the bonds in the substrate molecule are broken, new bonds are formed, and the product molecule is formed.

3. The role of the active site in enzymatic catalysis

As a result of research, it was shown that the enzyme molecule, as a rule, is many times larger than the substrate molecule undergoing chemical transformation by this enzyme. Only a small part of the enzyme molecule comes into contact with the substrate, usually from 5 to 10 amino acid residues, forming the active site of the enzyme. The role of the remaining amino acid residues is to ensure the correct conformation of the enzyme molecule for the optimal occurrence of the chemical reaction.

The active site at all stages of enzymatic catalysis cannot be considered as a passive site for substrate binding. It is a complex molecular "machine" that uses a variety of chemical mechanisms to convert a substrate into a product.

In the active site of the enzyme, the substrates are arranged in such a way that the functional groups of the substrates involved in the reaction are in close proximity to each other. This property of the active center is called the effect of convergence and orientation of reagents. This ordered arrangement of substrates causes a decrease in entropy and, as a consequence, a decrease in activation energy (Ea), which determines the catalytic efficiency of enzymes.

The active center of the enzyme also contributes to the destabilization of interatomic bonds in the substrate molecule, which facilitates the occurrence of a chemical reaction and the formation of products. This property of the active site is called the substrate deformation effect (Fig. 2-12).

Rice. 2-12. Stages of enzymatic catalysis.

I - stage of approaching and orienting the substrate relative to the active center of the enzyme; II - formation of an enzyme-substrate complex (ES) as a result of induced compliance; III - deformation of the substrate and formation of an unstable enzyme-product complex (EP); IV - decomposition of the complex (EP) with the release of reaction products from the active center of the enzyme and release of the enzyme.

B. Molecular mechanisms of enzymatic catalysis

The mechanisms of enzymatic catalysis are determined by the role of the functional groups of the active center of the enzyme in the chemical reaction of converting the substrate into the product. There are 2 main mechanisms of enzymatic catalysis: acid-base catalysis and covalent catalysis.

1. Acid-base catalysis

The concept of acid-base catalysis explains enzymatic activity by the participation of acidic groups (proton donors) and/or basic groups (proton acceptors) in a chemical reaction. Acid-base catalysis is a common phenomenon. The amino acid residues that make up the active center have functional groups that exhibit the properties of both acids and bases.

The amino acids involved in acid-base catalysis primarily include Cys, Tyr, Ser, Lys, Glu, Asp and His. The radicals of these amino acids in the protonated form are acids (proton donors), in the deprotonated form they are bases (proton acceptors). This property of active site functional groups makes enzymes unique biological catalysts, in contrast to nonbiological catalysts that can exhibit either acidic or basic properties.

An example of acid-base catalysis, in which the cofactors are Zn 2+ ions, and the NAD + molecule is used as a coenzyme, is the liver alcohol dehydrogenase enzyme, which catalyzes the oxidation reaction of alcohol (Fig. 2-13):

C 2 H 5 OH + NAD + > CH 3 -SON + NADH + H

2. Covalent catalysis

Covalent catalysis is based on the attack of nucleophilic (negatively charged) or electrophilic (positively charged) groups of the active center of the enzyme by substrate molecules with the formation of a covalent bond between the substrate and the coenzyme or the functional group of the amino acid residue (usually one) of the active center of the enzyme.

The action of serine proteases, such as trypsin, chymotrypsin and thrombin, is an example of the mechanism of covalent catalysis, when a covalent bond is formed between the substrate and the serine amino acid residue of the active site of the enzyme. The term “serine proteases” is due to the fact that the amino acid residue serine is part of the active center of all these enzymes and is directly involved in catalysis. Let us consider the mechanism of covalent catalysis using the example of chymotrypsin, which hydrolyzes peptide bonds during the digestion of proteins in the duodenum (see section 9). Chymotrypsin substrates are peptides containing amino acids with aromatic and cyclic

Rice. 2-13. The mechanism of acid-base catalysis using the example of liver alcohol dehydrogenase.

I - the ethyl alcohol molecule has a binding center that provides hydrophobic interaction between the active center and the methyl group of the alcohol; II - a positively charged zinc atom promotes the abstraction of a proton from the alcohol group of ethanol to form a negatively charged oxygen atom. The negative charge is redistributed between the oxygen atom and the neighboring hydrogen atom, which is then transferred in the form of hydrithione to the fourth carbon atom of the nicotinamide coenzyme NAD+; III - as a result, a reduced form of NADH and acetaldehyde are formed.

Hydrophobic radicals (Phen, Tyr, Tri), which indicates the participation of hydrophobic forces in the formation of the enzyme-substrate complex. The mechanism of covalent catalysis of chymotrypsin is discussed in Fig. 2-14.

Radicals Asp 102, His 57 and Ser 195 are directly involved in the act of catalysis. Due to the nucleophilic attack of the peptide bond of the substrate, this bond is broken with the formation of covalently modified serine - acyl-chymotrypsin. Another peptide fragment is released as a result of the rupture of the hydrogen bond between the peptide fragment and the His 57 active site of chymotrypsin. The final stage of hydrolysis of the peptide bond of proteins is deacylation of chymotrypsin in the presence of a water molecule with the release of the second fragment of the hydrolyzed protein and the original form of the enzyme.

enzyme biological catalysis transamination

The discovery of the spatial structure of a number of enzymes using X-ray diffraction analysis provided a reliable basis for constructing rational schemes of the mechanism of their action.

Establishing the mechanism of action of enzymes is of key importance for uncovering structure-function relationships in a variety of biologically active systems.

Lysozyme is found in various tissues of animals and plants; it is found, in particular, in tear fluid and egg white. Lysozyme functions as an antibacterial agent, catalyzing the hydrolysis of the cell walls of a number of bacteria. This polysaccharide is formed by alternating N-acetylmuranic acid (NAM) residues linked by a β-1,4-glycosidic bond (the polysaccharide chains are cross-linked by short peptide fragments).

Bacterial polysaccharide is a very complex insoluble compound, and therefore highly hydrolyzable oligosaccharides formed by NAG residues are often used as lysozyme substrates.

Chicken egg white lysozyme is formed by one polypeptide chain containing 129 amino acid residues; its molecular weight is 14,600. The high stability of the enzyme is ensured by the presence of four disulfide bridges.

Information about the active center and type of catalytic process was obtained by D. Phillips in 1965. based on X-ray diffraction studies of lysozyme and its complexes with inhibitors. The lysozyme molecule has the shape of an ellipsoid with axes of 4.5*3*3 nm; Between the two halves of the molecule there is a “gap” in which the binding of oligosaccharides occurs. The walls of the gap are formed mainly by side chains of non-polar amino acids, which ensure the binding of non-polar molecules of the substrate, and also include side chains of polar amino acids, which are capable of forming hydrogen bonds with the acylamine and hydroxyl groups of the substrate. The size of the gap allows accommodation of an oligosaccharide molecule containing 6 monosaccharide residues. It is not possible to determine the nature of binding of a substrate, for example, NAG 6 hexasaccharide, by X-ray diffraction analysis. At the same time, complexes of the enzyme with the inhibitor trisaccharide NAG 3 are stable and well studied. NAG 3 binds to clefts on the surface of the enzyme, forming hydrogen bonds and van der Waals contacts; at the same time, it fills only half of the gap in which three more monosaccharide residues can bind. The non-reducing end (sugar A) is at the beginning of the gap, and the reducing end (sugar C) is in its central part; Sugar residues A, B and C have a chair conformation. The construction of the model of the enzyme-substrate complex was based on the assumption that when the substrate NAG 6 binds, the same interactions are realized as during the binding of NAG 3. In the enzyme model, three sugar residues (referred to as residues D, E, and F) were placed inside the cleft; each subsequent sugar was added in such a way that its conformation was the same (as far as possible) as that of the first three sugars. As part of the model complex, all sugar residues implement effective non-covalent interactions with side and peptide groups of amino acid residues forming the cleft.

When identifying catalytic groups, it was natural to focus on those of them that are located near the cleavable glycosidic bond in the enzyme-substrate complex and can serve as proton donors or acceptors. It turned out that it was on one side of the bond being split, at a distance? 0.3 nm (from the oxygen of the glycosidic bond), there is a carboxyl group of Glu-35, and on the other (at the same distance) there is a carboxyl group of Asp-52, their environment is very different. Glu-35 is surrounded by hydrophobic residues; it can be assumed that at the optimum pH of the enzyme this group is in a non-ionized state. The environment of Asp-52 is clearly polar; its carboxyl group participates as a hydrogen acceptor in a complex network of hydrogen bonds and probably functions in an ionized state.

The following scheme of the catalytic process for the hydrolysis of oligosaccharide is proposed. The non-ionized carboxyl group of Glu-35 acts as a proton donor, supplying it to the glycosidic oxygen atom between the C (1) atom of sugar D and the C (4) atom of sugar E (stage of general acid catalysis); this leads to cleavage of the glycosidic bond. As a result, the sugar residue D goes into the state of a carbocation with a positively charged carbon atom C (1) and takes on a half-chair conformation. The negative charge of the carboxylate group of Asp-52 stabilizes the carbocation. The NAG 2 residue (E+F sugars) diffuses from the active site region. Then a water molecule reacts; its proton goes to Glu-35, and the OH - - group goes to the C atom (1) of residue D (stage of main catalysis). The NAG 4 residue (sugars A+B+C+D) leaves the active site region, and the enzyme returns to its original state.

Ribonuclease (RNase) of the bovine pancreas hydrolyzes internucleotide bonds in RNA near pyrimilyne units, which remain esterified at the 3" position. The enzyme, along with other nucleases, is widely used in analyzing the structure of RNA.

RNase is formed by one polypeptide chain containing 124 amino acid residues, and its molecular weight is 13,680; the molecule has four disulfide bonds. RNase is the first enzyme for which the primary structure has been determined.

Based on the results of a study of ribonuclease renaturation, K. Afinsen was the first to clearly formulate the idea that the spatial structure of a protein is determined by its primary structure.

In 1958, F. Richards showed that, under certain conditions, subtilisin cleaves the Ala-20 - Ser-21 peptide bond in RNase. The resulting fragments were named S-peptide (residues 1-20) and S-protein (residues 21-124); due to non-covalent interactions, the fragments form a complex called RNase S. This complex has almost complete catalytic activity of the native enzyme; in isolated form, S-peptide and S-protein are inactive. It was further found that a synthetic peptide identical in sequence to a fragment of the S-peptide, containing residues 1 to 13, restores the activity of the S protein, but a shorter peptide containing residues 1 to 11 does not have this ability. The data obtained allowed us to conclude that the corresponding His-12 or Met-13 residues (or both of these residues) are included in the active center of the enzyme.

When studying the effect of pH on RNase activity, the important role of protein functional groups with pK 5.2 and 6.8 was revealed; this suggested the participation of histidine residues in the catalytic process.

When RNase is carboxylated with iodoacetate at pH 5.5, i.e. under conditions in which modification of histidine residues predominantly occurs, a complete loss of activity was observed; the modified enzyme contains 1 mole of carboxymethyl groups per 1 mole of protein. As a result, two monocarboxymethylene forms of the enzyme are formed. In one form, residue His-12 is carboxymethylated, and in the other, His-119 is carboxymethylated. His-119 was predominantly modified.

These data suggested that His-12 and His-119 are located in the active site and that modification of one of them prevents modification of the other.

As a result of X-ray diffraction studies, the spatial structure of RNase S and the RNase S complex with inhibitors was elucidated. The molecule has a kidney shape, the active center is localized in the recess where the residues His-12, His-119 and Lys-41 are located.

Hydrolysis occurs as a result of the conjugate action of residues His-12 and His-119, which carry out acid-base catalysis. The diagram below shows the stages of the catalytic process:

1. The substrate is located in the active site; His-12, His-119 and Lys-41 are located near the negatively charged phosphate.

2. As a result of the action of His-12 as a base that accepts a proton from the 2"-OH group of ribose, and His-119 as an acid that donates a proton to the oxygen atom of the phosphate, first an intermediate complex is formed, and then a 2,3"-cyclic phosphate .

3. In place of the lost product, water enters, donating a proton to His-119 and OH to the phosphate, at the same time the proton from His-12 goes to the oxygen atom of ribose, a second product is formed, and the enzyme returns to its original state.

Chymotrypsin is secreted in the form of a proenzyme - chymotrypsinogen by the pancreas of vertebrates; activation of the proenzyme occurs in the duodenum under the influence of trypsin. The physiological function of chymotrypsin is the hydrolysis of proteins and polypeptides. Chymotrypsin attacks predominantly peptide bonds formed by carboxyl residues of tyrosine, tryptophan, cenylalanine and methionane. It also effectively hydrolyzes esters of the corresponding amino acids. The molecular weight of chymotrypsin is 25,000, the molecule contains 241 amino acid residues. Chymotrypsin is formed by three polypeptide chains that are linked by disulfide bridges.

The functional groups of the active site of chymotrypsin have been identified using irreversible inhibitors. The Ser-195 residue was modified with diisopropyl fluorophosphate and phenylmethyl sulfofluoride, and the His-122 residue was modified with N-tosyl-L-phenylalanine chloromethyl ketone. The two-stage process of chymotrypsin hydrolysis was discovered when studying the kinetics of p-nitrophenylacetate hydrolysis.

A characteristic feature of the process under consideration is the formation of a covalent intermediate - an acyl enzyme. The acylatable catalytic group was identified as residue Ser-195. The mechanism of catalysis carried out by the enzyme was proposed even before the spatial structure of the protein was established, but was later refined. In particular, studies using 18 H 2 O made it possible to prove the formation of an acyl enzyme during the hydrolysis of peptides.

The three-dimensional structure with a resolution of 0.2 nm was established by X-ray diffraction analysis by D. Blow. in 1976 The molecule has the shape of an ellipsoid with axes of 5.4*4*4 nm. The results of crystallographic studies confirmed the assumption that the Ser-195 and His-57 residues are close together. The hydroxyl group of Ser-195 is located at a distance of ∼0.3 nm from the nitrogen atom of the imidazole ring of His-57. The most interesting fact was that the nitrogen atom in position 1 of the ring is located at a distance of ∼0.28 nm from the oxygen atom of the carboxyl group of the side chain of Asp-102 and occupies a position favorable for the formation of a hydrogen bond.

It should be noted that chemical studies could not reveal the participation of Asp-102 in the functioning of the active site, since this residue is buried deep in the molecule.

It is currently believed that the three residues Asp-102, His-57 and Ser-195 form a charge transfer system that plays a crucial role in the catalysis process. The functioning of the system ensures the effective participation of His-57 in catalysis as an acid-base catalyst and increases the reactivity of Ser-195 to the carboxyl carbon of the attacked bond.

The key element of catalysis is the transfer of a proton from Ser-195 to His-57. At the same time, an attack by the serine oxygen atom on the carbonyl carbon atom of the substrate occurs, first forming an intermediate tetrahedral compound (1) and then an acyl enzyme (2). The next step is deacylation. A water molecule enters the charge transfer system, and the OH ion simultaneously attacks the carbonyl carbon atom of the acyl group of the acyl enzyme. As in the acylation step, a tetrahedral intermediate (4) is formed. His-57 then supplies a proton to the oxygen atom of Ser-195, resulting in the release of the acyl product; it diffuses into the solution, and the enzyme returns to its original state.

Carboxypeptidase A is secreted as a proenzyme by the pancreas of vertebrates. The formation of the active enzyme occurs in the small intestine with the participation of chymotrypsin. The enzyme sequentially cleaves off C-terminal amino acid residues from the peptide chain, i.e. is an exopeptidase.

Carboxypeptidase A is formed by a single polypeptide chain containing 307 amino acid residues; molecular weight is 34,470. The amino acid sequence of the protein was established in 1969 by R. Bredschow.

Elucidation of the mechanism of action of the enzyme was possible only after X-ray diffraction studies. The spatial structure of the enzyme and its complex with the Gly-Tyr dipeptide (substrate model) was established by W. Lipscomb. The enzyme molecule has the shape of an ellipsoid with axes of 5.0 * 4.2 * 3.8 nm; the active site is located in a recess that turns into a deep nonpolar pocket. In the zone of the active center, a zinc ion is localized (its ligands are the side chains of residues Glu-72, His196, His-69 and a water molecule), as well as functional groups involved in substrate binding and catalysis - residues Arg-145, Glu-270 and Tyr-248.

A comparative analysis of the structures of the enzyme and its complex with Gly-Tyr provided important information about the structure of the enzyme-substrate complex. In particular, it was found that during complex formation the hydroxyl group of Tyr-248 moves 1.2 nm relative to its position in the free enzyme (i.e., approximately 1/3 of the diameter of the molecule).

According to the scheme of the catalytic process, the carboxylate group of Glu-270 activates a water molecule located in the reaction sphere, withdrawing a proton from it; the resulting OH- ion carries out a nucleophilic attack on the carbonyl carbon of the bond being cleaved. At the same time, the hydroxyl group of Tyr-248, located near the nitrogen atom of the cleaved peptide bond, donates a proton to it. As a result, the attacked peptide bond is cleaved and the resulting products leave the active center zone. The diagram below illustrates general basic catalysis.

Aspartate aminotransferase catalyzes the reversible transamination reaction.

The enzymatic transamination reaction was discovered by A.E. Braunstein and M.G. Kritsman in 1937 when studying an enzyme preparation from pigeon muscle. Subsequent studies showed that transamination reactions are widespread in living nature and play an important role in coupling nitrogen and energy metabolism.

In 1945, it was found that pyridoxal-5"-phosphate (PLP) is a coenzyme of aminotransferases. The AAT molecule is a dimer formed by identical subunits. In the cardiac muscle of the studied vertebrates there are two isoenzymes - cytoplasmic (cAAT0 and mitochondrial (mAAT) aminotransferases.

The primary structure of cAAT from cardiac muscle was established in 1972. Yu.A. Ovchinnikov and A.E. Brainstein. The polypeptide chain of a protein contains 412 amino acid residues; molecular weight is 46,000.

The general theory of pyridoxal catalysis was developed by A.E. Braunstein and M.M. Shemyakin in 1952-1953, and somewhat later - D.E. Metzler and E.E. Snell. According to this theory, the catalytic effect of pyridoxal enzymes is determined by the ability of the aldehyde group of pyridoxal phosphate to form aldimines (Schiff bases) when interacting with amines, including amino acids.

In the resulting phosphopyridoxyldenamino acid there is a system of conjugated double bonds, along which the displacement of electrons from the b-carbon atom facilitates the breaking of bonds formed by this atom.

Modern ideas about the mechanism of enzymatic transamination, developed by A.E. Braunstein and his collaborators are a development of the theory discussed above. In the initial state, the aldehyde group of pyridoxal phosphate forms an aldimine bond with the e-amino group of the Lys-258 residue of the active site (I). When an amino acid binds, a Michaelis complex (II) is formed, followed by an aldimine between the pyridoxal phosphate and the substrate (III). As a result of subsequent transformations through intermediate stages (IV) and (V), oxoacid (VI) is formed. This completes the first half-reaction of transamination. Repeating these same steps in the “reverse direction” with a new hydroxy acid constitutes the second half-reaction, completing the catalytic transamination cycle.

Myoglobin and hemoglobin

These two proteins are often called respiratory enzymes. Their interaction with the substrate - oxygen - has been studied in detail, primarily on the basis of high-resolution X-ray diffraction analysis. The three-dimensional structure of myoglobin was determined by J. Kendrew in 1961, and the three-dimensional structure of hemoglobin by M. Perutz in 1960.

The myoglobin molecule has a compact shape - 4.5 * 3.5 * 2.5 nm, the polypeptide chain forms 8 helical sections, designated by the letters A to H. It is arranged in a specialized manner around a large flat iron-containing heme ring. Heme is a complex of porphyrin with divalent iron.

The polar propionic acid chains of heme are on the surface of the molecule, the rest of the heme is immersed in the globule. The connection of heme with the protein is carried out due to the coordination bond between the iron atom and the histidine atom localized in helix F; this is the so-called proximal histidine. Another important histidine residue, distal histidine, is localized in the heme pocket within helix E; it is located on the opposite side of the iron atom at a greater distance than the proximal histidine. The region between the gene iron and the distal histidine in deoxymyoglobin is free, and the lipophilic O 2 molecule can bind to the heme iron, occupying the sixth coordination position. A unique feature of myoglobin, as well as hemoglobin, is their ability to reversibly bind O 2 without oxidizing heme Fe 2+ to Fe 3+. This is possible because an environment with a low dielectric constant is created in the hydrophobic heme pocket, from which water is displaced.

When O2 binds to the iron atom, the latter moves by approximately 0.06 nm and ends up in the plane of the porphyrin ring, i.e. in an energetically more favorable position. It is believed that this movement is due to the fact that the Fe 2+ ion in deoxymyoglobin is in a high-spin state and its radius is too large to fit in the plane of the heme porphyrin ring. When O 2 binds, the Fe 2+ ion goes into a low-min state and its radius decreases; Now the Fe 2+ ion can move into the plane of the porphyrin ring.

Hemoglobin is the main component of red blood cells, delivering oxygen from the lungs to the tissues, and carbon dioxide from the tissues to the lungs. Hemoglobins of different types differ in crystal shape, solubility, and affinity for oxygen. This is due to differences in the amino acid sequence of proteins; the heme component is the same in hemoglobins of all species of vertebrates and some invertebrates.

Human hemoglobin is a tetramer consisting of four subunits, two b-subunits and two b-subunits, containing 141 and 146 amino acid residues, respectively. There is significant homology between the primary structures of the b and b subunits, and the conformation of their polypeptide chains is also similar.

The hemoglobin molecule has a spherical shape with a diameter of 5.5 nm. The four subunits are packaged in a tetrahedron shape.

X-ray diffraction data showed that oxygenation of hemoglobin is accompanied by a number of changes. At low resolution, it was found that in this case the structure becomes more compact (the Fe atoms of the β-chains come closer to each other by approximately 0.6-0.7 nm), the subunits rotate relative to each other and the second-order axes by 10-15 o. The results of the study at high resolution indicate that particularly significant changes occur in the area of ​​\u200b\u200bthe contacts.

To date, based on X-ray diffraction studies and a number of other methodological approaches, significant progress has been made in elucidating the mechanism of action of enzymes with desired properties based on advances in the field of genetic engineering. This opens up broad opportunities for testing the validity of modern ideas about the mechanism of action of enzymes and creating a fundamental theory of enzymatic catalysis.

The first enzymatic reaction of starch saccharification with malt was investigated by the domestic scientist K. Menten developed the theory of enzymatic catalysis. Sumner was the first to isolate a purified preparation of the urease enzyme in a crystalline state. Merrifield succeeded in artificially synthesizing the enzyme RNase.

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Essay

STRUCTURE, PROPERTIES AND MECHANISM OF ENZYME ACTION

A Brief History of Fermentology

The experimental study of enzymes in the 19th century coincided with the study of yeast fermentation processes, which was reflected in the terms “enzymes” and “enzymes”. The name enzymes comes from the Latin word fermentatio fermentation. The term enzymes comes from the concept en zyme - from yeast. At first these names were given different meanings, but nowadays they are synonymous.

The first enzymatic reaction of starch saccharification with malt was studied by the domestic scientist K.S. Kirchhoff in 1814. Subsequently, attempts were made to isolate enzymes from yeast cells (E. Buchner, 1897). At the beginning of the twentieth century, L. Michaelis and M. Menten developed the theory of enzymatic catalysis. In 1926, D. Sumner first isolated a purified preparation of the urease enzyme in a crystalline state. In 1966, B. Merrifield succeeded in artificially synthesizing the RNase enzyme.

Enzyme structure

Enzymes are highly specialized proteins that can increase the speed of reactions in living organisms. Enzymes are biological catalysts.

All enzymes are proteins, usually globular. They can refer to both simple and complex proteins. The protein part of the enzyme can consist of one polypeptide chain monomeric proteins enzymes (for example, pepsin). A number of enzymes are oligomeric proteins and include several protomers or subunits. Protomers, combining into an oligomeric structure, are connected spontaneously by weak non-covalent bonds. During the process of unification (cooperation), structural changes of individual protomers occur, as a result of which the activity of the enzyme noticeably increases. The separation (dissociation) of protomers and their association into an oligomeric protein is a mechanism for regulating enzyme activity.

Subunits (protomers) in oligomers can be either the same or different in primary - tertiary structure (conformation). When different protomers combine into the oligomeric structure of an enzyme, multiple forms of the same enzyme arise isozymes.

Isoenzymes catalyze the same reaction, but differ in the set of subunits, physicochemical properties, electrophoretic mobility, and affinity for substrates, activators, and inhibitors. For example,lactate dehydrogenase (LDH)the enzyme that oxidizes lactic acid into pyruvic acid is a tetramer. It consists of four protomers of two types. One type of protomer is designated H (isolated from cardiac muscle), the second protomer is designated M (isolated from skeletal muscle). There are 5 possible combinations of these protomers in LDH: N 4, N 3 M, N 2 M 2, N 1 M 3, M 4.

Biological role of isozymes.

• Isoenzymes ensure the occurrence of chemical reactions in accordance with the conditions in different organs. Thus, the LDH isoenzyme 1 has a high affinity for oxygen, so it is active in tissues with a high rate of oxidative reactions (erythrocytes, myocardium). LDH isoenzyme 5 active in the presence of high concentrations of lactate, most characteristic of liver tissue
• Pronounced organ specificity is used to diagnose diseases of various organs.
• Isoenzymes change their activity with age. Thus, in a fetus with a lack of oxygen, LDH predominates 3 , and with increasing age and increasing oxygen supply, the proportion of LDH increases 2 .

If an enzyme is a complex protein, then it consists of a protein and a non-protein part. The protein part is a high molecular weight, thermolabile part of the enzyme and is called apoenzyme . It has a unique structure and determines the specificity of enzymes.

The non-protein part of the enzyme is calledcofactor (coenzyme) . Cofactors are most often metal ions that can bind tightly to the apoenzyme (for example, Zn in the enzyme carbonic anhydrase, C u in the enzyme cytochrome oxidase). Coenzymes are most often organic substances less tightly bound to the apoenzyme. The coenzymes are the nucleotides NAD and FAD. Coenzymelow molecular weight, thermostable part of the enzyme. Its role is that it determines the spatial arrangement (conformation) of the apoenzyme and determines its activity. Cofactors can transfer electrons, functional groups, and participate in the formation of additional bonds between the enzyme and the substrate.

In terms of functionality, it is customary to distinguish two important sections in the enzyme molecule: the active center and the allosteric section.

Active center this is a section of the enzyme molecule that interacts with the substrate and participates in the catalytic process. The active site of the enzyme is formed by amino acid radicals that are distant from each other in the primary structure. The active center has a three-dimensional arrangement; most often it contains

OH groups of serine

SHcysteine

NH 2 lysine

- γ -COOH of glutamic acid

In the active center, two zones are distinguished: the substrate binding zone and the catalytic zone.

Bonding area usually has a rigid structure to which the reaction substrate is complementarily attached. For example, trypsin cleaves proteins in areas rich in the positively charged amino acid lysine, since its binding zone contains residues of negatively charged aspartic acid.

Catalytic zone -This is a region of the active center that directly affects the substrate and performs a catalytic function. This zone is more mobile; the relative position of functional groups can change in it.

In a number of enzymes (usually oligomeric), in addition to the active center, there isallosteric sitea section of the enzyme molecule that is distant from the active center and interacts not with the substrate, but with additional substances (regulators, effectors). In allosteric enzymes, one subunit may contain the active center, and the other - the allosteric site. Allosteric enzymes change their activity as follows: an effector (activator, inhibitor) acts on the allosteric subunit and changes its structure. Then, a change in the conformation of the allosteric subunit, according to the principle of cooperative changes, indirectly changes the structure of the catalytic subunit, which is accompanied by a change in enzyme activity.

Mechanism of action of enzymes.

Enzymes have a number of general catalytic properties:

• do not shift the catalytic equilibrium
• are not consumed during the reaction
• catalyze only thermodynamically real reactions. Such reactions are those in which the initial energy reserve of the molecules is greater than the final one.

During the reaction, a high energy barrier is overcome. The difference between the energy of this threshold and the initial energy level is the activation energy.

The rate of enzymatic reactions is determined by the activation energy and a number of other factors.

The rate constant of a chemical reaction is determined by the equation:

K = P*Z*e - (Ea / RT)

K - reaction rate constant

P spatial (steric) coefficient

Z number of interacting molecules

E a activation energy

R gas constant

T universal absolute temperature

e base of natural logarithms

In this equation Z, e, R, T constant values, and P and Ea are variables. Moreover, there is a direct relationship between the reaction rate and the steric coefficient, and an inverse and power-law relationship between the rate and activation energy (the lower Ea, the higher the reaction rate).

The mechanism of action of enzymes is reduced to an increase in the steric coefficient by enzymes and a decrease in activation energy.

Reduction of activation energy by enzymes.

For example, the splitting energy H 2 O 2 without enzymes and catalysts 18,000 kcal per mole. If platinum and high temperature are used, it is reduced to 12,000 kcal/mol. With the participation of an enzyme catalase the activation energy is only 2,000 kcal/mol.

A decrease in Ea occurs as a result of the formation of intermediate enzyme-substrate complexes according to the following scheme: F+S<=>FS complex → F + reaction products.The possibility of forming enzyme-substrate complexes was first proven by Michaelis and Menten. Subsequently, many enzyme-substrate complexes were isolated. To explain the high selectivity of enzymes when interacting with a substrate, it was proposedFisher's "key and lock" theory. According to it, the enzyme interacts with the substrate only if they are in absolute agreement with each other (complementarity), like a key and a lock. This theory explained the specificity of enzymes, but did not reveal the mechanisms of their action on the substrate. Later, the theory of induced correspondence between enzyme and substrate was developed - Koshland theory (the “rubber glove” theory). Its essence is as follows: the active center of the enzyme is formed and contains all functional groups even before interaction with the substrate. However, these functional groups are in an inactive state. At the moment of attachment of the substrate, it induces changes in the position and structure of radicals in the active center of the enzyme. As a result, the active center of the enzyme, under the influence of the substrate, enters an active state and, in turn, begins to affect the substrate, i.e. is happening interaction active site of the enzyme and substrate. As a result, the substrate goes into an unstable, unstable state, which leads to a decrease in activation energy.

The interaction between enzyme and substrate can involve reactions of nucleophilic substitution, electrophilic substitution, and dehydration of the substrate. Short-term covalent interaction of the functional groups of the enzyme with the substrate is also possible. Basically, a geometric reorientation of the functional groups of the active site occurs.

Increase in steric coefficient by enzymes.

The steric coefficient is introduced for reactions that involve large molecules that have a spatial structure. The steric coefficient shows the proportion of successful collisions between active molecules. For example, it is equal to 0.4 if 4 out of 10 collisions of active molecules resulted in the formation of a reaction product.

Enzymes increase the steric coefficient because they change the structure of the substrate molecule in the enzyme-substrate complex, as a result of which the complementarity of the enzyme and substrate increases. In addition, enzymes, due to their active centers, order the arrangement of substrate molecules in space (before interaction with the enzyme, the substrate molecules are located chaotically) and facilitate the reaction.

Enzyme nomenclature

Enzymes have several types of names.

1. Trivial names (trypsin, pepsin)
2. Working nomenclature. This enzyme name contains the ending aza, which is added:
   • to the name of the substrate (sucrase, amylase),
   • to the type of bond on which the enzyme acts (peptidase, glycosidase),
   • to the type of reaction, process (synthetase, hydrolase).

3) Each enzyme has a classification name, which reflects the type of reaction, type of substrate and coenzyme. For example: LDH L lactate-NAD+ - oxidoreductase.

Classification of enzymes.

The classification of enzymes was developed in 1961. According to the classification, each enzyme is located in a certain class, subclass, subsubclass and has a serial number. In this regard, each enzyme has a digital code in which the first digit indicates the class, the second subclass, the third subclass, the fourth serial number (LDG: 1,1,1,27). All enzymes are classified into 6 classes.

1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases
5. Isomerases
6. Synthetases (ligases)

Oxidoreductases.

Enzymes that catalyze redox processes. General type of reaction: A ok + B sun = A east + B ok . This class of enzymes includes several subclasses:

1. Dehydrogenases, catalyze reactions by removing hydrogen from the substance being oxidized. They can be aerobic (transfer hydrogen to oxygen) and anaerobic (transfer hydrogen not to oxygen, but to some other substance).

2. Oxygenases - enzymes that catalyze oxidation by adding oxygen to the substance being oxidized. If one oxygen atom is added, monooxygenases are involved, if two oxygen atoms are added, dioxygenases are involved.

3. Peroxidases enzymes that catalyze the oxidation of substances involving peroxides.

Transferases.

Enzymes that carry out intramolecular and intermolecular transfer of functional groups from one substance to another according to the scheme: AB + C = A + BC. Subclasses of transferases are distinguished depending on the type of transferred groups: aminotransferases, methyltransferases, sulfotransferases, acyltransferases (transfer fatty acid residues), phosphotransferases (transfer phosphoric acid residues).

Hydrolases.

Enzymes of this class catalyze the breaking of a chemical bond with the addition of water at the site of the break, that is, the hydrolysis reaction according to the scheme: AB + HOH = AN + BOH. Subclasses of hydrolases are distinguished depending on the type of bonds being broken: peptidases break down peptide bonds (pepsin), glycosidases break down glycosidic bonds (amylase), esterases break down ester bonds (lipase).

Lyases.

Lyases catalyze the breaking of a chemical bond without adding water at the site of the break. In this case, double bonds are formed in the substrates according to the scheme: AB = A + B. Subclasses of lyases depend on which atoms the bond is broken between and which substances are formed. Aldolases break the bond between two carbon atoms (for example, fructose 1,6-di-phosphate aldolase “cuts” fructose and two trioses). Lyases include decarboxylase enzymes (they remove carbon dioxide), while dehydratases “cut out” water molecules.

Isomerases.

Isomerases catalyze the interconversions of different isomers. For example, phosphohexoimerase converts fructose to glucose. Subclasses of isomerases include mutases (phosphoglucomutase converts glucose-1-phosphate to glucose-6-phosphate), epimerases (for example, converts ribose to xylulose), tautomerases

Synthetases (ligases).

Enzymes of this class catalyze reactions for the synthesis of new substances using the energy of ATP according to the scheme: A+B+ATP = AB. For example, glutamine synthetase combines glutamic acid, NH 3 + with the participation of ATP with the formation of glutamine.

Properties of enzymes.

Enzymes, in addition to properties common to inorganic catalysts, have certain differences from inorganic catalysts. These include:

• higher activity
• higher specificity
• milder conditions for catalysis
• ability to regulate activity

High catalytic activity of enzymes.

Enzymes are characterized by high catalytic activity. For example, one molecule of carbonic anhydrase catalyzes the formation (or breakdown) of 36 million molecules of carbonic acid (H 2 CO 3 ). The high activity of enzymes is explained by the mechanism of their action: they reduce the activation energy and increase the spatial (steric coefficient). High enzyme activity has an important biological significance in that it ensures a high rate of chemical reactions in the body.

High enzyme specificity.

All enzymes have specificity, but the degree of specificity varies from enzyme to enzyme. There are several types of enzyme specificity.

Absolute substrate specificity, in which the enzyme acts only on one specific substance. For example, the enzyme urease breaks down only urea.

Absolute groupspecificity, in which an enzyme has the same catalytic effect on a group of compounds that are similar in structure. For example, the enzyme alcohol dehydrogenase oxidizes not only C 2 N 5 OH, but also its homologues (methyl, butyl and other alcohols).

Relative groupspecificity in which the enzyme catalyzes different classes of organic substances. For example, the enzyme trypsin exhibits peptidase and esterase activity.

Stereochemicalspecificity (optical specificity), in which only a certain form of isomers is cleaved ( D, L forms, α, β, cis - trans isomers). For example, LDH only acts on L-lactate, L -amino acid oxidases act on L -isomers of amino acids.

High specificity is explained by the unique structure of the active center for each enzyme.

Thermolability of enzymes.

Thermolability is the dependence of enzyme activity on temperature. When the temperature rises from 0 to 40 degrees, enzyme activity increases according to Van't Hoff's rule (with an increase in temperature by 10 degrees, the reaction rate increases by 2 4 times). With a further increase in temperature, the activity of enzymes begins to decrease, which is explained by thermal denaturation of the protein molecule of the enzyme. Graphically, the temperature dependence of enzymes has the form:

Inactivation of the enzyme at 0 degrees is reversible, and at high temperatures the inactivation becomes irreversible. This property of enzymes determines the maximum reaction rate under human body temperature conditions. The thermolability of enzymes must be taken into account in practical medical practice. For example, when carrying out an enzymatic reaction in a test tube, it is necessary to create an optimal temperature. This property of enzymes can be used in cryosurgery, when a complex long-term operation is performed with a decrease in body temperature, which slows down the rate of reactions occurring in the body and reduces oxygen consumption by tissues. Enzyme preparations must be stored at low temperatures. To neutralize and disinfect microorganisms, high temperatures are used (autoclaving, boiling of instruments).

Photolability.

Photolability is the dependence of enzyme activity on the action of ultraviolet rays. UV rays cause photodenaturation of protein molecules and reduce enzyme activity. This property of enzymes is used in the bactericidal effect of ultraviolet lamps.

Dependence of activity on pH.

All enzymes have a certain pH range in which enzyme activity is maximum - pH optimum. For many enzymes, the optimum is about 7. At the same time, for pepsin the optimal environment is 1-2, for alkaline phosphatase it is about 9. When the pH deviates from the optimum, the activity of the enzyme decreases, as can be seen from the graph. This property of enzymes is explained by a change in the ionization of ionogenic groups in enzyme molecules, which leads to a change in ionic bonds in the protein molecule of the enzyme. This is accompanied by a change in the conformation of the enzyme molecule, and this, in turn, leads to a change in its activity. Under the body's conditions, pH-dependence determines the maximum activity of enzymes. This property also finds practical application. Enzymatic reactions outside the body are carried out at an optimum pH. In case of reduced acidity of gastric juice, a solution of NS is prescribed for therapeutic purposes. l.

Dependence of the rate of enzymatic reaction on the concentration of the enzyme and the concentration of the substrate

The dependence of the reaction rate on the enzyme concentration and substrate concentration (kinetics of enzymatic reactions) is presented in the graphs.

Schedule 1 schedule 2

In an enzymatic reaction ( F + S 2  1 FS → 3 F + P ) The speeds of three component stages are distinguished:

1- formation of an enzyme-substrate complex F.S.

2- reverse decomposition of the enzyme substrate complex,

3 decomposition of the enzyme-substrate complex with the formation of reaction products. The rate of each of these reactions obeys the law of mass action:

V 1 = K 1 [F]* [S]

V 2 = K 2 * [FS]

V 3 = K 3 *[ FS ]

At the moment of equilibrium, the rate of formation reaction FS equal to the sum of its decay rates: V 1 = V 2 + V 3. Of the three stages of an enzymatic reaction, the third is the most important and slowest., since it is associated with the formation of reaction products. Using the above formula, find the speed V 3 impossible, since the enzyme-substrate complex is very unstable, measuring its concentration is difficult. In this regard, Michaelis-Menten introduced K m Michaelis constant and transformed the equation to measure V 3 into a new equation that contains actually measurable quantities:

V 3 = K 3 * [ F 0 ] * [S] / Km + [S] or V 3 =V max * [S] / Km+[S]

[F0] initial enzyme concentration

K m Michaelis constant.

Physical meaning of K m: K m = (K 2 + K 3) / K 1 . It shows the ratio of the rate constants for the decomposition of the enzyme-substrate complex and the rate constant for its formation.

The Michaelis-Menten equation is universal. It illustrates the dependence of the reaction rate on [ F 0 ] from [ S ]

1. Dependence of reaction rate on substrate concentration.This dependence is revealed at low substrate concentrations [ S]< Km . In this case, the substrate concentration in the equation can be neglected and the equation takes the form: V 3 = K 3* [F 0] * [S] / Km. In this equation K 3 , F 0 ], Km constants and can be replaced by a new constant K*. Thus, at a low substrate concentration, the reaction rate is directly proportional to this concentration V 3 = K * * [ S ]. This dependence corresponds to the first section of graph 2.
2. Dependence of speed on enzyme concentrationappears at high substrate concentrations. S ≥ Km . In this case we can neglect Km and the equation becomes the following: V 3 = K 3* (([ F 0 ] * [ S ]) / [ S ]) = K 3 * [ F 0 ] = V max . Thus, at high substrate concentrations, the reaction rate is determined by the enzyme concentration and reaches its maximum value V 3 = K 3 [ F 0 ] = V max . (third section of graph 2).
3. Allows you to determine a numerical value Km provided V 3 = V max /2. In this case, the equation takes the form:

V max /2 = ((V max *[ S ])/ Km +[ S ]), whence it follows that Km =[ S ]

Thus, K m is numerically equal to the substrate concentration at a reaction rate equal to half the maximum. TO m is a very important characteristic of an enzyme, it is measured in moles (10-2 10 -6 mol) and characterize the specificity of the enzyme: the lower Km , the higher the specificity of the enzyme.

Graphical definition of the Michaelis constant.

It is more convenient to use a graph that represents a straight line. Such a graph was proposed by Lineweaver Burke (graph of double reciprocals), which corresponds to the inverse Michaelis-Menten equation

Dependence of the rate of enzymatic reactions on the presence of activators and inhibitors.

Activators substances that increase the rate of enzymatic reactions. There are specific activators that increase the activity of one enzyme (NS l - pepsinogen activator) and nonspecific activators that increase the activity of a number of enzymes (ions Mg hexokinase activators, K, Na ATPase and other enzymes). Metal ions, metabolites, and nucleotides can serve as activators.

Mechanism of action of activators.

1. Completion of the active center of the enzyme, as a result of which the interaction of the enzyme with the substrate is facilitated. This mechanism occurs mainly in metal ions.
2. An allosteric activator interacts with the allosteric site (subunit) of the enzyme, through its changes indirectly changes the structure of the active center and increases the activity of the enzyme. Metabolites of enzymatic reactions, ATP, have an allosteric effect.
3. The allosteric mechanism can be combined with a change in the oligomericity of the enzyme. Under the influence of the activator, several subunits are combined into an oligomeric form, which sharply increases the activity of the enzyme. For example, isocitrate is an activator of the enzyme acetyl-CoA carboxylase.
4. Phospholylation - dephosphorylation of enzymes refers to the reversible modification of enzymes. Connection H 3 RO 4 most often sharply increases the activity of the enzyme. For example, two inactive dimers of the enzyme phosphorylase combine with four molecules of ATP to form the active tetrameric phosphorylated form of the enzyme. Phospholylation of enzymes can be combined with a change in their oligomerity. In some cases, phosphorylation of an enzyme, on the contrary, reduces its activity (for example, phosphorylation of the enzyme glycogen synthetase)
5. Partial proteolysis (irreversible modification). With this mechanism, a fragment of the molecule is split off from the inactive form of the enzyme (proenzyme), blocking the active center of the enzyme. For example, inactive pepsinogen under the influence HCL turns into active pepsin.

Inhibitors substances that reduce enzyme activity.

By specificitydistinguish specific and nonspecific inhibitors

By reversibility effect, a distinction is made between reversible and irreversible inhibitors.

By locationThere are inhibitors acting on the active center and outside the active center.

By mechanism of actiondistinguished into competitive and non-competitive inhibitors.

Competitive inhibition.

Inhibitors of this type have a structure close to the structure of the substrate. Because of this, inhibitors and substrate compete for binding to the active site of the enzyme. Competitive inhibition is reversible inhibition. The effect of a competitive inhibitor can be reduced by increasing the concentration of the reaction substrate.

An example of competitive inhibition is the inhibition of the activity of succinate dehydrogenase, which catalyzes the oxidation of dicarboxylic succinic acid, by dicarboxylic malonic acid, which is similar in structure to succinic acid.

The principle of competitive inhibition is widely used in the development of drugs. For example, sulfonamide drugs have a structure close to that of para-aminobenzoic acid, which is necessary for the growth of microorganisms. Sulfonamides block microbial enzymes necessary for the absorption of para-aminobenzoic acid. Some anticancer drugs are analogues of nitrogenous bases and thereby inhibit the synthesis of nucleic acids (fluorouracil).

Graphically, competitive inhibition has the form:

Non-competitive inhibition.

Noncompetitive inhibitors are not structurally similar to the reaction substrates and therefore cannot be displaced at high substrate concentrations. There are several options for the action of non-competitive inhibitors:

1. Blocking the functional group of the active center of the enzyme and, as a result, reducing activity. For example, activity S H - groups can bind thiol poisons reversibly (metal salts, mercury, lead) and irreversibly (moniodoacetate). The inhibitory effect of thiol inhibitors can be reduced by the introduction of additives rich in SH groups (for example, unithiol). Serine inhibitors that block the OH groups of the active center of enzymes are found and used. Organic phosphofluorine-containing substances have this effect. These substances can, in particular, inhibit OH groups in the enzyme acetylcholinesterase, which destroys the neurotransmitter acetylcholine.
2. Blocking of metal ions that are part of the active site of enzymes. For example, cyanides block iron atoms, EDTA (ethylenediaminetetraacetate) blocks Ca ions, Mg.
3. An allosteric inhibitor interacts with the allosteric site, indirectly through it according to the principle of cooperativity, changing the structure and activity of the catalytic site. Graphically, non-competitive inhibition has the form:

The maximum reaction rate in noncompetitive inhibition cannot be achieved by increasing the substrate concentration.

Regulation of enzyme activity during metabolism.

Adaptation of the body to changing conditions (diet, environmental influences, etc.) is possible due to changes in enzyme activity. There are several possibilities for regulating the rate of enzyme reactions in the body:

1. Changing the rate of enzyme synthesis (this mechanism requires a long period of time).
2. Increasing substrate and enzyme availability by changing the permeability of cell membranes.
3. Changing the activity of enzymes already present in cells and tissues. This mechanism occurs at high speed and is reversible.

In multi-stage enzymatic processes,regulatory, keyenzymes that limit the overall rate of the process. Most often these are enzymes of the initial and final stages of the process. Changes in the activity of key enzymes occur through various mechanisms.

1. Allosteric mechanism:

1. Change in enzyme oligomerity:

Monomers are not active ↔ oligomers are active

1. Phospholylation - dephosphorylation:

Enzyme (inactive) + H 3 RO 4 ↔ phosphorylated active enzyme.

The autoregulatory mechanism is widespread in cells. The autoregulatory mechanism is, in particular, retroinhibition, in which the products of the enzymatic process inhibit the enzymes of the initial stages. For example, high concentrations of purine and pyrimidine nucleotides inhibit the initial stages of their synthesis.

Sometimes the initial substrates activate the final enzymes, in the diagram: substrate A activates F 3 . For example, the active form of glucose (glucose-6-phosphate) activates the final enzyme in the synthesis of glycogen from glucose (glycogen synthetase).

Structural organization of enzymes in the cell

The coherence of metabolic processes in the body is possible due to the structural unity of enzymes in cells. Individual enzymes are located in certain intracellular structurescompartmentalization.For example, the enzyme potassium - sodium ATPase - is active in the plasma membrane. Enzymes of oxidative reactions (succinate dehydrogenase, cytochrome oxidase) are active in mitochondria. Enzymes for the synthesis of nucleic acids (DNA polymerase) are active in the nucleus. Enzymes that break down various substances (RNAase, phosphatase, and others) are active in lysosomes.

The enzymes that are most active in a given cellular structure are called indicator or marker enzymes. Their definition in clinical practice reflects the depth of structural tissue damage. Some enzymes are combined into multienzyme complexes, for example, the pyruvate dehydrogenase complex (PDC), which carries out the oxidation of pyruvic acid.

Principles of enzyme detection and quantitation:

Detection of enzymes is based on their high specificity. Enzymes are identified by the action they produce, i.e. based on the occurrence of the reaction that this enzyme catalyzes. For example, amylase is detected by the reaction that breaks down starch into glucose.

Criteria for the occurrence of an enzymatic reaction can be:

• disappearance of the reaction substrate
• appearance of reaction products
• change in the optical properties of the coenzyme.

Enzyme quantification

Since the concentration of enzymes in cells is very low, their true concentration is not determined, but the amount of enzyme is judged indirectly, by the activity of the enzyme.

Enzyme activity is assessed by the rate of the enzymatic reaction occurring under optimal conditions (optimum temperature, pH, excessively high substrate concentration). Under these conditions, the reaction rate is directly proportional to the enzyme concentration ( V = K 3 [F 0 ]).

Units of enzyme activity (amount)

In clinical practice, several units of enzyme activity are used.

1. International unit is the amount of enzyme that catalyzes the conversion of 1 micromole of substrate per minute at a temperature of 25 0 C.
   1. Catal (in SI system) is the amount of enzyme that catalyzes the conversion of 1 mole of substrate per second.
   2. Specific activity is the ratio of enzyme activity to the mass of enzyme protein.
   3. The molecular activity of an enzyme shows how many molecules of substrate are converted under the action of 1 molecule of enzyme.

Clinical enzymology

The application of information about enzymes in medical practice is a branch of medical enzymology. It includes 3 sections:

1. Enzymodiagnostics
   1. Enzymopotology
      1. Enzyme therapy

Enzymodiagnosticssection exploring the possibilities of studying enzyme activity for diagnosing diseases. To assess damage to individual tissues, organ-specific enzymes and isoenzymes are used.

In pediatric practice, when carrying out enzyme diagnostics, it is necessary to take into account children's characteristics. In children, the activity of some enzymes is higher than in adults. For example, high LDH activity reflects the predominance of anaerobic processes in the early postnatal period. The content of transaminases in the blood plasma of children is increased as a result of increased vascular-tissue permeability. Glucose-6-phosphate dehydrogenase activity is increased as a result of increased breakdown of red blood cells. The activity of other enzymes, on the contrary, is lower than in adults. For example, the activity of pepsin and pancreatic enzymes (lipase, amylase) is reduced due to the immaturity of secretory cells.

With age, redistribution of individual isoenzymes is possible. Thus, LDH predominates in children 3 (more anaerobic form), and in adults LDH 2 (more aerobic form).

Enzymopathologya branch of enzymology that studies diseases, the leading mechanism of development of which is a violation of enzyme activity. These include metabolic disorders of carbohydrates (galactosemia, glycogenosis, mucopolysaccharidosis), amino acids (phenylketonuria, cystinuria), nucleotides (orotataciduria), porphyrins (porphyria).

Enzyme therapy a section of enzymology that studies the use of enzymes, coenzymes, activators, and inhibitors for medicinal purposes. Enzymes can be used for replacement purposes (pepsin, pancreatic enzymes), for lytic purposes to remove necrotic masses, blood clots, and to liquefy viscous exudates.

Literature

1. Avdeeva, L.V. Biochemistry: Textbook / L.V. Avdeeva, T.L. Aleynikova, L.E. Andrianova; Ed. E.S. Severin. - M.: GEOTAR-MED, 2013. - 768 p.

2. Auerman, T.L. Fundamentals of biochemistry: Textbook / T.L. Auerman, T.G. Generalova, G.M. Suslyanok. - M.: NIC INFRA-M, 2013. - 400 p.

3. Bazarnova, Yu.G. Biochemical principles of processing and storage of raw materials of animal origin: Textbook / Yu.G. Bazarnova, T.E. Burova, V.I. Marchenko. - St. Petersburg: Prosp. Sciences, 2011. - 192 p.

4. Baishev, I.M. Biochemistry. Test questions: Textbook / D.M. Zubairov, I.M. Baishev, R.F. Baykeev; Ed. D.M. Zubairov. - M.: GEOTAR-Media, 2008. - 960 p.

5. Bokut, S.B. Biochemistry of phylogenesis and ontogenesis: Textbook / A.A. Chirkin, E.O. Danchenko, S.B. Bokut; Under general ed. A.A. Chirkin. - M.: NIC INFRA-M, Nov. knowledge, 2012. - 288 p.

6. Gidranovich, V.I. Biochemistry: Textbook / V.I. Gidranovich, A.V. Gidranovich. - Mn.: TetraSystems, 2012. - 528 p.

7. Goloshchapov, A.P. Genetic and biochemical aspects of human adaptation to the conditions of a city with a developed chemical industry / A.P. Goloshchapov. - M.: KMK, 2012. - 103 p.

8. Gunkova, P.I. Biochemistry of milk and dairy products / K.K. Gorbatova, P.I. Gunkova; Under general ed. K.K. Gorbatova. - St. Petersburg: GIORD, 2010. - 336 p.

9. Dimitriev, A.D. Biochemistry: Textbook / A.D. Dimitriev, E.D. Ambrosieva. - M.: Dashkov and K, 2013. - 168 p.

10. Ershov, Yu.A. General biochemistry and sports: Textbook / Yu.A. Ershov. - M.: MSU, 2010. - 368 p.

11. Ershov, Yu.A. Fundamentals of biochemistry for engineers: Textbook / Yu.A. Ershov, N.I. Zaitseva; Ed. S.I. Shchukin. - M.: MSTU im. Bauman, 2010. - 359 p.

12. Kamyshnikov, V.S. Handbook of clinical and biochemical laboratory diagnostics: In 2 volumes. In 2 volumes. Handbook of clinical and biochemical laboratory diagnostics: In 2 volumes / V.S. Kamyshnikov. - Mn.: Belarus, 2012. - 958 p.

13. Klopov, M.I. Biologically active substances in physiological and biochemical processes in the animal’s body: Textbook / M.I. Klopov, V.I. Maksimov. - St. Petersburg: Lan, 2012. - 448 p.

14. Mikhailov, S.S. Sports biochemistry: Textbook for universities and colleges of physical education / S.S. Mikhailov. - M.: Sov. sport, 2012. - 348 p.

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	As a result of the thesis, the reaction of volutin granules to geoheliophysical influences was studied. Graphs were obtained expressing the dependence of the type of metachromasia reaction on various heliophysical factors. Observation of the 3rd type of metachromasia reaction often occurs 2-3 days after the maxima of the Ap and Kp indices of geomagnetic disturbance. Pearson correlation data was obtained
3643.		Operating principles angle. law in space	2.96 KB
	This is a question of determining the territory in which KM is applied. A person who has committed a crime on the territory of the Russian Federation is subject to criminal liability. Citizens of the Russian Federation and stateless persons permanently residing in the Russian Federation who have committed a crime outside the Russian Federation are subject to criminal justice under the Criminal Code if the act they committed is recognized as a crime in the state on the territory of which it was committed and if these persons were not convicted in a foreign state. When convicting these persons, the punishment cannot exceed the upper limit of the sanction provided for by the law of the foreign state in the territory where the crime was committed.
17448.		Study of batch vegetable peeling machine MOK-250	364.1 KB
	Food is one of the foundations in people’s lives; as a source of energy for the functioning of the body, a person should eat from 1 to 5 times a day. A complete food, her diet contains all the essential elements of food, these are the elements that food must include in order to ensure the normal functioning of the human body. The Life Safety section pays special attention to safety precautions and all measures taken to ensure that work on the machine under study causes as little harm and danger as possible...

Until recently, it was believed that absolutely all enzymes are substances of protein nature. But in the 80s, catalytic activity was discovered in some low-molecular RNAs. These enzymes were named ribozymes. The rest, over 2000 currently known enzymes, are protein in nature and are characterized by all the properties of proteins.

According to their structure, enzymes are divided into:

1.simple or one-component;

2. complex or two-component (holoenzymes).

Simple enzymes are simple proteins and, when hydrolyzed, break down into only amino acids. Simple enzymes include hydrolytic enzymes (pepsin, trypsin, urease, etc.).

Complex proteins are complex proteins and, in addition to polypeptide chains, contain a non-protein component ( cofactor). Most enzymes are complex proteins. The protein part of a two-component enzyme is called apoenzyme. Cofactors may have varying strengths of binding to the apoenzyme. If a cofactor is tightly bound to a polypeptide chain, it is called prosthetic group. There is a covalent bond between the prosthetic group and the apoenzyme.

If a cofactor is easily separated from the apoenzyme and is capable of independent existence, then such a cofactor is called coenzyme.

The connections between the apoenzyme and the coenzyme are weak - hydrogen, electrostatic, etc.

The chemical nature of cofactors is extremely diverse. The role of cofactors in two-component enzymes is played by:

1 – most vitamins (E, K, Q, C, H, B1, B2, B6, B12, etc.);

2 compounds of nucleotide nature (NAD, NADP, ATP, CoA, FAD, FMN), as well as a number of other compounds;

3 – lipolic acid;

4 – many divalent metals (Mg 2+, Mn 2+, Ca 2+, etc.).

Active site of enzymes.

Enzymes are high-molecular substances, the molecular weight of which reaches several million. The molecules of substrates that interact with enzymes usually have a much smaller size. Therefore, it is natural to assume that not the entire enzyme molecule as a whole interacts with the substrate, but only some part of it - the so-called “active center” of the enzyme.

The active center of an enzyme is a part of its molecule that directly interacts with substrates and participates in the act of catalysis.

The active center of the enzyme is formed at the level of the tertiary structure. Therefore, during denaturation, when the tertiary structure is disrupted, the enzyme loses its catalytic activity!

The active center in turn consists of:

1. catalytic center, which carries out the chemical transformation of the substrate;

2.substrate center (“anchor” or contact area), which ensures the attachment of the substrate to the enzyme, the formation of an enzyme-substrate complex.

It is not always possible to draw a clear line between the catalytic and substrate centers; in some enzymes they coincide or overlap.

In addition to the active center, the enzyme molecule contains a so-called allosteric center. This is a section of the enzyme molecule, as a result of the addition of a certain low-molecular substance (effector) to which the tertiary structure of the enzyme changes. This leads to a change in the configuration of the active site and, consequently, to a change in the activity of the enzyme. This is a phenomenon of allosteric regulation of enzyme activity.

Many enzymes are multimers (or oligomers), i.e. consist of two or more protomer subunits (similar to the quaternary structure of a protein).

The bonds between subunits are generally non-covalent. The enzyme exhibits maximum catalytic activity in the form of a multimer. Dissociation into protomers sharply reduces enzyme activity.

Enzymes - multimers usually contain a clear number of subunits (2-4), i.e. are di- and tetramers. Although hexa- and octamers (6-8) are known, trimers and pentamers (3-5) are extremely rare.

Multimer enzymes can be constructed from either the same or different subunits.

If multimer enzymes are formed from different types of subunits, they can exist as several isomers. Multiple forms of an enzyme are called isoenzymes (isoenzymes or isozymes).

For example, an enzyme consists of 4 subunits of types A and B. It can form 5 isomers: AAAA, AAAB, AABB, ABBB, BBBB. These isomeric enzymes are isoenzymes.

Isoenzymes catalyze the same chemical reaction, usually act on the same substrate, but differ in some physicochemical properties (molecular weight, amino acid composition, electrophoretic mobility, etc.), and localization in organs and tissues.

A special group of enzymes consists of the so-called. multimeric complexes. These are systems of enzymes that catalyze successive stages of the transformation of a substrate. Such systems are characterized by strong bonds and strict spatial organization of enzymes, which ensures a minimum path through the substrate and a maximum rate of its conversion.

An example is a multienzyme complex that carries out the oxidative decarboxylation of pyruvic acid. The complex consists of 3 types of enzymes (M.v. = 4,500,000).

Mechanism of action of enzymes

The mechanism of action of enzymes is as follows. When a substrate combines with an enzyme, an unstable enzyme-substrate complex is formed. It activates the substrate molecule due to:

1. polarization of chemical bonds in the substrate molecule and redistribution of electron density;

2. deformation of bonds involved in the reaction;

3. approach and necessary mutual orientation of substrate molecules (S).

The substrate molecule is fixed in the active center of the enzyme in a stressed configuration, in a deformed state, which leads to a weakening of the strength of chemical bonds and reduces the level of the energy barrier, i.e. the substrate is activated.

There are 4 stages in the enzymatic reaction process:

1 – attachment of a substrate molecule to the enzyme and formation of an enzyme-substrate complex;

2 – change in the substrate under the action of an enzyme, making it available for a chemical reaction, i.e. substrate activation;

3 – chemical reaction;

4 – separation of reaction products from the enzyme.

This can be written as a diagram:

E + SESES* EPE + P

where: E – enzyme, S – substrate, S* – activated substrate, P – reaction product.

At the 1st stage, that part of the substrate molecule that does not undergo chemical transformations is attached to the substrate center using weak interactions. For the formation of an enzyme-substrate complex (ES), three conditions must be met, which determine the high specificity of the enzyme action.

Conditions for the formation of the enzyme-substrate complex:

1.structural compliance between the substrate and the active site of the enzyme. As Fischer puts it, they must fit together “like a key to a lock.” This similarity is ensured at the level of the tertiary structure of the enzyme, i.e. spatial arrangement of functional groups of the active center.

2.electrostatic compliance the active center of the enzyme and the substrate, which is caused by the interaction of oppositely charged groups.

3. flexibility of the tertiary structure of the enzyme - “induced compliance”. According to the theory of forced or induced conformity, the catalytically active configuration of the enzyme molecule can arise only at the moment of attachment of the substrate as a result of its deforming effect according to the “hand-glove” principle.

The mechanism of action of one-component and two-component enzymes is similar.

Both apoenzyme and coenzyme take part in the formation of the enzyme-substrate complex in complex enzymes. In this case, the substrate center is usually located on the apoenzyme, and the coenzyme takes part directly in the act of chemical transformation of the substrate. At the last stage of the reaction, the apoenzyme and coenzyme are released unchanged.

At stages 2 and 3, the transformation of the substrate molecule is associated with the breaking and closing of covalent bonds.

After chemical reactions have occurred, the enzyme returns to its original state and the reaction products are separated.

The ability of an enzyme to catalyze a specific type of reaction is called specificity.

There are three types of specificity:

1.relative or group specificity– the enzyme acts on a certain type of chemical bond (for example, the enzyme pepsin cleaves a peptide bond);

2.absolute specificity - the enzyme acts only on one strictly defined substrate (for example, the enzyme urease cleaves the amide bond only in urea);

3.stoichiometric specificity– the enzyme acts only on one of the stereoisomers (for example, the enzyme glucosidase ferments only D-glucose, but does not act on L-glucose).

The specificity of the enzyme ensures the orderliness of metabolic reactions.