Enzyme inhibitors are reversible and irreversible. B

Orenburg – 2010

1.1 Reversible inhibition

1.1.2 Non-competitive inhibition

1.1.3 Non-competitive inhibition

1.2 Irreversible inhibition

1.3 Allosteric inhibition

2. A new type of enzyme activity inhibition

3. Use of enzyme inhibitors

CONCLUSION

List of used literature

1. Enzyme inhibitors. Types of enzyme activity inhibition

It is known that enzyme activity can be reduced relatively easily using a variety of influences. This reduction in the rate of enzymatic reactions is usually called inhibition of activity, or inhibition of enzymes.

Fig. 1. Scheme of activation and inhibition of the action of the enzyme (according to Yu. B. Filippovich): a. – allosteric center of the enzyme; K - catalytic center; c - substrate center

Enzymes are proteins; accordingly, their activity can be reduced or completely eliminated by effects leading to denaturation of proteins (heating, the action of concentrated acids, alkalis, salts of heavy metals, etc.) This is a nonspecific suppression of enzyme activity, which is important in the study of enzymatic reactions, is not of particular interest for studying their mechanism. Much more important is the study of inhibition using substances that specifically and usually in small quantities interact with enzymes - enzyme inhibitors. Deciphering the mechanisms of many biological processes, such as glycolysis, the Krebs cycle and others, became possible only as a result of the use of specific inhibitors of various enzymes (N.E. Kucherenko, Yu.D. Babenyuk et al., 1988).

Some enzyme inhibitors are effective medicinal substances for the animal and human body, others are deadly poisons (V.P. Komov, V.N. Shvedova, 2004).

Inhibitors interact with the active centers of the enzyme molecule, inactivating the functional groups of proteins. They can interact with metals that are part of enzyme molecules and enzyme-substrate complexes, inactivating them. High concentrations of inhibitors destroy the quaternary, tertiary and secondary structures of the enzyme molecule, causing its denaturation (A.I. Kononsky, 1992).

Recently, antienzymes (antienzymes, or antizymes) have been discovered, which are proteins that act as enzyme inhibitors. Such substances include, for example, trypsin inhibitor, found in soybeans, and serum antitrypsin. The antienzyme ornithine decarboxylase was recently discovered in animal liver. Antizymes most likely form difficult-to-dissociate complexes with the corresponding enzymes, excluding them from chemical reactions. Sometimes the inhibitor is an integral component of an enzyme precursor, or is part of complex enzyme complexes. However, it has not yet been clarified whether such antienzymes are true inhibitors or regulatory subunits.

If an inhibitor causes persistent changes in the spatial tertiary structure of the enzyme molecule or modification of the functional groups of the enzyme, then this type of inhibition is called irreversible. More often, however, reversible inhibition occurs, which can be quantified using the Michaelis-Menten equation. Reversible inhibition, in turn, is divided into competitive and non-competitive

In practice, many inhibitors do not exhibit the properties characteristic of purely competitive or purely noncompetitive inhibition. Another way to classify inhibitors is based on the nature of their binding site. Some of them bind to the enzyme in the same place as the substrate (in the catalytic center), while others bind at a considerable distance from the active center (in the allosteric center) (R. Murray, D. Grenner et al., 1993).

1.1 Reversible inhibition

There are three types of reversible enzyme inhibition: competitive, non-competitive and non-competitive, depending on whether the inhibition of the enzymatic reaction can or cannot be overcome by increasing the concentration of the substrate.

1.1.1 Competitive inhibition

A competitive inhibitor competes with a substrate for binding to the active site, but unlike a substrate, an enzyme-bound competitive inhibitor does not undergo enzymatic conversion. The great thing about competitive inhibition is that it can be eliminated or reduced simply by increasing the substrate concentration. For example, if at given concentrations of substrate and competitive inhibitor, enzyme activity is inhibited by 50%, then we can reduce the degree of inhibition by increasing the concentration of substrate.

In their three-dimensional structure, competitive inhibitors usually resemble the substrate of a given enzyme. Thanks to this similarity, the competitive inhibitor manages to “deceive” the enzyme and contact it. Competitive inhibition can be studied quantitatively based on the Michaelis-Menten theory. Competitive inhibitor I simply reversibly attaches to enzyme E, forming a complex with it

Competitive inhibition can most easily be recognized experimentally by determining the effect of inhibitor concentration on the dependence of the initial reaction rate on the substrate concentration. To clarify the question of what type - competitive or non-competitive - reversible inhibition of the enzyme occurs, the method of double reciprocals is used. From graphs constructed in double inverse coordinates, it is also possible to determine the value of the dissociation constant of the enzyme inhibitor complex (see Fig. 1) (A. Leninger, 1985)

Competitive inhibition can be caused by substances that have a structure similar to that of the substrate, but slightly different from the structure of the true substrate. This inhibition is based on the binding of the inhibitor to the substrate-binding (active) site (see Fig. 2).

Rice. 2. General principle of competitive inhibition (scheme according to V.L. Kretovich). E - enzyme; S - substrate; P 1 and P 2 - reaction products; I - inhibitor.

An example is the effect of malonic acid on a reaction that is catalyzed by succinate dehydrogenase and is associated with the conversion of succinic acid to fumaric acid. Adding malonic acid to the reaction mixture reduces or completely stops the enzymatic reaction because it is a competitive inhibitor of succinate dehydrogenase. The similarity of malonic acid to succinic acid is sufficient to form a complex with the enzyme, but the decomposition of this complex does not occur. When the concentration of succinic acid increases, it displaces malonic acid from the complex, as a result, the activity of succinate dehydrogenase is restored.

Rice. 3. Competitive inhibition of the reaction of conversion of succinic acid into fumaric acid under the influence of malonic acid.

The structures of the substrate (succinate) and inhibitor (malonate) are still somewhat different. Therefore, they compete for binding to the active site, and the degree of inhibition will be determined by the ratio of the concentrations of malonate and succinate, and not by the absolute concentration of the inhibitor. Thus, the inhibitor can reversibly bind to the enzyme, forming an enzyme-inhibitor complex. This type of inhibition is sometimes called metabolic antagonism inhibition (see Fig. 3).

In general form, the reaction between an inhibitor and an enzyme can be represented by the following equation:

The resulting complex, called the enzyme-inhibitor complex EI, unlike the enzyme-substrate complex ES, does not decompose to form reaction products.

Many drugs inhibit human and animal enzymes competitively. For example, sulfonamide drugs are used to treat certain infectious diseases caused by bacteria. It turned out that these drugs are structurally similar to para-aminobenzoic acid, which the bacterial cell uses to synthesize folic acid, which is an integral part of bacterial enzymes. Due to this structural similarity, sulfonamide blocks the action of the enzyme by displacing para-aminobenzoic acid from the complex with the enzyme that synthesizes folic acid, which leads to inhibition of bacterial growth.

The peptidoglycan structure of the bacterial cell wall includes D-alanine, which is absent in the body of animals and humans. To synthesize the cell wall, bacteria use the enzyme alanine racemase to convert animal L-alanine into the D form. Alanine racemase is characteristic of bacteria and is not found in mammals. Therefore, it represents a good target for drug inhibition. Substitution of one of the protons of the methyl group with fluorine produces fluoroalanine, to which alanine racemase binds, resulting in its inhibition.

2. Heterotrophic and autotrophic organisms: differences in nutrition and energy sources. Catabolism and anabolism.
3. Multimolecular systems (metabolic chains, membrane processes, biopolymer synthesis systems, molecular regulatory systems) as the main objects of biochemical research.
4. Levels of structural organization of living things. Biochemistry as the molecular level of studying life phenomena. Biochemistry and medicine (medical biochemistry).
5. Main sections and directions in biochemistry: bioorganic chemistry, dynamic and functional biochemistry, molecular biology.
6. History of the study of proteins. An idea of proteins as the most important class of organic substances and a structural and functional component of the human body.
7. Amino acids that make up proteins, their structure and properties. Peptide bond. Primary structure of proteins.
8. Dependence of the biological properties of proteins on the primary structure. Species specificity of the primary structure of proteins (insulins from different animals).
9. Conformation of peptide chains in proteins (secondary and tertiary structures). Weak intramolecular interactions in the peptide chain; disulfide bonds.
11. Domain structure and its role in the functioning of proteins. Poisons and drugs as protein inhibitors.
12. Quaternary structure of proteins. Features of the structure and functioning of oligomeric proteins using the example of heme-containing protein - hemoglobin.
13. Lability of the spatial structure of proteins and their denaturation. Factors causing denaturation.
14.Chaperones are a class of proteins that protect other proteins from denaturation under cellular conditions and facilitate the formation of their native conformation.
15.Variety of proteins. Globular and fibrillar proteins, simple and complex. Classification of proteins according to their biological functions and families: (serine proteases, immunoglobulins).
17. Physico-chemical properties of proteins. Molecular weight, size and shape, solubility, ionization, hydration
18.Methods for isolating individual proteins: precipitation with salts and organic solvents, gel filtration, electrophoresis, ion exchange and affinity chromatography.
19.Methods for quantitative measurement of proteins. Individual characteristics of the protein composition of organs. Changes in the protein composition of organs during ontogenesis and diseases.
21. Classification and nomenclature of enzymes. Isoenzymes. Units for measuring enzyme activity and quantity.
22. Enzyme cofactors: metal ions and coenzymes. Coenzyme functions of vitamins (for example, vitamins B6, pp, B2).
23.Enzyme inhibitors. Reversible and irreversible inhibition. Competitive inhibition. Drugs as enzyme inhibitors.
25. Regulation of enzyme activity by phosphorylation and dephosphorylation. Participation of enzymes in the conduction of hormonal signals.
26. Differences in the enzyme composition of organs and tissues. Organ-specific enzymes. Changes in enzymes during development.
27. Changes in enzyme activity in diseases. Hereditary enzymopathies. The origin of blood enzymes and the significance of their determination in diseases.
29. Metabolism: nutrition, metabolism and excretion of metabolic products. Organic and mineral food components. Major and minor components.
30. Basic nutrients: carbohydrates, fats, proteins, daily requirement, digestion; partial interchangeability when feeding.
31. Essential components of basic nutrients. Essential amino acids; nutritional value of various food proteins. Linoleic acid is an essential fatty acid.
32. History of the discovery and study of vitamins. Classification of vitamins. Functions of vitamins.
34. Mineral substances of food. Regional pathologies associated with insufficiency of microelements in food and water.
35. The concept of metabolism and metabolic pathways. Enzymes and metabolism. The concept of metabolic regulation. Major end products of human metabolism
36. Research on whole organisms, organs, tissue sections, homogenates, subcellular structures and at the molecular level
37.Endergonic and exergonic reactions in a living cell. Macroergic compounds. Examples.
39. Oxidative phosphorylation, p/o ratio. The structure of mitochondria and the structural organization of the respiratory chain. Transmembrane electrochemical potential.
40.Regulation of the electron transport chain (respiratory control). Dissociation of tissue respiration and oxidative phosphorylation. Thermoregulatory function of tissue respiration
42. Formation of toxic forms of oxygen, the mechanism of their damaging effect on cells. Mechanisms for eliminating toxic forms of oxygen.
43. Catabolism of basic nutrients - carbohydrates, fats, proteins. The concept of specific pathways of catabolism and general pathways of catabolism.
44. Oxidative decarboxylation of pyruvic acid. Sequence of reactions. Structure of the pyruvate decarboxylase complex.
45.Citric acid cycle: sequence of reactions and characteristics of enzymes. Relationship between common catabolic pathways and the electron and proton transport chain.
46. Mechanisms of regulation of the citrate cycle. Anabolic functions of the citric acid cycle. Reactions that replenish the citrate cycle
47. Basic carbohydrates of animals, their content in tissues, biological role. Basic carbohydrates of food. Digestion of carbohydrates
49. Aerobic breakdown is the main pathway of glucose catabolism in humans and other aerobic organisms. Sequence of reactions leading to the formation of pyruvate (aerobic glycolysis).
50.Distribution and physiological significance of aerobic breakdown of glucose. The use of glucose for the synthesis of fats in the liver and adipose tissue.
52. Biosynthesis of glucose (gluconeogenesis) from amino acids, glycerol and lactic acid. The relationship between glycolysis in muscles and gluconeogenesis in the liver (Cori cycle).
54. Properties and distribution of glycogen as a reserve polysaccharide. Biosynthesis of glycogen. Glycogen mobilization.
55. Features of glucose metabolism in different organs and cells: red blood cells, brain, muscles, adipose tissue, liver.
56. An idea of the structure and functions of the carbohydrate part of glycolipids and glycoproteins. Sialic acids
57. Hereditary disorders of the metabolism of monosaccharides and disaccharides: galactosemia, intolerance to fructose and disaccharides. Glycogenoses and aglycogenoses
Glyceraldehyde-3–phosphate
58. The most important lipids of human tissues. Reserve lipids (fats) and membrane lipids (complex lipids). Fatty acids in human tissue lipids.
Fatty acid composition of human subcutaneous fat
59. Essential nutritional factors of lipid nature. Essential fatty acids: ω-3- and ω-6-acids as precursors for the synthesis of eicosanoids.
60.Biosynthesis of fatty acids, regulation of fatty acid metabolism
61. Chemistry of reactions of β-oxidation of fatty acids, energy summary.
6Z. Dietary fats and their digestion. Absorption of digestion products. Digestion and absorption disorders. Resynthesis of triacylglycerols in the intestinal wall.
64. Formation of chylomicrons and transport of fats. The role of apoproteins in the composition of chylomicrons. Lipoprotein lipase.
65.Biosynthesis of fats in the liver from carbohydrates. Structure and composition of transport lipoproteins in the blood.
66. Deposition and mobilization of fats in adipose tissue. Regulation of fat synthesis and mobilization. The role of insulin, glucagon and adrenaline.
67.Main phospholipids and glycolipids of human tissues (glycerophospholipids, sphingophospholipids, glycoglycerolipids, glycosphygolipids). An idea of the biosynthesis and catabolism of these compounds.
68.Disturbance of the metabolism of neutral fat (obesity), phospholipids and glycolipids. Sphingolipidoses
Sphingolipids, metabolism: sphingolipidosis diseases, table
69.Structure and biological functions of eicosanoids. Biosynthesis of prostaglandins and leukotrienes.
70.Cholesterol as a precursor to a number of other steroids. Concept of cholesterol biosynthesis. Write the course of reactions before the formation of mevalonic acid. The role of hydroxymethylglutaryl-CoA reductase.
71. Synthesis of bile acids from cholesterol. Conjugation of bile acids, primary and secondary bile acids. Removing bile acids and cholesterol from the body.
72. LDL and HDL - transport, forms of cholesterol in the blood, role in cholesterol metabolism. Hypercholesterolemia. Biochemical basis for the development of atherosclerosis.
73. The mechanism of gallstone disease (cholesterol stones). The use of chenodesokeicholic acid for the treatment of cholelithiasis.
75. Digestion of proteins. Proteinases - pepsin, trypsin, chymotrypsin; proenzymes of proteinases and mechanisms of their conversion into enzymes. Substrate specificity of proteinases. Exopeptidases and endopeptidases.
76. Diagnostic value of biochemical analysis of gastric and duodenal juice. Give a brief description of the composition of these juices.
77. Pancreatic proteinases and pancreatitis. The use of proteinase inhibitors for the treatment of pancreatitis.
78. Transamination: aminotransferases; coenzyme function of vitamin B6. Specificity of aminotransferases.
80. Oxidative deamination of amino acids; glutamate dehydrogenase. Indirect deamination of amino acids. Biological significance.
82. Kidney glutaminase; formation and excretion of ammonium salts. Activation of renal glutaminase during acidosis.
83. Biosynthesis of urea. Relationship between the ornithine cycle and the TCA cycle. Origin of the nitrogen atoms of urea. Disturbances in the synthesis and excretion of urea. Hyperammonemia.
84. Metabolism of the nitrogen-free residue of amino acids. Glycogenic and ketogenic amino acids. Synthesis of glucose from amino acids. Synthesis of amino acids from glucose.
85. Transmethylation. Methionine and s-adenosylmethionine. Synthesis of creatine, adrenaline and phosphatidylcholines
86. DNA methylation. Concept of methylation of foreign and medicinal compounds.
88. Folic acid antivitamins. The mechanism of action of sulfonamide drugs.
89. Exchange of phenylalanine and tyrosine. Phenylketonuria; biochemical defect, manifestation of the disease, methods of prevention, diagnosis and treatment.
90. Alkaptonuria and albinism: biochemical defects in which they develop. Impaired dopamine synthesis, parkinsonism.
91. Decarboxylation of amino acids. Structure of biogenic amines (histamine, serotonin, γ-aminobutyric acid, catecholamines). Functions of biogenic amines.
92. Deamination and hydroxylation of biogenic amines (as reactions of neutralization of these compounds).
93. Nucleic acids, chemical composition, structure. Primary structure of DNA and RNA, bonds forming the primary structure
94. Secondary and tertiary structure of DNA. Denaturation, renativation of DNA. Hybridization, species differences in the primary structure of DNA.
95. RNA, chemical composition, levels of structural organization. RNA types, functions. The structure of the ribosome.
96. Structure of chromatin and chromosomes
97. Decay of nucleic acids. Nucleases of the digestive tract and tissues. Breakdown of purine nucleotides.
98. Idea about the biosynthesis of purine nucleotides; initial stages of biosynthesis (from ribose-5-phosphate to 5-phosphoribosylamine).
99. Inosinic acid as a precursor of adenylic and guanylic acids.
100. Concept of the breakdown and biosynthesis of pyrimidine nucleotides.
101. Nucleotide metabolism disorders. Gout; use of allopurinol for the treatment of gout. Xanthinuria. Orotaciduria.
102. Biosynthesis of deoxyribonucleotides. The use of deoxyribonucleotide synthesis inhibitors for the treatment of malignant tumors.
104. DNA synthesis and phases of cell division. The role of cyclins and cyclin-dependent proteinases in cell progression through the cell cycle.
105. DNA damage and repair. Enzymes of the DNA repair complex.
106. Biosynthesis of RNA. RNA polymerase. The concept of the mosaic structure of genes, primary transcript, post-transcriptional processing.
107. Biological code, concepts, properties of the code, collinearity, termination signals.
108. The role of transport RNAs in protein biosynthesis. Biosynthesis of aminoacyl-t-RNA. Substrate specificity of aminoacyl-tRNA synthetases.
109. Sequence of events on the ribosome during the assembly of a polypeptide chain. Functioning of polyribosomes. Post-translational processing of proteins.
110. Adaptive regulation of genes in pro- and eukaryotes. Operon theory. Functioning of operons.
111. The concept of cell differentiation. Changes in the protein composition of cells during differentiation (using the example of the protein composition of hemoglobin polypeptide chains).
112. Molecular mechanisms of genetic variability. Molecular mutations: types, frequency, significance
113. Genetic heterogeneity. Polymorphism of proteins in the human population (variants of hemoglobin, glycosyltransferase, group-specific substances, etc.).
114. Biochemical basis of the occurrence and manifestation of hereditary diseases (diversity, distribution).
115. Basic systems of intercellular communication: endocrine, paracrine, autocrine regulation.
116. The role of hormones in the metabolic regulation system. Target cells and cellular hormone receptors
117. Mechanisms of hormonal signal transmission into cells.
118. Classification of hormones by chemical structure and biological functions
119. Structure, synthesis and metabolism of iodothyronines. Effect on metabolism. Changes in metabolism during hypo- and hyperthyroidism. Causes and manifestations of endemic goiter.
120. Regulation of energy metabolism, the role of insulin and counter-insular hormones in ensuring homeostasis.
121. Changes in metabolism in diabetes mellitus. Pathogenesis of the main symptoms of diabetes mellitus.
122. Pathogenesis of late complications of diabetes mellitus (macro- and microangiopathies, nephropathy, retinopathy, cataracts). Diabetic coma.
123. Regulation of water-salt metabolism. Structure and functions of aldosterone and vasopressin
124. Renin-angiotensin-aldosterone system. Biochemical mechanisms of renal hypertension, edema, dehydration.
125. The role of hormones in the regulation of calcium and phosphate metabolism (parathyroid hormone, calcitonin). Causes and manifestations of hypo- and hyperparathyroidism.
126. Structure, biosynthesis and mechanism of action of calcitriol. Causes and manifestations of rickets
127. Structure and secretion of corticosteroids. Changes in catabolism during hypo- and hypercortisolism.
128. Regulation of hormone secretion by synthesis based on the feedback principle.
129. Sex hormones: structure, influence on metabolism and function of the gonads, uterus and mammary glands.
130. Growth hormone, structure, functions.
131. Metabolism of endogenous and foreign toxic substances: microsomal oxidation reactions and conjugation reactions with glutathione, glucuronic acid, sulfuric acid.
132. Metallothionein and neutralization of heavy metal ions. Heat shock proteins.
133. Oxygen toxicity: formation of reactive oxygen species (superoxide anion, hydrogen peroxide, hydroxyl radical).
135. Biotransformation of medicinal substances. The effect of drugs on enzymes involved in the neutralization of xenobiotics.
136. Basics of chemical carcinogenesis. An idea of some chemical carcinogens: polycyclic aromatic hydrocarbons, aromatic amines, dioxides, mitoxins, nitrosamines.
137. Features of the development, structure and metabolism of red blood cells.
138. Transport of oxygen and carbon dioxide by blood. Fetal hemoglobin (HbF) and its physiological significance.
139. Polymorphic forms of human hemoglobins. Hemoglobinopathies. Anemic hypoxia
140. Heme biosynthesis and its regulation. Synthesis disorders topic. Porphyria.
141. Heme breakdown. Neutralization of bilirubin. Disorders of bilirubin metabolism - jaundice: hemolytic, obstructive, hepatocellular. Jaundice of newborns.
142. Diagnostic value of determining bilirubin and other bile pigments in blood and urine.
143. Iron metabolism: absorption, blood transport, deposition. Iron metabolism disorders: iron deficiency anemia, hemochromatosis.
144. The main protein fractions of blood plasma and their functions. The significance of their definition for the diagnosis of diseases. Enzymodiagnostics.
145. Blood coagulation system. Stages of fibrin clot formation. Internal and external coagulation pathways and their components.
146. Principles of formation and sequence of functioning of enzyme complexes of the procoagulant pathway. The role of vitamin K in blood clotting.
147. Basic mechanisms of fibrinolysis. Plasminogen activators as thrombolytic agents. Basic blood anticoagulants: antithrombin III, macroglobulin, anticonvertin. Hemophilia.
148. Clinical significance of biochemical blood test.
149. Main cell membranes and their functions. General properties of membranes: fluidity, transverse asymmetry, selective permeability.
150. Lipid composition of membranes (phospholipids, glycolipids, cholesterol). The role of lipids in the formation of the lipid bilayer.
151. Membrane proteins - integral, surface, “anchored”. The importance of post-translational modifications in the formation of functional membrane proteins.
Reversible inhibition Reversible inhibitors bind to the enzyme with weak non-covalent bonds and, under certain conditions, are easily separated from the enzyme. Reversible inhibitors can be competitive or non-competitive.
Competitive inhibition Competitive inhibition refers to a reversible decrease in the rate of an enzymatic reaction caused by an inhibitor that binds to the active site of the enzyme and prevents the formation of an enzyme-substrate complex. This type of inhibition is observed when the inhibitor is a structural analogue of the substrate, resulting in competition between substrate and inhibitor molecules for a place in the active center of the enzyme. In this case, either the substrate or the inhibitor interacts with the enzyme, forming enzyme-substrate (ES) or enzyme-inhibitor (EI) complexes. When an enzyme-inhibitor (EI) complex is formed, no reaction product is formed. For the competitive type of inhibition, the following equations are valid:
E + S ⇔ ES → E + P,
Drugs as competitive inhibitors Many drugs exert their therapeutic effect through the mechanism of competitive inhibition. For example, quaternary ammonium bases inhibit acetylcholinesterase, which catalyzes the hydrolysis of acetylcholine into choline and acetic acid. When inhibitors are added, the activity of acetylcholinesterase decreases, the concentration of acetylcholine (substrate) increases, which is accompanied by an increase in the conduction of nerve impulses. Cholinesterase inhibitors are used in the treatment of muscular dystrophies. Effective anticholinesterase drugs - prozerin, endrophonium, etc.
Non-competitive inhibition Non-competitive inhibition of an enzymatic reaction is called when the inhibitor interacts with the enzyme at a site other than the active site. Noncompetitive inhibitors are not structural analogues of the substrate. A noncompetitive inhibitor can bind to either the enzyme or the enzyme-substrate complex, forming an inactive complex. The addition of a noncompetitive inhibitor causes a change in the conformation of the enzyme molecule in such a way that the interaction of the substrate with the active center of the enzyme is disrupted, which leads to a decrease in the rate of the enzymatic reaction.
Irreversible inhibition Irreversible inhibition is observed in the case of the formation of stable covalent bonds between the inhibitor molecule and the enzyme. Most often, the active center of the enzyme is modified. As a result, the enzyme cannot perform a catalytic function. Irreversible inhibitors include heavy metal ions, such as mercury (Hg 2+), silver (Ag +) and arsenic (As 3+), which in low concentrations block the sulfhydryl groups of the active center. The substrate cannot undergo chemical transformation. In the presence of reactivators, the enzymatic function is restored. In high concentrations, heavy metal ions cause denaturation of the protein molecule of the enzyme, i.e. lead to complete inactivation of the enzyme.
Irreversible enzyme inhibitors as drugs. An example of a drug whose action is based on irreversible enzyme inhibition is the widely used drug aspirin. The anti-inflammatory nonsteroidal drug aspirin provides a pharmacological effect by inhibiting the enzyme cyclooxygenase, which catalyzes the formation of prostaglandins from arachidonic acid. As a result of a chemical reaction, the acetyl residue of aspirin is attached to the free terminal NH 2 group of one of the subunits of cyclooxygenase. This causes a decrease in the formation of prostaglandin reaction products, which have a wide range of biological functions, including mediators of inflammation.
24. Regulation of enzyme action: allosteric inhibitors and activators. Catalytic and regulatory centers. Quaternary structure of allosteric enzymes and cooperative changes in the conformation of enzyme protomers.
Allosteric regulation . In many strictly biosynthetic reactions, the main type of rate regulation of a multistep enzymatic process is feedback inhibition. This means that the final product of the biosynthetic chain inhibits the activity of the enzyme catalyzing the first stage of synthesis, which is key for this reaction chain. Since the final product is structurally different from the substrate, it binds to the allosteric (non-catalytic) center of the enzyme molecule, causing inhibition of the entire synthetic reaction chain.
Let us assume that a multi-stage biosynthetic process takes place in cells, each stage of which is catalyzed by its own enzyme:
The rate of such a total sequence of reactions is largely determined by the concentration of the final product P, the accumulation of which above the permissible level has a powerful inhibitory effect on the first stage of the process and, accordingly, on enzyme E1.
It should, however, be borne in mind that modulators of allosteric enzymes can be both activators and inhibitors. It often turns out that the substrate itself has an activating effect. Enzymes for which both the substrate and the modulator are represented by identical structures are called homotropic, in contrast to heterotropic enzymes, for which the modulator has a different structure from the substrate. Interconversion of active and inactive allosteric enzymes in a simplified form, as well as conformational changes observed upon attachment of substrate and effectors. The attachment of a negative effector to the allosteric center causes significant changes in the configuration of the active center of the enzyme molecule, as a result of which the enzyme loses affinity for its substrate (formation of an inactive complex).
Allosteric interactions are manifested in the nature of the curves of the dependence of the initial reaction rate on the concentration of the substrate or effector, in particular in the S-shape of these curves (deviation from the hyperbolic Michaelis-Menten curve). The S-shaped nature of the dependence of v on [S] in the presence of a modulator is due to the cooperativity effect. This means that the binding of one molecule of the substrate facilitates the binding of a second molecule at the active site, thereby increasing the rate of the reaction. In addition, allosteric regulatory enzymes are characterized by a nonlinear dependence of the reaction rate on the substrate concentration.

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With irreversible inhibition, the binding or destruction of functional groups of the enzyme necessary for the manifestation of its activity occurs.

For example, a substance diisopropyl fluorophosphate binds strongly and irreversibly to the hydroxy group of serine in the active site of the enzyme acetylcholinesterase, hydrolyzing acetylcholine at nerve synapses. Inhibition of this enzyme prevents the breakdown of acetylcholine in the synaptic cleft, as a result of which the transmitter continues to act on its receptors, which uncontrollably increases cholinergic regulation. Combat weapons operate in a similar way. organophosphates(sarin, soman) and insecticides(karbofos, dichlorvos).

Mechanism of irreversible inhibition of acetylcholinesterase

Another example involves inhibition acetylsalicylic acid(aspirin) the key enzyme in the synthesis of prostaglandins - cyclooxygenase. This acid is part of anti-inflammatory drugs and is used for inflammatory diseases and febrile conditions. The addition of an acetyl group to the amino group in the active center of the enzyme causes inactivation of the latter and cessation of prostaglandin synthesis.

Mechanism of irreversible inhibition of cyclooxygenase

Reversible inhibition

With reversible inhibition, weak binding of the inhibitor to the functional groups of the enzyme occurs, as a result of which the activity of the enzyme is gradually restored.

An example of a reversible inhibitor is prozerin, binding to enzyme acetylcholinesterase at its active center. A group of cholinesterase inhibitors (prozerin, distigmine, galantamine) is used for myasthenia gravis, after encephalitis, meningitis, and central nervous system injuries.

Competitive inhibition

With this type of inhibition, the inhibitor is similar in structure to the enzyme substrate. Therefore, it competes with the substrate for the active site, which leads to decreased binding of the substrate to the enzyme and disruption of catalysis. This is a feature of competitive inhibition - the ability to strengthen or weaken inhibition by changing the concentration of the substrate.

For example:

1. Competitive interaction ethanol And methanol for the active center alcohol dehydrogenase.

2. Inhibition succinate dehydrogenase malonic acid, the structure of which is similar to the structure of the substrate of this enzyme - succinic acid (succinate).

Nonspecific inhibitors. Inhibitors to influenza viruses in normal blood sera of humans and animals were discovered in 1942 by Hirst.

The cells of the body produce special virus-tropic substances - inhibitors that can interact with viruses and suppress their activity. Thus, serum inhibitors have a wide range of action: some suppress the hemagglutinating properties of viruses, others suppress their infectious activity. Serum inhibitors are divided into: thermolabile (Chu-inhibitors, β-inhibitors), which are inactivated at a temperature of 60-62 ° C. They are able to neutralize the infectious and hemagglutinating activity of influenza viruses, measles, Newcastle disease, etc.; thermostable (Francis, α- and γ-inhibitors). They block the hemagglutinating activity of the virus.

Different viruses (even of the same species) vary in sensitivity to inhibitors. There are inhibitor-sensitive and inhibitor-resistant strains.

Profound differences in the biochemical nature of inhibitors and their quantitative content in the blood serum of animals of various species have been established.

There is a difference between inhibitors and antibodies in their interaction with the virus. Thus, unlike antibodies, the inhibitor-virus complex does not fix complement; the virus binds to antibodies in the simultaneous presence of antibodies and inhibitors; the virus forms a stronger bond with antibodies.

In addition to serum inhibitors, inhibitors have been described in tissues, secretions and excreta of animals, including birds, as well as in cell cultures.

Interferon system (IFN). In 1957 English virologists A. Isaacs and J. Lindeman discovered that cells infected with a virus produce a special substance that inhibits the reproduction of both homologous and heterologous viruses, which they called interferon. It has been established that there is not just one interferon, but a whole system of them, in which three main types are distinguished.

The nomenclature of interferons was developed by a special WHO commission in 1980.

Within each type there are subtypes, for example, α-interferon has about 20 of them. By nature, interferons are glycoproteins. They are encoded in the genetic apparatus of the cell. In humans, interferon genes are localized on chromosomes 2, 5, 9 and 11.

The interferon system does not have a central organ, since all cells of the body of vertebrates have the ability to produce interferon, although white blood cells (leukocytes, T-lymphocytes, NK, macrophages, etc.) produce it most actively.

Interferon is not spontaneously produced by cells. For its formation, an inducer is needed (viruses, bacterial toxins, synthetic substances, double-stranded viral RNA).

Induction of interferon occurs due to derepression of its gene (the operon for α-interferon has 12 structural genes). Transcription of mRNA for interferon and its translation on cell ribosomes occur.

The time interval between the interaction of the inducer and the cell and the appearance of interferon (lag period) usually lasts 4-8 hours. Interferon does not interact directly with the virus and does not interfere with the adsorption of the virus on the cell and its penetration into it.

The antiviral effect of interferon is not associated with the synthesis of any new protein, but is manifested in an increase in the activity of a number of key enzymes of cellular metabolism (protein kinases and synthetases). As a result, the stages of initiation and translation are blocked and viral mRNAs are destroyed - this determines the universal mechanism of action of interferon in infections caused by different viruses. The most characteristic properties of interferon: tissue specificity. It is active in homologous systems and sharply reduces activity in heterogeneous organisms (therefore, interferons of human origin are used to treat humans);

universality against a wide range of viruses, i.e. it does not have specificity for viruses, although different viruses have unequal sensitivity to interferon;

high efficiency. Small doses of it have antiviral activity.

A study of the properties of interferons has shown that they also have antibacterial properties (especially against gram-positive bacteria), antitumor effects and immunomodulatory properties. Interferons stimulate the activity of natural killer cells and cytotoxic T-lymphocytes, increase the sensitivity of target cells to them, stimulate phagocytosis, antibody formation, complement fixation, etc.

The biological activity of different interferons can be expressed to different degrees; for example, α- and β-interferons have higher antiviral activity than γ-interferons, which have many times greater immunomodulatory activity.

One of the factors determining the body's resistance is the ability of its tissues to produce interferon. It is different in different animals and is determined by the congenital characteristics of the body, age (interferon in newborns exhibits less antiviral effect compared to interferon in adult animals). In addition, the production of interferon by body tissues is also influenced by external conditions, for example, weather, air temperature (in winter and autumn the body produces less interferons than in the warm season), ionizing radiation of animals leads to a decrease in the production of endogenous interferon.

In practice, there are two ways to use interferon: the use of ready-made exogenous homologous interferon for the prevention and treatment of a number of viral infections (influenza, hepatitis B, herpes and malignant neoplasms). The drug is more effective in the early stages of the disease; induction of endogenous interferon in the body. Its manifestation is well known when birds are injected with vaccine strains of the Newcastle disease virus, as well as the lapinized strain L3 and LT of the rinderpest virus.

Currently, interferons are produced by genetic engineering.

Killer cells. In 1976, natural killer cells - NK cells (from the English Natural killer - natural killer) were discovered in lymphoid tissue; they are also referred to as natural killer cells (NK cells). They originate from bone marrow progenitor cells. The content of NK cells in the blood is 5-20% of the total number of lymphocytes, in the liver - 42%, in the spleen - 36, in the lymph nodes - 3, in the lungs - 5, in the small intestine - 3 and in the bone marrow - 2%. Unlike T-cytotoxic lymphocytes, the killer activity of NK cells does not depend on the presentation of foreign antigens to them by molecules of the major histocompatibility complex class I.

Recognition and destruction of target cells by NK cells does not require prior sensitization (immunization) and is not accompanied by the formation of memory cells. However, NK cells play an important role in protecting the body against tumor growth, tumor metastases and viral infections - in the elimination of mutated and virus-infected cells, and transplant rejection. Essentially, natural killer cells are involved in the body's first defense reaction before other, specific immune mechanisms are activated. NK cells cause lysis of target cells, independent of antibodies and complement, and at the same time do not have the ability to phagocytose. The cytotoxic factor of NK cells is a special protein, which in its physicochemical and immunological properties is similar to the protein perforin, which causes the formation of pores in the membrane of target cells. NK cells also contain granzymes that cause the induction of apoptosis (programmed cell death) upon penetration into target cells.

After lysis of target cells, NK cells remain viable, are released from targets and can interact with a new target cell (recycled NK cells). NK cells kill target cells quickly (1-2 hours) without preparation in the form of an immune response, this distinguishes them from T lymphocytes.

In addition to NK cells, antibody-dependent K cells (antibody-dependent cell-mediated cytotoxicity - ADCC) exhibit natural cytotoxicity not caused by previous immunization.

Thanks to the well-coordinated interaction of the systems of macrophages, interferons, complement, the major histocompatibility complex, T-lymphocytes and natural killer cells, even before the acquisition of specific immunity, timely recognition and destruction of all genetically foreign substances (microorganisms and mutant cells) is ensured. As a result, the structural and functional integrity of the body is preserved.

At the same time, these systems serve as the basis for the formation of acquired (specific) immunity, and at their level, species and acquired immunity merge, forming a single and most effective system of self-defense of the body.

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Enzyme inhibition

Medicines are more likely to inhibit enzyme activity

Covalent (chemical) modification

Activation of protein kinase A by cAMP

Covalent modification involves the reversible addition or removal of a specific group, thereby changing the activity of the enzyme. Most often, such a group is phosphoric acid, less often methyl and acetyl groups. Phosphorylation of the enzyme occurs at serine and tyrosine residues. The addition of phosphoric acid to protein is carried out by enzymes protein kinases, splitting – protein phosphatases.

Change in enzyme activity
during phosphorylation-dephosphorylation

Enzymes can be active in both phosphorylated and dephosphorylated states. For example, the enzymes glycogen phosphorylase and glycogen synthase are phosphorylated when the body needs glucose, and glycogen phosphorylase becomes active and begins the breakdown of glycogen, and glycogen synthase inactive. When it is necessary to synthesize glycogen, both enzymes are dephosphorylated, the synthase becomes active, and the phosphorylase becomes inactive.

Dependence of metabolic enzyme activity
glycogen from the presence of phosphoric acid in the structure

In medicine, compounds are actively being developed and used that change the activity of enzymes in order to regulate the rate of metabolic reactions and reduce the synthesis of certain substances in the body.

Inhibition of enzyme activity is usually called inhibition, however this is not always correct. Inhibitor is a substance that causes a specific decrease in enzyme activity. Thus, inorganic acids and heavy metals are not inhibitors, but are inactivators, since they reduce the activity of any enzymes, i.e. act nonspecific.

Two main directions of inhibition can be distinguished

Based on the strength of binding of the enzyme to the inhibitor, inhibition occurs reversible And irreversible.

Based on the ratio of the inhibitor to the active center of the enzyme, inhibition is divided into competitive And non-competitive.

With irreversible inhibition, the binding or destruction of functional groups of the enzyme necessary for the manifestation of its activity occurs.