B. Irreversible inhibition

Enzyme inhibition. An inhibitor is a substance that causes specific decrease in enzyme activity. A distinction should be made between inhibition and inactivation. Inactivation is, for example, the denaturation of a protein as a result of the action of denaturing agents.

By bonding strength Inhibitors with enzyme Inhibitors are divided into reversible and irreversible.

irreversible inhibitors are strongly bound and destroy the functional groups of the enzyme molecule, which are necessary for the manifestation of its catalytic activity. All protein purification procedures do not affect the binding of the inhibitor and the enzyme. Ex: the action of organophosphorus compounds on the enzyme - cholinesterase. Chlorophos, sarin, soman, and other organophosphorus compounds bind to the active center of cholinesterase. As a result, the catalytic groups of the active center of the enzyme are phosphorylated. As a result, the enzyme molecules associated with the inhibitor cannot bind to the substrate and severe poisoning occurs.

Also allocate reversible inhibitors, such as proserin for cholinesterase. Reversible inhibition depends on the concentration of substrate and inhibitor and is removed by an excess of substrate.

According to the mechanism of action allocate:

Competitive inhibition;

Noncompetitive inhibition;

Substrate inhibition;

Allosteric.

1) Competitive (isosteric) inhibition- this is the inhibition of the enzymatic reaction caused by the binding of the inhibitor to the active site of the enzyme. In this case, the inhibitor is similar to the substrate. In the process, there is competition for the active center: enzyme-substrate and inhibitor-enzyme complexes are formed. E+S®ES® EP® E+P; E+I® E. Ex: succinate dehydrogenase reaction [Fig. COOH-CH 2 -CH 2 -COOH® (above the arrow LDH, under FAD®FADH 2) COOH-CH=CH-COOH]. The true substrate of this reaction is succinate (amber to-ta). Inhibitors: malonic acid (COOH-CH 2 -COOH) and oxaloacetate (COOH-CO-CH 2 -COOH). [rice. 3-hole enzyme + substrate + inhibitor = inhibitor-enzyme complex]

Ex: the cholinesterase enzyme catalyzes the conversion of acetylcholine to choline: (CH 3) 3 -N-CH 2 -CH 2 -O-CO-CH 3 ® (above the ChE arrow, under - water) CH 3 COOH + (CH 3) 3 - N-CH 2 -CH 2 -OH. Competitive inhibitors are prozerin, sevin.

2) Noncompetitive inhibition– inhibition associated with the effect of the inhibitor on the catalytic conversion, but not on the binding of the enzyme to the substrate. In this case, the inhibitor can bind both to the active center (catalytic site) and outside it.

Attachment of an inhibitor outside the active center leads to a change in the conformation (tertiary structure) of the protein, as a result of which the conformation of the active center changes. This affects the catalytic site and interferes with the interaction of the substrate with the active site. In this case, the inhibitor is not similar to the substrate, and this inhibition cannot be removed by an excess of the substrate. The formation of triple enzyme-inhibitor-substrate complexes is possible. The speed of such a reaction will not be maximum.

Non-competitive inhibitors include:

cyanides. They bind to the iron atom in cytochrome oxidase and as a result, the enzyme loses its activity, and since. is an enzyme of the respiratory chain, then the respiration of cells is disturbed and they die.

Ions of heavy metals and their organic compounds (Hg, Pb, etc.). The mechanism of their action is associated with their connection with various SH-groups. [rice. enzyme with SH-groups, mercury ion, substrate. All this combines into a triple complex]

A number of pharmacological agents that should affect the enzymes of malignant cells. This includes inhibitors used in agriculture, household poisonous substances.

3) Substrate inhibition (uncompetitive)- inhibition of the enzymatic reaction caused by an excess of the substrate. Occurs as a result of the formation of an enzyme-substrate complex that is unable to undergo catalytic transformation. It can be removed and the substrate concentration reduced. [rice. enzyme binding to 2 substrates at once]

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

For example, substance diisopropylfluorophosphate 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 mediator continues to act on its receptors, which uncontrollably enhances cholinergic regulation. Combat works in the same way. organophosphates(sarin, soman) and insecticides(karbofos, dichlorvos).

Mechanism of irreversible inhibition of acetylcholinesterase

Another example is related to the inhibition acetylsalicylic acid(aspirin) a key enzyme in the synthesis of prostaglandins - cyclooxygenases. This acid is part of anti-inflammatory drugs and is used in inflammatory diseases and feverish conditions. Attachment of the acetyl group to the amino group in the active site of the enzyme causes inactivation of the latter and cessation of prostaglandin synthesis.

Mechanism of irreversible inhibition of cyclooxygenase

Reversible inhibition

With reversible inhibition, the inhibitor is not firmly bound to the functional groups of the enzyme, as a result of which the activity of the enzyme is gradually restored.

An example of a reversible inhibitor is prozerin that binds to an enzyme acetylcholinesterase in its active center. A group of cholinesterase inhibitors (prozerin, distigmine, galantamine) is used for myasthenia gravis, after encephalitis, meningitis, and CNS injuries.

Competitive inhibition

In this type of inhibition, the inhibitor is structurally similar to the substrate of the enzyme. Therefore, it competes with the substrate for the active site, which leads to a decrease in the binding of the substrate to the enzyme and disruption of catalysis. This is the feature of competitive inhibition, i.e., the ability to enhance or weaken inhibition through a change in 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).

Irreversible inhibition is observed in the case of the formation of covalent stable bonds between the inhibitor molecule and the enzyme. Most often, the active center of the enzyme undergoes modification. 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 block the sulfhydryl groups of the active center in low concentrations. In this case, 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.

1. Specific and non-specific
inhibitors

The use of irreversible inhibitors is of great interest for elucidating the mechanism of enzyme action. For this purpose, substances are used that block certain groups of the active center of enzymes. Such inhibitors are called specific. A number of compounds readily react with certain chemical groups. If these groups are involved in catalysis, then complete inactivation of the enzyme occurs.

2. Irreversible enzyme inhibitors like
medications

Example medicinal product, whose action is based on the irreversible inhibition of enzymes, is a widely used drug aspirin. The anti-inflammatory non-steroidal drug aspirin provides a pharmacological effect by inhibiting the cyclooxygenase enzyme, 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 cyclooxygenase subunits.

This causes a decrease in the formation of prostaglandin reaction products, which have a wide range of biological functions, including mediators of inflammation.

Allosteric regulation of enzyme activity. The role of allosteric enzymes in cell metabolism. Allosteric effectors and inhibitors. Features of the structure and functioning of allosteric enzymes and their localization in metabolic pathways. Regulation of enzyme activity by the principle of negative feedback. Give examples.

The most subtle and widespread way to regulate enzyme activity is allosteric regulation . In this case, the regulatory factor binds not to the catalytic center of the enzyme, but to another part of it ( regulatory center), which leads to a change in the activity of the enzyme. Enzymes regulated in this way are called allosteric, they often occupy a key position in metabolism. The substance that binds to the regulatory center is called effector , the effector can be inhibitor , maybe activator . Typically, effectors are either end products of biosynthetic pathways (feedback inhibition) or substances whose concentration reflects the state of cellular metabolism (ATP, AMP, NAD+, etc.). As a rule, allosteric enzymes catalyze one of the reactions that begins the formation of some kind of metabolite. Usually this stage limits the speed of the whole process. In catabolic processes accompanied by the synthesis of ATP from ADP, as an allosteric inhibitor of one of early stages catabolism is often the end product itself - ATP. An allosteric inhibitor of one of the early stages of anabolism is often the end product of biosynthesis, for example, some amino acid.

The activity of some allosteric enzymes is stimulated by specific activators. An allosteric enzyme that regulates one of the catabolic reaction sequences can, for example, be subject to the stimulatory effect of positive effectors, ADR or AMP, and the inhibitory effect of a negative effector, ATP. Cases are also known when an allosteric enzyme of one metabolic pathway reacts in a specific way to intermediate or end products of other metabolic pathways. This makes it possible to coordinate the rate of action of various enzyme systems.

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 the living. Biochemistry as a molecular level of studying the phenomena of life. 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. The idea of proteins as the most important class of organic substances and structural and functional component of the human body.
7. Amino acids that make up proteins, their structure and properties. peptide bond. The 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 of 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 on the example of heme-containing protein - hemoglobin.
13. Lability of the spatial structure of proteins and their denaturation. Factors causing denaturation.
14. Chaperones - a class of proteins that protect other proteins from denaturation under cell 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. Physical and 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 the quantitative measurement of proteins. Individual features 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 of measurement of activity and quantity of enzymes.
22. Enzyme cofactors: metal ions and coenzymes. Coenzyme functions of vitamins (on the example of 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 enzymatic composition of organs and tissues. organ-specific enzymes. Changes in enzymes during development.
27. Change in the activity of enzymes 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 components of food. Major and minor components.
30. Basic nutrients: carbohydrates, fats, proteins, daily requirement, digestion; partial interchangeability in nutrition.
31. Essential components of essential nutrients. Essential amino acids; nutritional value of various food proteins. Linoleic acid is an essential fatty acid.
32. History of discovery and study of vitamins. Classification of vitamins. Functions of vitamins.
34. Minerals of food. Regional pathologies associated with micronutrient deficiencies in food and water.
35. The concept of metabolism and metabolic pathways. Enzymes and metabolism. The concept of regulation of metabolism. 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 coefficient. The structure of mitochondria and the structural organization of the respiratory chain. Transmembrane electrochemical potential.
40. Regulation of the electron transport chain (respiratory control). Uncoupling 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 oxygen species.
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. The sequence of reactions. The structure of the pyruvate decarboxylase complex.
45. Citric acid cycle: sequence of reactions and characteristics of enzymes. Relationship between common pathways of catabolism and the electron and proton transport chain.
46. Mechanisms of regulation of the citrate cycle. Anabolic functions of the citric acid cycle. Reactions replenishing the citrate cycle
47. Basic carbohydrates of animals, their content in tissues, biological role. The main carbohydrates in food. Digestion of carbohydrates
49. Aerobic breakdown is the main route of glucose catabolism in humans and other aerobic organisms. The sequence of reactions until 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 in adipose tissue.
52. Biosynthesis of glucose (gluconeogenesis) from amino acids, glycerol and lactic acid. The relationship of glycolysis in muscles and gluconeogenesis in the liver (Cori cycle).
54. Properties and distribution of glycogen as a reserve polysaccharide. biosynthesis of glycogen. Mobilization of glycogen.
55. Features of glucose metabolism in different organs and cells: erythrocytes, brain, muscles, adipose tissue, liver.
56. The 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 of lipids in human tissues.
Fatty acid composition of human subcutaneous fat
59. Essential nutritional factors of a lipid nature. Essential fatty acids: ω-3- and ω-6-acids as precursors for the synthesis of eicosanoids.
60. Fatty acid biosynthesis, regulation of fatty acid metabolism
61. Chemistry of reactions of β-oxidation of fatty acids, energy total.
63. Dietary fats and their digestion. Absorption of products of digestion. Violation of digestion and absorption. Resynthesis of triacylglycerols in the intestinal wall.
64. Formation of chylomicrons and transport of fats. Role of apoproteins in chylomicrons. Lipoprotein lipase.
65. Biosynthesis of fats in the liver from carbohydrates. Structure and composition of blood transport lipoproteins.
66. Deposition and mobilization of fats in adipose tissue. Regulation of synthesis and mobilization of fats. The role of insulin, glucagon and adrenaline.
67. Basic phospholipids and glycolipids of human tissues (glycerophospholipids, sphingophospholipids, glycoglycerolipids, glycosphygolipids). The idea of the biosynthesis and catabolism of these compounds.
68. Violation of the exchange 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 of a number of other steroids. Introduction to cholesterol biosynthesis. Write the course of reactions until the formation of mevalonic acid. The role of hydroxymethylglutaryl-CoA reductase.
71. Synthesis of bile acids from cholesterol. Bile acid conjugation, primary and secondary bile acids. Removal of bile acids and cholesterol from the body.
72.Lpnp 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 occurrence of cholelithiasis (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 transformation 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 in acidosis.
83. Biosynthesis of urea. Relationship of the ornithine cycle with the cts. Origin of urea nitrogen atoms. Violations of the synthesis and excretion of urea. Hyperammonemia.
84. Exchange of 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. The concept of methylation of foreign and medicinal compounds.
88. Folic acid antivitamins. Mechanism of action of sulfa drugs.
89. Metabolism 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. Violation of the synthesis of dopamine, parkinsonism.
91. Decarboxylation of amino acids. The 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. The primary structure of dna and rna, the bonds that form 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 chromosome
97. Decay of nucleic acids. Nucleases of the digestive tract and tissues. The breakdown of purine nucleotides.
98. The idea of 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. The idea of the breakdown and biosynthesis of pyrimidine nucleotides.
101. Violations of nucleotide metabolism. Gout; 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. Synthesis of DNA 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-repairing complex.
106. Biosynthesis of RNA. RNA polymerase. The concept of the mosaic structure of genes, the primary transcript, post-transcriptional processing.
107. Biological code, concepts, code properties, collinearity, termination signals.
108. The role of transport RNA in protein biosynthesis. Biosynthesis of aminoacyl-t-RNA. Substrate specificity of aminoacyl-t-RNA synthetases.
109. The sequence of events on the ribosome during the assembly of the 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 (on 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 bases for the occurrence and manifestation of hereditary diseases (diversity, distribution).
115. Main 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 transmission of hormonal signals to cells.
118. Classification of hormones by chemical structure and biological functions
119. Structure, synthesis and metabolism of iodothyronines. Influence on metabolism. Changes in metabolism in hypo- and hyperthyroidism. Causes and manifestation of endemic goiter.
120. Regulation of energy metabolism, the role of insulin and contrainsular hormones in homeostasis.
121. Changes in metabolism in diabetes mellitus. The pathogenesis of the main symptoms of diabetes mellitus.
122. Pathogenesis of late complications of diabetes mellitus (macro- and microangiopathy, nephropathy, retinopathy, cataract). diabetic coma.
123. Regulation of water-salt metabolism. Structure and function 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 (parathormone, calcitonin). Causes and manifestations of hypo- and hyperparathyroidism.
126. Structure, biosynthesis and mechanism of action of calcitriol. Causes and manifestation of rickets
127. Structure and secretion of corticosteroids. Changes in catabolism in hypo- and hypercortisolism.
128. Regulation by syntheses of secretion of hormones on the principle of feedback.
129. Sex hormones: structure, influence on metabolism and functions of sex glands, 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. Fundamentals of chemical carcinogenesis. Introduction to some chemical carcinogens: polycyclic aromatic hydrocarbons, aromatic amines, dioxides, mitoxins, nitrosamines.
137. Features of development, structure and metabolism of erythrocytes.
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. Disorders of synthesis theme. Porfiria.
141. Disintegration of heme. Neutralization of bilirubin. Disorders of bilirubin-jaundice metabolism: hemolytic, obstructive, hepatocellular. Jaundice of newborns.
142. Diagnostic value of determination of bilirubin and other bile pigments in blood and urine.
143. Exchange of iron: absorption, transport by blood, deposition. Iron metabolism disorders: iron deficiency anemia, hemochromatosis.
144. Main protein fractions of blood plasma and their functions. The value of their definition for the diagnosis of diseases. Enzymodiagnostics.
145. Blood coagulation system. Stages of fibrin clot formation. Intrinsic and extrinsic 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. Main mechanisms of fibrinolysis. Plasminogen activators as thrombolytic agents. Based blood anticoagulants: antithrombin III, macroglobulin, anticonvertin. Hemophilia.
148. Clinical significance of a biochemical blood test.
149. Basic 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". Significance of post-translational modifications in the formation of functional membrane proteins.
Reversible inhibition Reversible inhibitors bind to the enzyme by weak non-covalent bonds and, under certain conditions, are easily separated from the enzyme. Reversible inhibitors are either 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 the enzyme-substrate complex. This type of inhibition is observed when the inhibitor is a structural analogue of the substrate, resulting in competition between the substrate and inhibitor molecules for a place in the active site 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 the complex of the enzyme and the inhibitor (EI) is formed, the reaction product is not 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 to 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 a nerve impulse. Cholinesterase inhibitors are used in the treatment of muscular dystrophies. Effective anticholinesterase drugs - prozerin, endrophonium, etc.
Noncompetitive inhibition Such inhibition of an enzymatic reaction is called non-competitive, in which the inhibitor interacts with the enzyme in a site other than the active site. Non-competitive inhibitors are not structural analogues of the substrate. A non-competitive inhibitor can bind to either the enzyme or the enzyme-substrate complex to form an inactive complex. The addition of a non-competitive inhibitor causes a change in the conformation of the enzyme molecule in such a way that the interaction of the substrate with the active site 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 covalent stable bonds between the inhibitor molecule and the enzyme. Most often, the active center of the enzyme undergoes modification. 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 block the sulfhydryl groups of the active center in low concentrations. In this case, 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 non-steroidal drug aspirin provides a pharmacological effect by inhibiting the cyclooxygenase enzyme, 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 cyclooxygenase subunits. 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 the action of enzymes: 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 regulation of the rate of a multistage enzymatic process is feedback inhibition. This means that the end product of the biosynthetic chain inhibits the activity of the enzyme that catalyzes the first stage of synthesis, which is the key to this reaction chain. Since the end 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 multistage biosynthetic process is carried out 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 an acceptable level has a powerful inhibitory effect on the first stage of the process and, accordingly, on the E1 enzyme.
However, it should be borne in mind that both activators and inhibitors can be modulators of allosteric enzymes. 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. Mutual transformation of active and inactive allosteric enzymes in a simplified form, as well as conformational changes observed when the substrate and effectors are attached. 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 its affinity for its substrate (the 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 Michaelis-Menten hyperbolic curve). The S-shaped dependence of v on [S] in the presence of a modulator is due to the effect of cooperativity. This means that the binding of one molecule of the substrate facilitates the binding of the second molecule in the active site, thereby increasing the rate of the reaction. In addition, allosteric regulatory enzymes are characterized by a non-linear dependence of the reaction rate on the substrate concentration.

"

1. Under the term "inhibition enzyme activity" understand the specific reduction in catalytic activity caused by certain chemicals - inhibitors.

Inhibitors are of great interest for elucidating the mechanisms of enzymatic catalysis, helping to establish the role of individual enzymatic reactions in the metabolic pathways of the body. The action of many drugs and poisons is based on the principle of inhibition of enzymatic activity.

2. Inhibitors are able to bind to enzymes with varying degrees of strength. Based on this, distinguish reversible and irreversible inhibition. Reversible inhibitors bind to the enzyme by weak non-covalent bonds and, under certain conditions, are easily separated from the enzyme:

E+I↔ E.I.

irreversible inhibition observed in the case of the formation of covalent stable bonds between the inhibitor molecule and the enzyme:

E+I→ E-I.

3. According to the mechanism of action, reversible inhibitors are divided into competitive and non-competitive.

Competitive inhibition causes a reversible decrease in the rate of the enzymatic reaction as a result of the binding of the inhibitor to the active site of the enzyme, which prevents the formation of the enzyme-substrate complex. This type of inhibition occurs when the inhibitor is structural analogue of the substrate; as a result, there is competition between substrate and inhibitor molecules for binding to the active site 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 the complex of the enzyme and the inhibitor (EI) is formed, the reaction product is not formed (Fig. 2.19).

Rice. 2.19. Scheme of competitive inhibition of enzyme activity

For the competitive type of inhibition, the following equations are valid:

E+S↔ES→E+P; E+I↔ EI.

Distinctive feature competitive inhibition is the possibility of its weakening with increasing substrate concentration, since a reversible inhibitor does not change the structure of the enzyme. Therefore, at high substrate concentrations, the reaction rate does not differ from that in the absence of an inhibitor; competitive inhibitor does not change V max but increases K m .

A classic example of competitive inhibition is the inhibition of the succinate dehydrogenase reaction by malonic acid (Fig. 2.20). Malonate is a structural analogue of succinate (the presence of two carboxyl groups) and can also interact with the active site of succinate dehydrogenase. However, the transfer of two hydrogen atoms to the FAD prosthetic group from malonic acid is not possible and hence the reaction rate is reduced.

Rice. 2.20. Example of competitive inhibition of succinate dehydrogenase by malonic acid:

A - succinate binds to the active site of the enzyme succinate dehydrogenase by ionic bonds; B - during the enzymatic reaction, two hydrogen atoms are cleaved from succinate with their addition to the FAD coenzyme. As a result, fumarate is formed, which is removed from the active site of succinate dehydrogenase; B - malonate is a structural analogue of succinate, it also binds to the active site of succinate dehydrogenase, but chemical reaction does not go

4. Many drugs exert their therapeutic effect through the mechanism of competitive inhibition. For example, the reaction of hydrolysis of acetylcholine to choline and acetic acid is catalyzed by the enzyme acetylcholinesterase (AChE) (Fig. 2.21) and can be inhibited in the presence of competitive inhibitors of this enzyme (for example, prozerin, endrophonium etc.) (Fig. 2.22). When such inhibitors are added, the activity of acetylcholinesterase decreases, the concentration of acetylcholine (substrate) increases, which is accompanied by an increase in the conduction of a nerve impulse. Competitive acetylcholine esterase inhibitors are used in the treatment of muscular dystrophy, as well as for the treatment of movement disorders after trauma, paralysis, and poliomyelitis.

Rice. 2.21. Acetylcholine hydrolysis reaction under the action of AChE

Rice. 2.22. Binding in the AChE active site of competitive inhibitors

A - addition of a substrate (acetylcholine) to the active site of the enzyme.

The arrow indicates the site of hydrolysis of acetylcholine; B - attachment of a competitive inhibitor of proserin to the active center of the enzyme. The reaction does not go; B - attachment of a competitive inhibitor of endrophonium to the active site of the enzyme. Attachment of inhibitors to the active site of AChE prevents the attachment of acetylcholine

Another example of drugs whose mechanism of action is based on competitive inhibition of the enzyme is the use of peptide inhibitors of the proteolytic enzyme trypsin in diseases of the pancreas (acute pancreatitis, necrosis), such as aprotinin, trasylol, contrical. These drugs inhibit trypsin, which is released into the surrounding tissues and blood, and thereby prevent unwanted autolytic events in pancreatic diseases.

5. In some cases, competitive inhibitors, interacting with the active site of the enzyme, can be used by them as pseudosubstrates(antimetabolites), which leads to the synthesis of a product with an irregular structure. The resulting substances do not have the “necessary” structure and therefore lack functional activity. These drugs include sulfa drugs.

6. Non-competitive reversible is the inhibition of an enzymatic reaction, in which the inhibitor interacts with the enzyme in a site other than the active site. Non-competitive inhibitors are not structural analogues of the substrate; the attachment of a non-competitive inhibitor to an enzyme changes the conformation of the active site and reduces the rate of the enzymatic reaction, i.e. reduces enzymatic activity. An example of a non-competitive inhibitor can be the action of heavy metal ions, which interact with the functional groups of the enzyme molecule, preventing catalysis.

7. Irreversible inhibitors reduce enzymatic activity as a result of the formation of covalent bonds with the enzyme molecule. Most often, the active site of the enzyme undergoes modification. As a result, the enzyme cannot perform its catalytic function.

The use of irreversible inhibitors is of greater interest for elucidating the mechanism of enzyme action. Important information about the structure of the active center of the enzyme is provided by compounds that block certain groups of the active center. Such inhibitors are called specific. Specific inhibitors include diisopropylfluorophosphate (DFF). DPP forms a covalent bond with the OH group of serine, which is contained in the active center of the enzyme and is directly involved in catalysis, therefore DPP is classified as a specific irreversible inhibitor of "serine" enzymes (Fig. 2.23). DPP is used to study the structure of the active site of enzymes in enzymology.

Unlike specific inhibitors non-specific inhibitors form covalent bonds with certain enzyme groups located not only in the active center, but also in any part of the enzyme molecule. For example, iodine acetate (Fig. 2.24) interacts with any SH-groups of the protein. This interaction changes the conformation of the enzyme molecule and, accordingly, the conformation of the active center and reduces the catalytic activity.

Rice. 2.23. Specific inhibition of chymotrypsin activity by DPP

Rice. 2.24. Nonspecific inhibition of enzyme activity by iodine acetate.

Nonspecific inhibition occurs due to covalent modification of cysteine SH groups by iodine acetate molecules

8. An example of a drug whose action is associated with irreversible inhibition of enzymes is a widely used aspirin. The action of this anti-inflammatory non-steroidal drug is based on the inhibition of the cyclooxygenase enzyme, which catalyzes the formation of prostaglandins from arachidonic acid. As a result, the acetyl residue of aspirin is attached to the free terminal OH group of serine of one of the cyclooxygenase subunits (Fig. 2.25). This blocks the formation of prostaglandins (see module 8), which have a wide range of biological functions, including mediators of inflammation. Therefore, aspirin is classified as an anti-inflammatory drug. Inhibited enzyme molecules are destroyed, the synthesis of prostaglandins is restored only after the synthesis of new enzyme molecules.

Rice. 2.25. Mechanism of cyclooxygenase inactivation by an irreversible inhibitor - aspirin