REMEMBER

Question 1. What are the optimal conditions for digestion in the mouth, stomach and intestines?

In order for digestion to occur in the oral cavity, an alkaline environment is needed, and for digestion in the stomach, an acidic environment is needed.

Question 2. What enzymes do you know and what is their role in the human body?

Proteases break down proteins into peptides, peptones and amino acids.

Lipases – fats to glycerol and fatty acids.

QUESTIONS FOR THE PARAGRAPH

Question 1. What are enzymes? Give examples of enzymes known to you.

Enzymes are complex organic substances that are formed in a living cell and play an important role as a catalyst for all processes occurring in the body. Most of them consist of two components: protein (apoenzyme) and non-protein (coenzyme). Examples include enzymes such as pepsin, trypsin, and amylase.

Question 2. What is the mechanism of enzymes?

Enzyme activity is usually determined by a small part of the enzyme's protein molecule, called the active site. Sometimes the active centers, in addition to amino acids, include metal ions, vitamins and other non-protein compounds, which are called coenzymes.

The active center of an enzyme must have a structure that will enable it to momentarily contact a molecule of a strictly defined substance - the substrate of this enzyme. For example, the active site of lysozyme, found in saliva and tears, exactly corresponds to a site on one of the saccharides of the membrane of some bacteria. By decomposing this saccharide, lysozyme also kills bacteria, preventing them from entering the human body.

Explain the role of enzymes in the human body. Give examples.

Enzymes, due to their catalytic activity, are very important for the normal functioning of our body systems. Therefore, the absence or disruption of the activity of any enzyme can lead to illness and sometimes death.

Enzymes are necessary for protein synthesis, digestion and absorption of nutrients, energy metabolism reactions, muscle contraction, neuropsychic activity, reproduction, processes of removing substances from the body, etc.

To diagnose many human diseases, determination of enzyme activity in blood, urine, cerebrospinal fluid and other structures is used. For example, by analyzing enzymes in blood plasma, viral hepatitis can be detected, early stages myocardial infarction, kidney disease, etc.

THINK!

Why is a significant increase in body temperature (above 40 °C) dangerous for a person during illness?

Since all enzymes are proteins by nature, which begin to break down when the temperature rises above 40 degrees, an increase in human body temperature poses a great danger.

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Structure, properties and mechanism of action of enzymes

Content

• Enzyme structure
• Mechanism of action of enzymes
• Enzyme nomenclature
• Classification of enzymes
• Properties of enzymes
• Clinical enzymology
• Literature

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 managed to implement artificial synthesis 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 association (cooperation), structural changes occur in individual protomers, as a result of which the activity of the enzyme increases markedly. 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). In the case of combining different protomers into the oligomeric structure of an enzyme, multiple forms of the same enzyme arise - isoenzymes .

Isoenzymes catalyze the same reaction, but differ in the set of subunits, physical and chemical properties, electrophoretic mobility, affinity for substrates, activators, 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 flow chemical reactions according to conditions in different organs. Thus, the LDH 1 isoenzyme has a high affinity for oxygen, so it is active in tissues at a high speed oxidative reactions(erythrocytes, myocardium). Isoenzyme LDH 5 is 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 3 predominates, and with increasing age and increasing oxygen supply, the proportion of LDH 2 increases.

enzyme activator inhibitor energy

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 called cofactor ( coenzyme ). The cofactor is most often metal ions that can bind tightly to the apoenzyme (for example, Zn in the carbonic anhydrase enzyme, Cu in the cytochrome oxidase enzyme). Coenzymes are most often organic substances less tightly bound to the apoenzyme. The coenzymes are the nucleotides NAD and FAD. Coenzyme - low 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

SH - cysteine

NH 2 lysine

g-COOH of glutamic acid

There are two zones in the active center - the substrate binding zone and the catalytic zone.

Zone binding 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 is allosteric plot - a 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:

TO= 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 - constants, 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 energy of splitting H 2 O 2 without enzymes and catalysts is 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 + products reactions. For the first time, the possibility of forming enzyme-substrate complexes was 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 proposed theory " key And castle" Fisher. 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 - theory Koshlanda(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. interaction between the active center of the enzyme and the substrate occurs. 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 having 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 - the subclass, the third - the subclass, the fourth - the 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 form reactions: A ok + B ok = A ok + B ok. This class of enzymes includes several subclasses:

1 . Dehydrogenase, 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 rupture chemical bond with the addition of water at the break point, that is, the hydrolysis reaction according to the scheme: AB + NOH = AN + BOH. Subclasses of hydrolases are distinguished depending on the type of bonds being broken: peptidases cleave peptide bonds (pepsin), glycosidases - glycosidic bonds (amylase), esterases - ester bonds (lipase).

Lyases .

Lyases catalyze the breaking of a chemical bond without adding water at the site of the break. At the same time, in the substrates are formed double bonds according to the scheme: AB = A + B. Subclasses of lyases depend on which atoms the bond is broken between and what 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 (cleave off carbon dioxide), dehydratases - “cut out” water molecules.

Isomerases .

Isomerases catalyze the interconversions of different isomers. For example, phosphohexoimerase converts fructose into 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 due to ATP energy according to the scheme: A+B+ATP = AB. For example, glutamine synthetase combines glutamic acid, NH 3 + with the participation of ATP to form 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 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) in one minute. 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 is important biological significance, consisting in the fact that they provide high speed chemical reactions in the body.

High specificity enzymes .

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 group specificity, in which the 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 H 5 OH, but also its homologues (methyl, butyl and other alcohols).

Relative group specificity, in which the enzyme catalyzes different classes organic matter. For example, the enzyme trypsin exhibits peptidase and esterase activity.

Stereochemical specificity (optical specificity), in which only definite form isomers (D, L forms, b, c, cis - trans isomers). For example, LDH acts only 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 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 conducting enzymatic reaction in vitro, 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 - dependence of enzyme activity on action 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.

Addiction activity from pH.

All enzymes have a certain pH range in which enzyme activity is maximum - pH optimum. For many enzymes the optimum is around 7. At the same time, for pepsin optimal environment 1-2, for alkaline phosphatase 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 finds and practical use. Enzymatic reactions outside the body are carried out at an optimum pH. When the acidity of gastric juice is reduced, a solution of HCl is prescribed for therapeutic purposes.

Addiction speed enzymatic reactions from concentrations enzyme And concentrations 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 the enzyme-substrate complex FS,

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 *

V 3 = K 3 *

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

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

- initial enzyme concentration

Km is the Michaelis constant.

Physical meaning of Km: TOm = (TO 2 +K 3 ) /TO 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 [S]

1. Dependence of the reaction rate on the substrate concentration. This dependence is revealed at low substrate concentrations [S]

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. The dependence of the rate on the enzyme concentration appears at high substrate concentrations.

S?Km.

In this case, Km can be neglected and the equation becomes:

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 the numerical value of Km under the condition V 3 = V max /2. In this case, the equation takes the form:

V max /2 = ((V max * [S]) /Km+ [S]), which means that Km= [S]

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

Graphic definition constants Michaelis.

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 (HCl - pepsinogen activator) and nonspecific activators that increase the activity of a number of enzymes (Mg ions - activators of hexokinase, 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. The addition of H 3 PO 4 most often sharply increases the activity of the enzyme. For example, two inactive dimers of the enzyme phosphorylase combine with four ATP molecules 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 is converted into active pepsin under the influence of HCL.

Inhibitors - substances that reduce enzyme activity.

By specificity distinguish specific and nonspecific inhibitors

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

By place actions There are inhibitors acting on the active center and outside the active center.

By mechanism actions distinguished 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, the activity of SH 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 additional substances 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 and Mg ions.

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. Change in the rate of enzyme synthesis (this mechanism requires a long period of time).

2. Increasing the availability of substrate and enzyme by changing the permeability of cell membranes.

3. Changes in 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, key enzymes are isolated that limit the overall speed 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:

2. Change in enzyme oligomerity:

Monomers are not active - oligomers are active

3. Phospholyration - dephosphorylation:

Enzyme (inactive) + H 3 PO 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 stage 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 structures - compartmentalization . 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.

PrinciplesdetectionAndquantitativedefinitionsenzymes:

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 ).

Units activity ( quantities ) enzyme

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.

2. Catal (in the SI system) is the amount of enzyme that catalyzes the conversion of 1 mole of substrate per second.

3. Specific activity - the ratio of enzyme activity to the mass of enzyme protein.

4. The molecular activity of an enzyme shows how many molecules of the 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

2. Enzymopotology

3. Enzyme therapy

Enzymodiagnostics - section 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 conducting 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, in children LDH 3 (more anaerobic form) predominates, and in adults LDH 2 (more aerobic form) predominates.

Enzymopathology - a 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 branch 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; Edited by 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.

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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.

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The mechanism of action of simple and complex enzymes is the same, since the active centers in their molecules perform similar functions.

The action of enzymes is based on their ability to speed up reactions by reducing the activation energy of the substrate. Enzymes deform the electron shells of substrates, thus facilitating the interaction between them. The energy required to bring molecules into an active state is called activation energy. The role of a conventional catalyst (and even more so of a biological catalyst) is that it reduces the activation energy of the substrate.

The basic mechanisms of enzyme action were studied at the beginning of the 20th century. In 1902, the English chemist A. Brown suggested that an enzyme, acting on a substrate, should form an intermediate enzyme with it - a substrate complex. At the same time and independently of A. Brown, the same assumption was made by the French scientist V. Henri. In 1913, L. Michelis and M. Menten confirmed and developed ideas about the mechanism of action of enzymes, which can be represented in the form of a diagram:

E + S " " [E-P]  E + P,

where E is the enzyme, S is the substrate, P is the product.

At the first stage of enzymatic catalysis, the formation of an enzyme-substrate complex occurs, where the enzyme and substrate can be linked by ionic, covalent or other bonds. The formation of the E-S complex occurs almost instantly.

At the second stage, the substrate, under the influence of the enzyme associated with it, is modified and becomes more accessible for the corresponding chemical reaction. This stage determines the speed of the entire process. At these stages of enzymatic catalysis, repeated changes occur in the tertiary structure of the enzyme protein, leading to a sequential approach to the substrate and orientation in space of those active groups that interact with each other at various stages of transformation of substrates

At the third stage, a chemical reaction occurs, as a result of which a complex of the reaction product with the enzyme is formed.

The final process is the release of the reaction product from the complex.

In the body, the transformation of substances to final products occurs in several stages, each of which is catalyzed by a separate enzyme. The sum of the activation energies of intermediate reactions is lower than the activation energies required for simultaneous cleavage of the substrate.

4.3. Properties of enzymes

Enzymes have all the properties of proteins. However, in comparison with proteins that perform other functions in the cell, enzymes have a number of specific properties inherent only to them.

Dependence of enzyme activity on temperature. Temperature can affect enzyme activity in different ways. At high temperatures, denaturation of the protein part of the enzyme can occur, which negatively affects its activity. At certain (optimal) temperatures, temperature can affect the rate of formation of the enzyme-substrate complex, causing an increase in the reaction rate. The temperature at which the catalytic activity of an enzyme is maximum is called the temperature optimum of the enzyme. Various cellular enzymes have their own temperature optimums, which are determined experimentally. For enzymes of animal origin, the temperature optimum is in the range of 40-50°C.

Dependence of enzyme activity on pH environment. Most enzymes exhibit maximum activity at pH values ​​close to neutral. Only a few enzymes work in strongly acidic or strongly alkaline environments. For example, the activity of pepsin, an enzyme that hydrolyzes proteins in the stomach, is maximum at a pH of 1.5-2.5. Enzymes localized in the intestines “work” in an alkaline environment. A change in the optimal pH value for a given enzyme can lead to a change in the tertiary structure of the enzyme, which will affect its activity. On the other hand, when the pH changes, the ionization of the substrate may change, which will affect the formation of the enzyme-substrate complex.

Specificity of enzyme action -- one of their main properties. Specificity - is the selectivity of the enzyme towards its substrate (or substrates). The specificity of the action of enzymes is explained by the fact that the substrate must approach the active center like a “key to a lock.” This figurative comparison was made by E. Fisher in 1894. He considered the enzyme as a rigid structure, the active center of which is a “cast” of the substrate. However, it is difficult to explain the group specificity of enzymes with this hypothesis, since the configuration of the “keys” (substrates) that fit into one “lock” is too diverse. This discrepancy was explained in the 50s. XX century in D. Koshland's hypothesis. It is called the “forced correspondence” hypothesis.

According to D. Koshland's hypothesis, the enzyme molecule is not rigid, but flexible, elastic, therefore the information of the enzyme and its active center can change when a substrate or other ligands are attached. At the moment of attachment, the substrate “forces” the active site of the enzyme to take the appropriate shape. This can be compared to a "glove" and a "hand".

The “forced correspondence” hypothesis has received experimental confirmation. This hypothesis also allows us to explain the reason for the transformation of close analogues of substrates.

There are several types of specificity.

Stereochemical substrate specificity - enzyme

catalyzes the conversion of only one stereoisomer of the substrate. For example, fumarate hydratase catalyzes the addition of a water molecule to the multiple bond of fumaric acid, but not to its stereoisomer, maleic acid.

Absolute substrate specificity - the enzyme catalyzes the conversion of only one substrate. For example, urease catalyzes the hydrolysis of only urea.

Group substrate specificity - the enzyme catalyzes the transformation of a group of substrates of similar chemical structure. For example, alcohol dehydrogenase catalyzes the conversion of ethanol and other aliphatic alcohols, but at different rates.

Effect of activators and inhibitors on enzyme activity. Factors that increase enzyme activity include metal cations and some anions. Most often, enzyme activators are cations Mg 2+, Mn 2+, Zn 2+, K + and Co 2+, and among anions - Cl -. Cations act on enzymes in different ways. In some cases, they facilitate the formation of the enzyme-substrate complex, in others they facilitate the attachment of the coenzyme to the apoenzyme, or they attach to the allosteric center of the enzyme and change its tertiary structure, as a result of which the substrate and catalytic centers acquire the most favorable configuration for catalysis.

Inhibitors inhibit the action of enzymes. Inhibitors can be both endogenous and exogenous substances. The mechanisms of the inhibitory action of various chemical compounds are varied.

Nomenclature and classification of enzymes

Nomenclature of enzymes. At the first stages of the development of enzymology, names were given to enzymes by their discoverers based on random characteristics (trivial nomenclature). For example, the names of enzymes are trivial: pepsin, trypsin, chymotrypsin. The first attempt to introduce a rule for the names of enzymes was made by E. Duclos in 1898 (rational nomenclature). According to rational nomenclature, a simple enzyme was named after the name of the substrate with the addition of the ending -aza(DNase, RNase, amylase, urease). To name the holo-enzyme according to rational nomenclature, we used the name of the coenzyme (pyridoxal enzyme, heminenzyme). Later, the name of the enzyme began to use the name of the substrate and the type of reaction catalyzed (alcohol dehydrogenase).

In 1961, the V International Biochemical Congress, held in Moscow, approved the scientific nomenclature of enzymes. According to this nomenclature, the name of an enzyme consists of the chemical name of the substrate (substrates) on which the enzyme acts, the type of reaction catalyzed and the end -aza. For example, the enzyme that hydrolyzes urea (the rational name is urease) is called urea amide hydrolase according to scientific nomenclature.

If a chemical reaction involves a donor of some group of atoms and an acceptor, then the enzyme is named as follows: chemical name of the donor, chemical name of the acceptor, type of reaction catalyzed. For example, the enzyme that catalyzes the process of transamination of glutamic and urovic acids is called glutamate: pyruvate aminotransferase.

However, it should be noted that along with names according to scientific nomenclature, the use of trivial names of enzymes is allowed.

Classification of enzymes. Currently, more than 2000 enzymes are known. All enzymes are divided into six classes, each of which has a strictly defined number.

1.Oxidoreductases catalyze redox processes.

2.Transferases catalyze reactions of transfer of functional groups and molecular residues from one molecule to another.

3.Hydrolases catalyze hydrolysis reactions.

4.Lyases catalyze elimination reactions (except for hydrogen atoms) with the formation of a double bond or addition at a double bond, as well as non-hydrolytic decomposition of organic compounds or synthesis without the participation of high-energy substances.

5.Isomerases catalyze processes of change in the geometric or spatial configuration of molecules.

6.Ligases catalyze synthesis reactions accompanied by the hydrolysis of energy-rich bonds (usually ATP).

Enzyme classes are divided into subclasses, and the subclasses, in turn, into subsubclasses. Subclass clarifies the action of the enzyme, as it indicates in general terms the nature of the chemical group of the substrate. Subclass further specifies the action of the enzyme by specifying the nature of the substrate bond being attacked or the nature of the acceptor that is involved in the reaction.

The classification system provides for each enzyme a special code consisting of four code numbers separated by dots. The first digit in the code indicates the class number, the second - the subclass number, the third - the subsubclass and the fourth - the serial number in this subclass. Thus, lactate dehydrogenase has the code EC 1.1.1.27, i.e. it belongs to the first class, the first subclass, the first subsubclass and occupies 27th place in the list of enzymes of the mentioned subsubclass.

Let us give specific examples of biochemical processes catalyzed by enzymes belonging to a specific class and subclass.

1. Oxidoreductases. The general scheme of processes catalyzed by oxidoreductases can be expressed as follows:

Most often we will encounter oxidoreductases of the subclass of oxidases and dehydrogenases, so we will consider them in more detail.

Oxidases - These are oxidoreductases that transfer hydrogen atoms or electrons directly to oxygen atoms or introduce an oxygen atom into the substrate molecule.

Dehydrogenases - These are oxidoreductases that catalyze the process of abstraction of hydrogen atoms.

All dehydrogenases are holoenzymes, the coenzymes of which are the following compounds: nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide (NADP), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), quinones.

The most common dehydrogenases in nature contain NAD as a coenzyme.

Coenzymes FMN and FAD contain phosphorylated vitamin B2 (riboflavin phosphate), which is capable of abstracting two hydrogen atoms from the substrate.

2. Transferases. This is one of the most numerous classes of enzymes. Depending on the nature of the transferred groups, phosphotransferases, aminotransferases, glycosyltransferases, acyltransferases, etc. are distinguished.

Phosphotransferases - These are enzymes that catalyze the transfer of phosphoric acid residues. As a result of the action of phosphotransferases, phosphorus esters of various organic compounds are formed, many of which have increased reactivity and more easily enter into subsequent reactions. Therefore, phosphorylation of organic compounds can be considered a process of their activation. The most common donor of phosphate groups is the adenosine triphosphoric acid (ATP) molecule. Phosphotransferases that use the ATP molecule as a phosphate group donor are called kinases . Kinases include, for example, glycerol kinase, which accelerates the transfer of a phosphoric acid residue from an ATP molecule to a glycerol molecule.

Aminotransferases accelerate the transfer of amino groups. Aminotransferases are two-component enzymes whose coenzyme is pyridoxal phosphate (phosphorylated vitamin B 6).

Glycosyltransferases accelerate the transfer reactions of glycosyl residues, providing mainly reactions for the synthesis and breakdown of oligo- and polysaccharides. If a glycosyl residue is transferred to a phosphoric acid molecule, the process is called phosphorolysis, and the enzymes that ensure this process are called phosphorylases.

The donor of glycosyl residues in the synthesis of oligo- and polysaccharides are nucleoside diphosphate sugars (NDP-sugars), one of the representatives of which is uridine diphosphate glucose (UDP-glucose).

Acyltransferases catalyze the transfer of apils (carboxylic acid radicals) to alcohols, amines, amino acids and other compounds. The source of acyls is acyl-CoA, which can be considered as a cofactor in acyl group transfer reactions. An example of a transacylation reaction is the synthesis of phosphatidic acid, which involves phosphoglycerol and two acyl-CoA molecules.

3. Hydrolases. These enzymes accelerate the hydrolysis reactions of organic compounds; Water is an obligatory participant in these processes. Depending on the nature of the hydrolyzed bond, hydrolases are divided into a number of subclasses: esterases, glycosidases, peptide hydrolases, etc. A distinctive feature of all hydrolases is that they are single-component enzymes.

Esterases catalyze the hydrolysis reactions of ester bonds.

Lipase accelerates the hydrolysis of external ester bonds in the triglyceride molecule. Particularly widespread are esterases that catalyze the hydrolysis of phosphoric acid esters and carbohydrates. These enzymes are called phosphatases .

Glycosidases accelerate the hydrolysis reactions of glycosidic bonds. An example of a glycosidase is maltase (-glucosidase).

Of the glycosidases acting on polysaccharides, the most common are amylases. .

Peptide hydrolases . Enzymes of this subclass catalyze the hydrolysis of peptide bonds in peptide and protein molecules. Peptide hydrolases do not hydrolyze all peptide bonds in protein and peptide molecules, but only certain ones. amidases accelerate the hydrolysis of amides of dicarboxylic amino acids - asparagine and glutamine.

4. Lyases. Enzymes of this class catalyze a variety of decomposition and synthesis reactions. Depending on which bond is cleaved or, conversely, formed, carbon-carbon, carbon-oxygen, carbon-nitrogen lyases are distinguished. Let us give examples of processes catalyzed by enzymes of these subclasses.

Carbon-carbon lyase . Enzymes that accelerate the decarboxylation of keto and amino acids are widely present in nature. Decarboxylases or carboxylases are two-component enzymes, the coenzyme of which is the phosphorus ester of vitamin B 1 - in the case of decarboxylation of keto acids and vitamin B 6 - in the case of decarboxylation of amino acids.

Carbon-oxygen lyase (hydrolyases). Enzymes of this subclass accelerate the reactions of hydration and dehydration of organic compounds.

These reactions constantly occur during the breakdown and synthesis of carbohydrates and fatty acids, so hydratases play an important role in the life of organisms. An example is fumarate hydratase, which adds a water molecule to the multiple bond of fumaric acid.

Carbon-nitrogen lyases catalyze direct deamination reactions of some amino acids.

5. Isomerases. Isomerases accelerate the processes of transformation of some isomers of organic compounds into others.

6. Ligases (synthetases). Enzymes of this class provide the synthesis of various organic compounds. A characteristic feature of enzymes of this class is the use of compounds that can supply energy for biosynthesis. One of these compounds is adenosine triphosphoric acid - ATP. An example of the action of a ligase is the synthesis of oxaloacetic acid from pyruvic acid by carboxylation.

Attention should be paid to the fact that the ATP molecule does not participate in the formation of reaction products, but simply breaks down to ADP and

H 3 PO 4; this releases the energy necessary for biosynthesis.

An important reaction is the formation of acyl-coenzyme A (acyl-CoA), which is also accelerated by an enzyme of this class.

ChapterIV.3.

Enzymes

Metabolism in the body can be defined as the totality of all chemical transformations to which compounds coming from outside undergo. These transformations include all known types of chemical reactions: intermolecular transfer of functional groups, hydrolytic and non-hydrolytic cleavage of chemical bonds, intramolecular rearrangement, new formation of chemical bonds and redox reactions. Such reactions occur in the body at extremely high speed only in the presence of catalysts. All biological catalysts are substances of protein nature and are called enzymes (hereinafter F) or enzymes (E).

Enzymes are not components of reactions, but only accelerate the achievement of equilibrium by increasing the rate of both direct and reverse conversion. Acceleration of the reaction occurs due to a decrease in the activation energy - the energy barrier that separates one state of the system (the initial chemical compound) from another (the reaction product).

Enzymes speed up a variety of reactions in the body. So, quite simple from the point of view of traditional chemistry, the reaction of the elimination of water from carbonic acid with the formation of CO 2 requires the participation of an enzyme, because without it, it proceeds too slowly to regulate blood pH. Thanks to the catalytic action of enzymes in the body, it becomes possible for reactions to occur that without a catalyst would proceed hundreds and thousands of times slower.

Properties of enzymes

1. Influence on the rate of a chemical reaction: enzymes increase the rate of a chemical reaction, but are not consumed themselves.

The rate of a reaction is the change in the concentration of reaction components per unit time. If it goes in the forward direction, then it is proportional to the concentration of the reactants, if in the opposite direction, then it is proportional to the concentration of the reaction products. The ratio of the rates of forward and reverse reactions is called the equilibrium constant. Enzymes cannot change the values ​​of the equilibrium constant, but the state of equilibrium occurs faster in the presence of enzymes.

2. Specificity of enzyme action. 2-3 thousand reactions take place in the cells of the body, each of which is catalyzed by a specific enzyme. The specificity of an enzyme's action is the ability to accelerate the course of one specific reaction without affecting the speed of others, even very similar ones.

There are:

Absolute– when F catalyzes only one specific reaction ( arginase– breakdown of arginine)

Relative(group special) – F catalyzes a certain class of reactions (for example, hydrolytic cleavage) or reactions involving a certain class of substances.

The specificity of enzymes is due to their unique amino acid sequence, which determines the conformation of the active center that interacts with the reaction components.

A substance whose chemical transformation is catalyzed by an enzyme is called substrate ( S ) .

3. Enzyme activity – the ability to accelerate the reaction rate to varying degrees. Activity is expressed in:

1) International units of activity - (IU) the amount of enzyme that catalyzes the conversion of 1 µM of substrate in 1 minute.

2) Catalach (kat) - the amount of catalyst (enzyme) capable of converting 1 mole of substrate in 1 s.

3) Specific activity - the number of activity units (any of the above) in the test sample to the total mass of protein in this sample.

4) Less commonly used is molar activity - the number of substrate molecules converted by one enzyme molecule per minute.

Activity depends primarily on temperature . This or that enzyme exhibits its greatest activity at the optimal temperature. For F of a living organism, this value is in the range +37.0 - +39.0° C, depending on the type of animal. As the temperature decreases, the Brownian motion slows down, the diffusion rate decreases and, consequently, the process of complex formation between the enzyme and the reaction components (substrates) slows down. If the temperature rises above +40 - +50° The enzyme molecule, which is a protein, undergoes a process of denaturation. In this case, the rate of the chemical reaction noticeably drops (Fig. 4.3.1.).

Enzyme activity also depends on pH of the environment . For most of them, there is a certain optimal pH value at which their activity is maximum. Since a cell contains hundreds of enzymes and each of them has its own pH limits, pH changes are one of the important factors in the regulation of enzymatic activity. So, as a result of one chemical reaction with the participation of a certain enzyme, the pH value of which lies in the range of 7.0 - 7.2, a product is formed that is an acid. In this case, the pH value shifts to the region of 5.5 – 6.0. The activity of the enzyme decreases sharply, the rate of product formation slows down, but at the same time another enzyme is activated, for which these pH values ​​are optimal and the product of the first reaction undergoes further chemical transformation. (Another example about pepsin and trypsin).

Chemical nature of enzymes. The structure of the enzyme. Active and allosteric centers

All enzymes are proteins with a molecular weight from 15,000 to several million Da. According to their chemical structure they are distinguished simple enzymes (consisting only of AA) and complex enzymes (have a non-protein part or a prosthetic group). The protein part is called - apoenzyme, and non-protein, if it is covalently linked to the apoenzyme, it is called coenzyme, and if the bond is non-covalent (ionic, hydrogen) – cofactor . The functions of the prosthetic group are as follows: participation in the act of catalysis, contact between the enzyme and the substrate, stabilization of the enzyme molecule in space.

The role of cofactor is usually played by inorganic substances - ions of zinc, copper, potassium, magnesium, calcium, iron, molybdenum.

Coenzymes can be considered as an integral part of the enzyme molecule. These are organic substances, among which there are: nucleotides ( ATP, UMF, etc.), vitamins or their derivatives ( TDF– from thiamine ( IN 1), FMN– from riboflavin ( AT 2), coenzyme A– from pantothenic acid ( AT 3), NAD, etc.) and tetrapyrrole coenzymes - hemes.

In the process of catalyzing a reaction, not the entire enzyme molecule comes into contact with the substrate, but a certain part of it, which is called active center. This zone of the molecule does not consist of a sequence of amino acids, but is formed by twisting the protein molecule into a tertiary structure. Individual sections of amino acids come closer to each other, forming a specific configuration of the active center. An important feature of the structure of the active center is that its surface is complementary to the surface of the substrate, i.e. AK residues in this zone of the enzyme are capable of entering into chemical interactions with certain groups of the substrate. One can imagine that The active site of the enzyme coincides with the structure of the substrate like a key and a lock.

IN active center two zones are distinguished: binding center, responsible for substrate attachment, and catalytic center, responsible for the chemical transformation of the substrate. The catalytic center of most enzymes includes AAs such as Ser, Cys, His, Tyr, Lys. Complex enzymes have a cofactor or coenzyme at the catalytic center.

In addition to the active center, a number of enzymes are equipped with a regulatory (allosteric) center. Substances that affect its catalytic activity interact with this zone of the enzyme.

Mechanism of action of enzymes

The act of catalysis consists of three successive stages.

1. Formation of an enzyme-substrate complex upon interaction through the active center.

2. Binding of the substrate occurs at several points in the active center, which leads to a change in the structure of the substrate and its deformation due to changes in the bond energy in the molecule. This is the second stage and is called substrate activation. In this case, a certain chemical modification of the substrate occurs and it is converted into a new product or products.

3. As a result of this transformation, the new substance (product) loses its ability to be retained in the active center of the enzyme and the enzyme-substrate, or rather, enzyme-product complex dissociates (breaks up).

Types of catalytic reactions:

A+E = AE = BE = E + B

A+B +E = AE+B = ABE = AB + E

AB+E = ABE = A+B+E, where E is the enzyme, A and B are substrates or reaction products.

Enzymatic effectors - substances that change the rate of enzymatic catalysis and thereby regulate metabolism. Among them there are inhibitors - slow down the reaction rate and activators - accelerating the enzymatic reaction.

Depending on the mechanism of reaction inhibition, competitive and non-competitive inhibitors are distinguished. The structure of the competitive inhibitor molecule is similar to the structure of the substrate and coincides with the surface of the active center like a key and a lock (or almost coincides). The degree of this similarity may even be higher than with the substrate.

If A+E = AE = BE = E + B, then I+E = IE¹

The concentration of the enzyme capable of catalysis decreases and the rate of formation of reaction products drops sharply (Fig. 4.3.2.).

A large number of chemical substances of endogenous and exogenous origin (i.e., those formed in the body and coming from outside - xenobiotics, respectively) act as competitive inhibitors. Endogenous substances are regulators of metabolism and are called antimetabolites. Many of them are used in the treatment of oncological and microbial diseases, as. they inhibit key metabolic reactions of microorganisms (sulfonamides) and tumor cells. But with an excess of substrate and a low concentration of the competitive inhibitor, its effect is canceled.

The second type of inhibitors is non-competitive. They interact with the enzyme outside the active site and excess substrate does not affect their inhibitory ability, as is the case with competitive inhibitors. These inhibitors interact either with certain groups of the enzyme (heavy metals bind to the thiol groups of Cys) or most often with the regulatory center, which reduces the binding ability of the active center. The actual process of inhibition is the complete or partial suppression of enzyme activity while maintaining its primary and spatial structure.

A distinction is also made between reversible and irreversible inhibition. Irreversible inhibitors inactivate the enzyme by forming a chemical bond with its AK or other structural components. This is usually a covalent bond to one of the active site sites. Such a complex practically does not dissociate under physiological conditions. In another case, the inhibitor disrupts the conformational structure of the enzyme molecule and causes its denaturation.

The effect of reversible inhibitors can be removed when there is an excess of substrate or under the influence of substances that change the chemical structure of the inhibitor. Competitive and non-competitive inhibitors are in most cases reversible.

In addition to inhibitors, activators of enzymatic catalysis are also known. They:

1) protect the enzyme molecule from inactivating influences,

2) form a complex with the substrate that binds more actively to the active center of F,

3) interacting with an enzyme that has a quaternary structure, they separate its subunits and thereby open up access for the substrate to the active center.

Distribution of enzymes in the body

Enzymes involved in the synthesis of proteins, nucleic acids and energy metabolism enzymes are present in all cells of the body. But cells that perform special functions also contain special enzymes. Thus, the cells of the islets of Langerhans in the pancreas contain enzymes that catalyze the synthesis of the hormones insulin and glucagon. Enzymes that are characteristic only of the cells of certain organs are called organ-specific: arginase and urokinase- liver, acid phosphatase- prostate. By changing the concentration of such enzymes in the blood, the presence of pathologies in these organs is judged.

In a cell, individual enzymes are distributed throughout the cytoplasm, others are embedded in the membranes of mitochondria and the endoplasmic reticulum, such enzymes form compartments, in which certain, closely interconnected stages of metabolism occur.

Many enzymes are formed in cells and secreted into anatomical cavities in an inactive state - these are proenzymes. Proteolytic enzymes (that break down proteins) are often formed as proenzymes. Then, under the influence of pH or other enzymes and substrates, their chemical modification occurs and the active center becomes accessible to the substrates.

There are also isoenzymes - enzymes that differ in molecular structure, but perform the same function.

Nomenclature and classification of enzymes

The name of the enzyme is formed from the following parts:

1. name of the substrate with which it interacts

2. nature of the catalyzed reaction

3. name of the enzyme class (but this is optional)

4. suffix -aza-

pyruvate - decarboxyl - aza, succinate - dehydrogen - aza

Since about 3 thousand enzymes are already known, they need to be classified. Currently, an international classification of enzymes has been adopted, which is based on the type of reaction catalyzed. There are 6 classes, which in turn are divided into a number of subclasses (presented only selectively in this book):

1. Oxidoreductases. Catalyze redox reactions. They are divided into 17 subclasses. All enzymes contain a non-protein part in the form of heme or derivatives of vitamins B2, B5. The substrate undergoing oxidation acts as a hydrogen donor.

1.1. Dehydrogenases remove hydrogen from one substrate and transfer it to other substrates. Coenzymes NAD, NADP, FAD, FMN. They accept the hydrogen removed by the enzyme, transforming it into a reduced form (NADH, NADPH, FADH) and transfer it to another enzyme-substrate complex, where they release it.

1.2. Oxidases - catalyze the transfer of hydrogen to oxygen to form water or H 2 O 2. F. Cytochrome oxidase respiratory chain.

RH + NAD H + O 2 = ROH + NAD + H 2 O

1.3. Monoxidases - cytochrome P450. According to its structure, it is both a hemoprotein and a flavoprotein. It hydroxylates lipophilic xenobiotics (according to the mechanism described above).

1.4. PeroxidasesAnd catalase- catalyze the decomposition of hydrogen peroxide, which is formed during metabolic reactions.

1.5. Oxygenases - catalyze reactions of oxygen addition to the substrate.

2. Transferases - catalyze the transfer of various radicals from a donor molecule to an acceptor molecule.

A A+ E + B = E A+ A + B = E + B A+ A

2.1. Methyltransferase (CH 3 -).

2.2.Carboxyl- and carbamoyltransferases.

2.2. Acyltransferases – Coenzyme A (transfer of acyl group - R -C=O).

Example: synthesis of the neurotransmitter acetylcholine (see chapter “Protein Metabolism”).

2.3. Hexosyltransferases catalyze the transfer of glycosyl residues.

Example: the cleavage of a glucose molecule from glycogen under the influence of phosphorylases.

2.4. Aminotransferases - transfer of amino groups

R 1- CO - R 2 + R 1 - CH - N.H. 3 - R 2 = R 1 - CH - N.H. 3 - R 2 + R 1- CO - R 2

They play an important role in the transformation of AK. The common coenzyme is pyridoxal phosphate.

Example: alanine aminotransferase(ALT): pyruvate + glutamate = alanine + alpha-ketoglutarate (see chapter “Protein Metabolism”).

2.5. Phosphotransferase (kinase) - catalyze the transfer of a phosphoric acid residue. In most cases, the phosphate donor is ATP. Enzymes of this class mainly take part in the breakdown of glucose.

Example: Hexo(gluco)kinase.

3. Hydrolases - catalyze hydrolysis reactions, i.e. splitting of substances with addition at the site where the water bond is broken. This class includes mainly digestive enzymes; they are single-component (do not contain a non-protein part)

R1-R2 +H 2 O = R1H + R2OH

3.1. Esterases - break down ester bonds. This is a large subclass of enzymes that catalyze the hydrolysis of thiol esters and phosphoesters.
Example: NH 2 ).

Example: arginase(urea cycle).

4.Lyases - catalyze reactions of molecular splitting without adding water. These enzymes have a non-protein part in the form of thiamine pyrophosphate (B 1) and pyridoxal phosphate (B 6).

4.1. C-C bond lyases. They are usually called decarboxylases.

Example: pyruvate decarboxylase.

5.Isomerases - catalyze isomerization reactions.

Example: phosphopentose isomerase, pentose phosphate isomerase(enzymes of the non-oxidative branch of the pentose phosphate pathway).

6.Ligases catalyze reactions for the synthesis of more complex substances from simpler ones. Such reactions require the energy of ATP. “Synthetase” is added to the name of such enzymes.

REFERENCES FOR THE CHAPTER IV.3.

1. Byshevsky A. Sh., Tersenov O. A. Biochemistry for the doctor // Ekaterinburg: Uralsky Rabochiy, 1994, 384 pp.;

2. Knorre D. G., Myzina S. D. Biological chemistry. – M.: Higher. school 1998, 479 pp.;

3. Filippovich Yu. B., Egorova T. A., Sevastyanova G. A. Workshop on general biochemistry // M.: Enlightenment, 1982, 311 pp.;

4. Leninger A. Biochemistry. Molecular basis of cell structure and functions // M.: Mir, 1974, 956 pp.;

5. Pustovalova L.M. Workshop on biochemistry // Rostov-on-Don: Phoenix, 1999, 540 p.

Proteins of nature that perform a role in the body

Mechanism of action of enzymes

Elucidation of the mechanisms underlying the catalytic process is one of the fundamental tasks and pressing problems not only of enzymology, but also of modern molecular biochemistry and biology.

Long before pure enzymes became available and their nature was clarified, it was believed that the combination of the enzyme with the substrate was crucial for the implementation of the enzymatic process. Attempts to detect a complex compound of an enzyme with a substrate for a long time did not lead to success, since such a complex is labile and disintegrates very quickly. The use of spectroscopy made it possible to identify enzyme-substrate complexes for catalase, peroxidase, alcohol dehydrogenase, and flavin-dependent enzymes.

The method of X-ray diffraction analysis made it possible to obtain a lot of important information about the structure and catalytic mechanisms of action of enzymes. This method was used to establish the association of substrate analogues with the enzymes lysozyme and chymotrypsin.

Some direct evidence of the existence of enzyme-substrate complexes was obtained for cases when, at one of the stages of the catalytic cycle, the enzyme is connected to the substrate by a covalent bond. An example is n-nitrophenylacetate, catalyzed by chymotrypsin. When the enzyme is mixed with this ester, chymotrypsin is acetylated at the hydroxyl group of the reactive serine residue. This stage proceeds quickly, but the hydrolysis of acetylchymotrypsin with the formation of acetate and free chymotrypsin is much slower. Therefore, in the presence of n-nitrophenylacetate, acetylchymotrypsin accumulates, which is easy to detect.

The presence of a substrate in the enzyme can be “caught” by converting the unstable EC complex into an inactive form, for example, by treating the enzyme-substrate complex with sodium borohydride, which has a strong reducing effect. A similar complex in the form of a stable covalent derivative was discovered in the enzyme aldolase. It turned out that the e-amino group of lysine interacts with the substrate molecule.

The substrate interacts with the enzyme at a specific part called the active site, or active zone of the enzyme.

The active center, or active zone, is understood as that part of the enzyme protein molecule that combines with the substrate (and cofactors) and determines the enzymatic properties of the molecule. The active center determines the specificity and catalytic activity of the enzyme and must be a structure of a certain degree of complexity, adapted for close proximity and interaction with the substrate molecule or its parts directly involved in the reaction.

Among the functional groups, a distinction is made between those that are part of the “catalytically active” site of the enzyme and those that form a site that provides specific affinity (binding of the substrate to the enzyme) - the so-called contact or “anchor” (or adsorption site of the active center of the enzyme).

The mechanism of action of enzymes is explained by the Michaelis-Menten theory. According to this theory, the process occurs in four stages.

Mechanism of action of enzymes: Stage I

A bond arises between the substrate (C) and the enzyme (E) - an enzyme-substrate complex EC is formed, in which the components are connected to each other by covalent, ionic, water and other bonds.

Mechanism of action of enzymes: Stage II

The substrate, under the influence of the attached enzyme, is activated and becomes available for the corresponding EC catalysis reactions.

Mechanism of action of enzymes: Stage III

EC catalysis is taking place. This theory has been confirmed by experimental studies.

And finally, stage IV is characterized by the release of the enzyme molecule E and reaction products P. The sequence of transformations can be displayed as follows: E + C - EC - EC * - E + P.

Specificity of enzyme action

Each enzyme acts on a specific substrate or group of substances that are similar in structure. The specificity of the action of enzymes is explained by the similarity of the configuration of the active center and the substrate. During the interaction, an enzyme-substrate complex is formed.