Isoenzymes, or isoenzymes- this is multiple forms of the enzyme, catalyzing the same reaction, but differing from each other in physical and chemical properties, in particular by affinity to the substrate, the maximum rate of the catalyzed reaction (activity), electrophoretic mobility or regulatory properties.

In living nature, there are enzymes whose molecules consist of two or more subunits with the same or different primary, secondary or tertiary structure. Subunits are often called protomers, and the combined oligomeric molecule is multimer(Fig. 14.8 a-d).

It is believed that the oligomerization process imparts increased stability and resistance to the action of denaturing agents, including heating, the effect of proteinases, etc., to protein subunits. , allowing under "soft" conditions to destroy only the Quaternary structure. The commonly used severe treatment methods (extreme pH values, high concentrations of guanidine chloride or urea) lead to the destruction of not only the quaternary, but also the secondary and tertiary structures of a stable oligomeric enzyme, the protomers of which are denatured and, as a result, devoid of biological activity.

Rice. 14.8. Models of the structure of some oligomeric enzymes: a – glutamate dehydrogenase molecule consisting of 6 protomers (336 kDa); b – RNA polymerase molecule; (c) half of the catalase molecule; d – molecular complex of pyruvate dehydrogenase

The absence of covalent, principal valent bonds between subunits should be pointed out. The bonds are mostly non-covalent, so such enzymes dissociate quite easily into protomers. A surprising feature of these enzymes is the dependence of the activity of the entire complex on the way the individual subunits are packaged. If a genetically distinct subunits can exist in more than one form, then, accordingly, an enzyme formed from two or more types of subunits combined in different quantitative proportions can exist in several similar, but not identical, forms. These types of enzymes are called isoenzymes (isoenzymes or, less commonly, isozymes).

One of the most studied enzymes, the multiplicity of forms of which has been studied in detail by gel electrophoresis, is lactate dehydrogenase (LDH), which catalyzes the reversible conversion of pyruvic acid into lactic acid. It can consist of four subunits of two different H- and M-types (cardiac and muscle). The active enzyme is one of the following combinations: HHHH, HHHM, HHMM, HMMM, MMMM, or H 4 , H 3 M, H 2 M 2 , HM 3 , M 4 . They correspond to the isoenzymes LDH 1, LDH 2, LDH 3, LDH 4, and LDH 5. At the same time, the synthesis of H- and M-types is carried out by different genes and is expressed differently in different organs.

Since H-protomers at pH 7.0-9.0 carry a more pronounced negative charge than M-protomers, the H 4 isoenzyme during electrophoresis will migrate from top speed in an electric field to the positive electrode (anode). The isoenzyme M 4 will move to the anode at the lowest speed, while the remaining isoenzymes will occupy intermediate positions (Fig. 14.9).

Rice. 14.9. Distribution and relative amount of LDH isoenzymes in various organs

Each tissue normally has its own ratio of forms (isoenzyme spectrum) of LDH. For example, in the heart muscle, the H 4 type, i.e., LDH 1, predominates, and in the skeletal muscles and liver, the M 4 type, i.e. LDH 5 .

These circumstances are widely used in clinical practice, since the study of the appearance of LDH isoenzymes (and a number of other enzymes) in blood serum may be of interest for the differential diagnosis of organic and functional lesions of organs and tissues. By changing the content of isoenzymes in the blood serum, one can judge both the topography of the pathological process and the degree of damage to an organ or tissue.

In some cases, the subunits have an almost identical structure and each contains a catalytically active site (for example, β-galactosidase, which consists of four subunits). In other cases, the subunits are not identical. An example of the latter is tryptophan synthase, which consists of two subunits, each of which is endowed with its own (but not the main) enzymatic activity, however, only when combined into a macromolecular structure, both subunits exhibit tryptophan synthase activity.

The term " multiple forms of the enzyme» applies to proteins that catalyze the same reaction and occur naturally in organisms of the same species. The term " isoenzyme» applies only to those multiple forms of enzymes that appear due to genetically determined differences in the primary structure of the protein (but not in forms resulting from the modification of one primary sequence).

It has long been found that all enzymes are proteins and have all the properties of proteins. Therefore, like proteins, enzymes are divided into simple and complex.

simple enzymes are made up of only amino acids, for example, pepsin , trypsin , lysozyme.

Complex enzymes(holoenzymes) have in their composition a protein part, consisting of amino acids - apoenzyme, and a non-protein part - cofactor. An example of complex enzymes are succinate dehydrogenase(contains FAD), aminotransferases(contain pyridoxal phosphate), peroxidase(contains heme) lactate dehydrogenase(contains Zn 2+), amylase(contains Ca2+).

Cofactor, in turn, can be called a coenzyme (NAD+, NADP+, FMN, FAD, biotin) or a prosthetic group (heme, oligosaccharides, metal ions Fe2+, Mg2+, Ca2+, Zn2+).

The division into coenzymes and prosthetic groups is not always unambiguous:
if the connection of the cofactor with the protein is strong, then in this case they speak of the presence prosthetic group,
but if a vitamin derivative acts as a cofactor, then it is called coenzyme regardless of bond strength.

For the implementation of catalysis, a full-fledged complex of apoprotein and cofactor is required; they cannot catalyze separately. The cofactor is part of the active center, participates in the binding of the substrate or in its transformation.

Like many proteins, enzymes can be monomers, i.e. consist of one subunit, and polymers consisting of several subunits.

Structural and functional organization of enzymes

In the composition of the enzyme, regions are distinguished that perform various functions:

1. Active center - a combination of amino acid residues (usually 12-16) that provides direct binding to the substrate molecule and catalysis. Amino acid radicals in the active center can be in any combination, while amino acids are located nearby, which are significantly distant from each other in the linear chain. There are two sections in the active center:

• anchor(contact, binding) - responsible for the binding and orientation of the substrate in the active center,
• catalytic- directly responsible for the implementation of the reaction.

Diagram of the structure of enzymes

Enzymes that have several monomers in their composition may have several active centers according to the number of subunits. Also, two or more subunits can form one active site.

In complex enzymes, the functional groups of the cofactor are necessarily located in the active center.

Scheme of the formation of a complex enzyme

2. Allosteric center (allos- alien) - the center of regulation of enzyme activity, which is spatially separated from the active center and is not available for all enzymes. Binding to the allosteric center of any molecule (called an activator or inhibitor, as well as an effector, modulator, regulator) causes a change in the configuration of the enzyme protein and, as a result, the rate of the enzymatic reaction.

Allosteric enzymes are polymeric proteins, the active and regulatory centers are located in different subunits.

Scheme of the structure of an allosteric enzyme

The product of this or one of the subsequent reactions, the substrate of the reaction, or another substance can act as such a regulator (see "Regulation of enzyme activity").

Isoenzymes

Isoenzymes are molecular forms of the same enzyme that result from small genetic differences in the primary structure of the enzyme, but catalyze the same reaction. Isoenzymes are different affinity to the substrate, maximum speed catalyzed reaction sensitivity to inhibitors and activators, conditions work (optimum pH and temperature).

As a rule, isoenzymes have Quaternary structure, i.e. composed of two or more subunits. For example, the dimeric enzyme creatine kinase (CK) is represented by three isozyme forms, composed of two types of subunits: M (eng. muscle- muscle) and B (eng. brain- brain). Creatine kinase-1 (CK-1) consists of type B subunits and is localized in the brain, creatine kinase-2 (CK-2) has one M- and B-subunit, is active in the myocardium, two M-subunits, specific to skeletal muscle.

There are also five isoenzymes lactate dehydrogenase(role of LDH) - an enzyme involved in glucose metabolism. The differences between them are in the different ratio of H subunits (eng. heart- heart) and M (eng. muscle- muscle). Lactate dehydrogenase types 1 (H 4) and 2 (H 3 M 1) are present in tissues with aerobic metabolism (myocardium, brain, cortical layer of the kidneys), have a high affinity for lactic acid (lactate) and convert it into pyruvate. LDH-4 (H 1 M 3) and LDH-5 (M 4) are found in tissues prone to anaerobic metabolism (liver, skeletal muscle, skin, renal medulla), have a low affinity for lactate and catalyze the conversion of pyruvate to lactate. In fabrics with intermediate the type of metabolism (spleen, pancreas, adrenal glands, lymph nodes) is dominated by LDH-3 (H 2 M 2).

Another example of isoenzymes is the group hexokinase, which attach a phosphate group to hexose monosaccharides and involve them in the reactions of cellular metabolism. Of the four isoenzymes, hexokinase IV is released ( glucokinase), which differs from other isoenzymes in its high specificity for glucose, low affinity for it, and insensitivity to inhibition by the reaction product.

Multienzyme complexes

In a multienzyme complex, several enzymes are firmly linked together into a single complex and carry out a series of successive reactions in which the reaction product is directly transferred to the next enzyme and is only him substrate. Arises tunnel effect, i.e. the substrate enters the "tunnel" created by the enzymes. As a result, intermediate metabolites avoid contact with environment, the time of their transition to the next active center is reduced and the reaction rate is significantly accelerated.

) and catalyze specific reactions. This ability arises as a result of the formation of an intermediate product when an antibody binds to an antigen (imitation of a transitional complex E-X enzymatic reaction).

Enzymes that catalyze the same chemical reaction, but differ in the primary structure of the protein, are called isoenzymes, or isoenzymes. They catalyze the same type of reaction with a fundamentally identical mechanism, but differ from each other in kinetic parameters, activation conditions, and features of the relationship between apoenzyme and coenzyme.

The nature of the appearance of isoenzymes is diverse, but most often due to differences in the structure of the genes encoding these isoenzymes. Consequently, isoenzymes differ in the primary structure of the protein molecule and, accordingly, in physicochemical properties. Methods for determining isoenzymes are based on differences in physicochemical properties.

In their structure, isoenzymes are mainly oligomeric proteins. Moreover, this or that tissue predominantly synthesizes certain types of protomers. As a result of a certain combination of these protomers, enzymes are formed with different structure- isomeric forms. The discovery of certain isoenzyme forms of enzymes makes it possible to use them for diagnosing diseases.

Isoforms of lactate dehydrogenase. The enzyme lactate dehydrogenase (LDH) catalyzes the reversible oxidation of lactate (lactic acid) to pyruvate (pyruvic acid) (see Section 7).

lactate dehydrogenase- an oligomeric protein with a molecular weight of 134,000 D, consisting of 4 subunits of 2 types: M (from English, muscle - muscle) and H (from English, heart - heart). The combination of these subunits underlies the formation of 5 isoforms of lactate dehydrogenase (Fig. 2-35, A). LDH 1 and LDH 2 are most active in the heart muscle and kidneys, LDH4 and LDH5 - in skeletal muscles and liver. In other tissues there are various forms of this enzyme.

LDH isoforms are characterized by electrophoretic mobility, which makes it possible to establish the tissue affiliation of LDH isoforms (Fig. 2-35, B).

Isoforms of creatine kinase. Creatine kinase (CK) catalyzes the formation of creatine phosphate:

The KK molecule is a dimer consisting of two types of subunits: M (from English, muscle - muscle) and B (from English, brain - brain). From these subunits, 3 isoenzymes are formed - BB, MB, MM. The BB isoenzyme is found predominantly in the brain, MM in skeletal muscle, and MB in the heart muscle. KA isoforms have different electrophoretic mobility (Fig. 2-36).

The activity of CK in the norm should not exceed 90 IU / l. Determination of CK activity in blood plasma is of diagnostic value in myocardial infarction (there is an increase in the level of the MB isoform). The amount of the MM isoform may increase with injuries and damage to skeletal muscles. The BB isoform cannot penetrate the blood-brain barrier; therefore, it is practically not detected in the blood even in strokes and has no diagnostic value.

Isoenzymes- these are enzymes, the synthesis of which is encoded by different genes, they have a different primary structure and different properties, but they catalyze the same reaction. Types of isoenzymes:

Organic - enzymes of glycolysis in the liver and muscles.

Cellular - cytoplasmic and mitochondrial malate dehydrogenase (enzymes are different, but catalyze the same reaction).

Hybrid - enzymes with quaternary structure, are formed as a result of non-covalent binding of individual subunits (lactate dehydrogenase - 4 subunits of 2 types).

Mutant - are formed as a result of a single mutation of a gene.

Alloenzymes - encoded by different alleles of the same gene.

10. I. The use of enzymes with therapeutic purpose in turn is divided into two types: 1) application for the purpose of replacement therapy and 2) in order to influence the enzyme on the focus of the disease.

Most commonly used for replacement therapy digestive enzymes, when the patient is found to be deficient. Examples include gastric juice preparations or pure pepsin or acidin-pepsin, which is indispensable for gastritis with secretory insufficiency, for dyspepsia in children. Pancreatin - the drug, which is a mixture of pancreatic enzymes, is used for pancreatitis, mostly of a chronic nature. Known drugs have the same value cholenzim, panzinorm, etc.

Another field of application of substitution therapy is the treatment of diseases associated with the so-called enzymopathies. These are congenital or hereditary diseases in which the synthesis of any enzymes is impaired. These diseases are usually extremely severe, children with a hereditary lack of any enzyme do not live long, suffer from severe mental disorders, physical and mental retardation. mental development. Replacement therapy can sometimes help overcome these disorders.

A number of enzyme preparations are used in surgical practice for cleaning the wound surface from pus, microbes, excess granulation tissue; in the clinic of internal diseases they are used: in order to thin viscous secrets, exudates, blood clots, for example, in severe inflammatory diseases of the lungs and bronchi. these are mainly enzymes - hydrolases, capable of breaking down natural biopolymers - proteins, NA, polysaccharides. In connection with their anti-inflammatory action, they are also used for thrombophlebitis, inflammatory-dystrophic forms. steam about dontoza, osteomyelitis, sinusitis, otitis and other inflammatory diseases.

These include enzymes such as trypsin, chymotrypsin, RNA-za, DNA-ase, fibrinolysin. fibrinolysin also used to remove intravascular clots. RNase and DNase are successfully used to treat some viral infections, for example, to kill herpes virus.

Enzymes such as hyaluronidase, collagenase, lidase, used to deal with excess scar formations.

Asparaginase- an enzyme produced by some strains of Escherichia coli. It has a therapeutic effect in some forms of tumors. The therapeutic effect is associated with the property of the enzyme to disrupt the metabolism of the amino acid asparagine, which is necessary for tumor cells to grow.

The use of enzyme preparations for therapeutic purposes is still a very young area of ​​medical science. The limitation here is the complexity of technologies and the high cost of obtaining pure enzyme preparations in a crystalline form suitable for storage and use in humans. In addition, when using enzyme preparations, other circumstances must also be taken into account:

1) Enzymes are proteins, and therefore in some cases can cause an unwanted allergic reaction.

2) Rapid decomposition of the introduced enzymes (a protein preparation, therefore, it is immediately captured by "scavenger" cells - macrophages, fibroblasts, etc. Hence, large concentrations of preparations are required to achieve the desired effect.

3) However, with increasing concentration, enzyme preparations can be toxic.

And yet, in cases where these obstacles can be overcome, enzyme preparations have an excellent therapeutic effect.

For example, these shortcomings are partially eliminated by converting enzymes into the so-called "immobilized" form.

You will read more about the methods of immobilization of enzymes and how to use them in your teaching aids.

The maximum rate of catalyzed (), electrophoretic mobility or regulatory properties.

Rice. 4.5. Models of the structure of some oligomeric .

The absence of covalent, principal valent bonds between subunits should be pointed out. The bonds are mostly non-covalent, so they dissociate quite easily into protomers. A surprising feature of these is the dependence of the entire complex on the way the individual subunits are packed together. If genetically distinct subunits can exist in more than one form, then, respectively, and formed from two or more types of subunits combined in different quantitative proportions, can exist in several similar, but not identical, forms. Similar varieties are called (isoenzymes or, more rarely, isozymes). In particular, if it consists of 4 subunits of two different types - H and M (cardiac and muscle), then the active can be one of the following combinations: HHHH, HHHM, HHMM, HMMM, MMMM, or H 4, H 3 M, H 2 M 2 , NM 3 , M 4 corresponding to LDH 1 , LDH 2 , LDH 3 , LDH 4 and LDH 5 . At the same time, the synthesis of H- and M-types is carried out differently and is expressed differently in different organs.

In some cases, the subunits have an almost identical structure and each contains a catalytically active site (for example, β-galactosidase, consisting of 4 subunits). In other cases, the subunits are not identical. An example of the latter is tryptophan synthase, consisting of 2 subunits, each of which is endowed with its own (but not the main) enzymatic, however, only when combined into a macromolecular structure, both subunits exhibit tryptophan synthase.

The term "multiple forms" is applicable to catalyzing the same and occurring in nature in one species. The term "" applies only to those plural forms, which appear due to genetically determined differences in (but not to forms resulting from the modification of one primary sequence).

One of the most studied 4 , the multiplicity of forms of which has been studied in detail by gel electrophoresis, is LDH, which catalyzes the reversible transformation into lactic acid. Five LDHs are formed from 4 subunits of approximately the same size, but two different types. Since H-protomers carry a more pronounced negative charge at pH 7.0–9.0 than M-protomers, consisting of 4 H-type subunits (H 4), will migrate at the highest rate to electric field to positive (). With the lowest speed will move to M 4, while the rest of the iso-enzymes will occupy intermediate positions. It should be emphasized that

Question 2. Isoenzymes. Concept. Examples of isoforms of lactate dehydrogenase (LDH) and creatine kinase (CK). Reactions catalyzed by LDH and CK. The value of determining the activity of isoenzymes in blood serum

Isoenzymes, or isoenzymes, are enzymes that catalyze the same type of reaction with a fundamentally identical mechanism, but differ from each other in kinetic parameters, activation conditions, and features of the relationship between apoenzyme and coenzyme.

The nature of the appearance of isoenzymes is diverse, but most often due to differences in the structure of the genes encoding these isoenzymes. Consequently, isoenzymes differ in the primary structure of the protein molecule and, accordingly, in physicochemical properties.

In their structure, isoenzymes are mainly oligomeric proteins. Moreover, this or that tissue synthesizes mainly certain types of protomers. As a result of a certain combination of these protomers, enzymes with different structures are formed - isomeric forms. The detection of certain isozyme forms of enzymes makes it possible to use them for diagnosing diseases.

Lactate dehydrogenase (LDH) is an oligomeric protein with a molecular weight of 134,000 D. LDH consists of 4 peptide chains of two types - M (from the English muscle) and H (from the English heart). There are 5 isoforms of LDH, slightly different in chemical and physical properties. Unlike total LDH, enzyme isoforms are more or less tissue-specific.

• LDH-1 (HHHH, H 4) - prevails in the heart, kidneys and red blood cells;
• LDH-2 (HHHM, H 3 M) - in the heart, spleen and lymph nodes; krebs isoform blood metabolic
• LDH-3 (HHMM, H 2 M 2) - in the lungs;
• LDH-4 (HMMM, HM 3) - in the pancreas, placenta;
• LDH-5 (MMMM, M 4) - in the liver and skeletal muscles.

The appearance in evolution of various isoforms of LDH is due to the peculiarities of the oxidative metabolism of tissues. Isoenzymes LDH4 and LDH5 (m-types) work effectively under anaerobic conditions, LDH1 and LDH2 (H-types) - in aerobic conditions, when pyruvate is rapidly oxidized to CO2 and H2O, and not reduced to lactic acid.

The enzyme lactate dehydrogenase (LDH) catalyzes the reversible oxidation of lactate (lactic acid) to pyruvate (pyruvic acid)

In a number of diseases, the activity of LDH in blood plasma is examined. Normally, LDH activity is 170 - 520 IU / l. An increase in the activity of certain LDH isoforms is observed in lesions of the heart, liver, kidneys, as well as in megaloblastic and hemolytic anemia. To make a diagnosis, it is necessary to study LDH isoforms in blood plasma by electrophoresis. Detection of tissue-specific isoforms of LDH in blood plasma is widely used as a diagnostic test. With liver damage, the activity of LDH 5 increases in the blood, and with myocardial infarction - LDH 1.

Creatine kinase (CK) is an enzyme that catalyzes the transfer of a phosphoryl residue from ATP to creatinine to form creatine phosphate and ADP. ATP (adenosine triphosphate) is a molecule that is a source of energy in the biochemical reactions of the human body.

The reaction catalyzed by creatine kinase provides energy for muscle contractions. There are creatine kinase contained in the mitochondria and the cytoplasm of cells.

The creatine kinase molecule consists of two parts, which can be represented by one of two subunits: M, from the English muscle - "muscle", and B, brain - "brain". Thus, in the human body, creatine kinase is present in three isomers: MM, MV, BB. The MM-isomer is found in the skeletal muscles and myocardium, MB - mainly in the myocardium, BB - in the tissues of the brain, in a small amount in any cells of the body.

The activity of CK in the norm should not exceed 90 IU / l. Determination of CK activity in blood plasma is of diagnostic value in myocardial infarction (there is an increase in the level of the MB isoform). The amount of the MM isoform may increase with injuries and damage to skeletal muscles. The BB isoform cannot penetrate the blood-brain barrier; therefore, it is practically not detected in the blood even in strokes and has no diagnostic value.