Let us consider the features of the synthesis of those proteins that are formed by membrane-bound ribosomes. As already noted, these are “export”, membrane and lysosomal proteins. This also includes peroxisomal proteins.
In the formation of all these proteins, a key role is played by:
Firstly, granular or rough ER (endoplasmic reticulum),
Secondly, the Golgi complex.
Thanks to these structures, two additional processes (in addition to translation and folding) occur:
Specific sorting (together with directed transport) and
Modification of newly formed proteins.
2.1. Processes in granular EPS
2.1.1. Structure of granular EPS
As is known, the ER (endoplasmic reticulum), or ER (endoplasmic reticulum), looks like sets membrane tanks (sacs), tubes and vesicles.
In fact, almost all of these structures represent a single continuous compartment (compartment), delimited by a membrane from the hyaloplasm. This compartment forms all kinds of invaginations and folds, which are perceived in the section as separate tubes, vesicles and flat cisterns located parallel to each other.
Only some vesicles can be truly detached from a given compartment. These are “vehicles” that carry a portion of newly synthesized proteins to the Golgi complex for further processes of sorting and modification.
EPS is divided into smooth and granular (rough). The peculiarity of the latter is that on the hyaloplasm side it is covered with ribosomes, which gives it a characteristic rough appearance. These ribosomes are called membrane-bound, in contrast to the free ones found in the hyaloplasm.
Thus, during the formation of “export” and other proteins under consideration (membrane, lysosomal, peroxisomal) broadcast is happening on granular EPS.
According to some data, the localization of this EPS is also important: “export” proteins are synthesized in some areas of the granular EPS, membrane proteins in others, and lysosomal proteins in others.
At the same time, the ribosomes used are no different from free ribosomes. They become membrane-bound only during translation. Outside of this process, ribosomes exist in the form of separate subunits. And the association of the latter occurs only upon initiation of translation, i.e., upon the formation of a complex with mRNA and the initiating aa-tRNA.
Therefore, speaking of both free and membrane-bound ribosomes, it is implied that both are in a state of translation, and, therefore, associated with the mRNA and the synthesized peptide. In this case, ribosomes are often included in polysomes - also free or membrane-bound.
Individual ribosomal subunits are never associated with the EPS.
Let us consider the features of translation on granular EPS taking into account these comments.
2.1.2. Broadcast Features
When translating “export” and other similar (according to the mechanism of synthesis) proteins, two problems arise:
Binding of a free ribosome that has begun translation to the ER membrane (and, apparently, in a certain region of the ER);
Penetration of the synthesized peptide into the internal space of the EPS.
In solving both of these problems, the key role is played by the so-called. signal sequence (SP) (Fig. 2.1), with which the primary polypeptide chain of any protein considered here always begins.
The main processing reactions include:
1. Removal from the N-terminus of methionine or even several amino acids by specific aminopeptidases.
2. Education disulfide bridges between cysteine residues.
3. Partial proteolysis– removal of part of the peptide chain, as is the case with insulin or proteolytic enzymes of the gastrointestinal tract.
4. Joining chemical group to amino acid residues of the protein chain:
phosphorus acids - for example, phosphorylation of the amino acids Serine, Threonine, Tyrosine is used in the regulation of enzyme activity or for binding calcium ions,
carboxyl groups - for example, with the participation of vitamin K, γ-carboxylation of glutamate occurs in the composition of prothrombin, proconvertin, Stewart factor, Christmas, which allows the binding of calcium ions during the initiation of blood clotting,
methyl groups - for example, methylation of arginine and lysine in histones is used to regulate genome activity,
hydroxyl groups - for example, the formation of hydroxyproline and hydroxylysine is necessary for the maturation of collagen molecules with the participation of vitamin C,
iodine– for example, in thyroglobulin, the addition of iodine is necessary for the formation of iodothyronine precursors of thyroid hormones,
5. Turn on prosthetic groups:
carbohydrate residues - for example, glycation is required in the synthesis of glycoproteins.
heme– for example, in the synthesis of hemoglobin, myoglobin, cytochromes, catalase,
vitamin coenzymes - biotin, FAD, pyridoxal phosphate, etc.
6. Combining protomers into a single oligomeric protein, for example, hemoglobin, collagen, lactate dehydrogenase, creatine kinase.
Protein folding
Folding is the process of arranging an elongated polypeptide chain into a regular three-dimensional spatial structure. To ensure folding, a group of auxiliary proteins called chaperones(chaperon, French - companion, nanny). They prevent the interaction of newly synthesized proteins with each other, isolate the hydrophobic regions of proteins from the cytoplasm and “remove” them inside the molecule, and correctly position the protein domains.
In general, chaperones contribute to the transition of protein structure from the primary level to the tertiary and quaternary levels.
When the function of chaperones is impaired and there is no folding in the cell, protein deposits– develops amyloidosis. There are about 15 variants of amyloidosis.
Broadcast is a good drug target
Many substances have the ability to bind to ribosomal elements or other translation factors. Some of these substances are used as drugs that are able to act at different levels of translation, for example:
1. Inactivation of initiation factors
interferon activates intracellular protein kinases, which, in turn, phosphorylate the protein initiation factor IF-2 and suppress its activity.
2. Violation of codon-anticodon interaction
streptomycin binds to the small subunit and causes a misreading of the first base of the codon.
3. Blockade of the elongation stage
tetracyclines block the A-center of the ribosome and deprive it of the ability to bind to aminoacyl-tRNA,
chloramphenicol binds to the 50S ribosomal particle and inhibits peptidyl transferase,
erythromycin binds to the 50S ribosomal particle and inhibits translocase,
puromycin its structure is similar to tyrosyl-tRNA, it enters the A-center of the ribosome and participates in the peptidyl transferase reaction, forming a bond with the existing peptide. After this, the puromycin-peptide complex is separated from the ribosome, which stops protein synthesis.
1. Primary structure of the protein. Dependence of the properties and conformation of proteins on the primary structure. Examples of protein polymorphisms, hemoglobin A and F, structural and functional differences. The role of fetal hemoglobin in period of intrauterine development of the fetus . Hereditary changes in the primary structure - molecular diseases (sickle cell anemia).
The primary structure of a protein is the sequence of alternating amino acids in a polypeptide chain. This structure is formed by peptide bonds between the α-amino and α-carboxyl groups of amino acids (see 1.4.2). Keep in mind that even small changes in the primary structure of a protein can significantly change its properties. An example of diseases that develop as a result of changes in the primary structure of a protein are hemoglobinopathies (hemoglobinoses).
Hemoglobin A (Hb A) is present in the red blood cells of healthy adults. Some people's blood contains abnormal (altered) hemoglobin - hemoglobin (Hb S). The only difference between the primary structure of Hb S and Hb A is the replacement of the hydrophilic glutamic acid residue with a hydrophobic valine residue in the terminal region of their β-chains.
As you know, the main function of hemoglobin is to transport oxygen to tissues. Under conditions of reduced partial pressure of O2, the solubility of hemoglobin S in water and its ability to bind and transport oxygen decreases. The red blood cells take on a sickle shape and are quickly destroyed, resulting in anemia (sickle cell anemia).
It has been established that the sequence of amino acid residues of the polypeptide chain of a protein carries the information necessary for the formation of the spatial structure of the protein. It has been established that each polypeptide sequence corresponds to only one stable variant of the spatial structure. The process of folding a polypeptide chain into a regular three-dimensional structure is called folding
Until recently, it was believed that the formation of the spatial structure of a protein occurs spontaneously, without the participation of any components. However, relatively recently it was discovered that this is true only for relatively small proteins (about 100 amino acid residues). In the process of folding larger proteins, special proteins take part - chaperones, which create the possibility of rapid formation of the correct spatial structure of the protein.
An example of protein polymorphism is hemoglobin, which has many forms. Hemoglobin A is the normal hemoglobin of an adult. This protein is a tetramer consisting of two pairs of polypeptide chains - monomers: two α-chain monomers and two β-chain monomers, or two α monomers and two δ monomers. Hemoglobin F is the fetal type of human hemoglobin. Hemoglobin F is a heterotetramer protein of two α-chains and two γ-chains of globin. Hemoglobin F has an increased affinity for oxygen (it contains serine instead of lysine) and allows the relatively small volume of fetal blood to perform oxygen supply functions more efficiently. However, hemoglobin F is less resistant to degradation and less stable. During the last trimester of pregnancy and after birth, hemoglobin F is gradually replaced by “adult” hemoglobin A (HbA), a less active oxygen transporter, but more resistant to destruction and more stable. Molecular diseases are hereditary disorders in the primary structure of the bun. For example, replacing the sixth glutamine amino acid with valine in the β-subunit of hemoglobin leads to the formation of hemoglobin S and the fact that the hemoglobin molecule as a whole cannot perform its main function - oxygen transport; In such cases, the person develops a disease called sickle cell anemia.
2. Conformation of the protein molecule (secondary and tertiary structures). Types of intramolecular bonds in proteins. Fibrillar and globular proteins (examples). Quaternary structure of protein. Examples of the structure and functioning of oligomeric proteins.
Protein secondary structure represents a method of folding a polypeptide chain into a helical or other conformation. In this case, hydrogen bonds are formed between the CO and NH groups of the peptide backbone of one chain or adjacent polypeptide chains. Several types of secondary structure of peptide chains are known, among which the main ones are the α-helix and β-sheet layer.
α-Helix- rigid structure, looks like a rod. The inner part of this rod is created by a tightly twisted peptide backbone, the amino acid radicals are directed outward. In this case, the CO group of each amino acid residue interacts with the NH group of the fourth residue from it. There are 3.6 amino acid residues per turn of the helix, and the helix pitch is 0.54 nm (Figure 2.1).
Figure 2.1.α-Helix.
Some amino acids prevent the chain from folding into an α-helix, and at their location the continuity of the helix is disrupted. These amino acids include proline (in which the nitrogen atom is part of a rigid ring structure and rotation around the N - C α bond becomes impossible), as well as amino acids with charged radicals that electrostatically or mechanically prevent the formation of an α-helix. If there are two such radicals (or more) within one turn (about 4 amino acid residues), they interact and deform the helix.
β-fold layer differs from an α-helix in that it is flat rather than rod-shaped. Formed by hydrogen bonds within one or more polypeptide chains. The peptide chains can be arranged in the same direction (parallel) or in opposite directions (antiparallel), resembling the bellows of an accordion. Lateral radicals are located above and below the plane of the layer.
Figure 2.2.β-folded layer.
Note that the type of secondary structure of a protein is determined by its primary structure. For example, at the location of the proline residue (the atoms of the pyrrolidine ring in proline lie in the same plane), the peptide chain bends, and hydrogen bonds between amino acids are not formed. Therefore, proteins with a high proline content (for example, collagen) are not able to form an α-helix. Amino acid radicals, which carry an electrical charge, also prevent helicalization.
2.1.3. The tertiary structure of a protein is the distribution in space of all the atoms of the protein molecule, or in other words, spatial packing of a helical polypeptide chain. The main role in the formation of the tertiary structure of a protein is played by hydrogen, ionic, hydrophobic and disulfide bonds, which are formed as a result of the interaction between amino acid radicals.
- Hydrogen bonds are formed between two polar uncharged radicals or between an uncharged and a charged radical, for example, serine and glutamine radicals:
- Ionic bonds can occur between oppositely charged radicals, for example, glutamate and arginine radicals:
- Hydrophobic interactions are typical for non-polar radicals, for example, valine and leucine:
- Disulfide
bonds are formed between the SH groups of two cysteine radicals located in different parts of the polypeptide chain:
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Based on the shape of the molecule and the characteristics of the formation of the tertiary structure, proteins are divided into globular and fibrillar.
Globular proteins- have a spherical or ellipsoidal molecule shape (globule). During the formation of a globule, hydrophobic amino acid radicals are immersed in the internal regions, while hydrophilic radicals are located on the surface of the molecule. When interacting with the aqueous phase, polar radicals form numerous hydrogen bonds. Proteins are held in a dissolved state due to their charge and hydration shell. In the body, globular proteins perform dynamic functions (transport, enzymatic, regulatory, protective). Globular proteins include:
- Albumen - blood plasma protein; contains many glutamate and aspartate residues; precipitates at 100% saturation of the solution with ammonium sulfate.
- Globulins - blood plasma proteins; Compared to albumin, they have a higher molecular weight and contain fewer glutamate and aspartate residues; they precipitate at 50% saturation of the solution with ammonium sulfate.
- Histones - are part of cell nuclei, where they form a complex with DNA. Contains many arginine and lysine residues.
Fibrillar proteins- have a thread-like shape (fibrils), form fibers and bundles of fibers. There are many covalent cross-links between adjacent polypeptide chains. Insoluble in water. The transition into solution is prevented by non-polar amino acid radicals and cross-links between peptide chains. In the body they perform mainly a structural function, providing mechanical strength to tissues. Fibrillar proteins include:
- Collagen - connective tissue protein. Its composition is dominated by the amino acids glycine, proline, and hydroxyproline.
- Elastin - more elastic than collagen, it is part of the walls of arteries and lung tissue; its composition is dominated by the amino acids glycine, alanine, and valine.
- Keratin - protein of the epidermis and skin derivatives; the amino acid cysteine predominates in its structure.
3. Hemoglobin is an allosteric protein. Conformational changes in the hemoglobin molecule. Cooperative effect. Regulators of hemoglobin affinity for oxygen. Structural and functional differences between myoglobin and hemoglobin.
Hemoproteins include: hemoglobin, myoglobin, cytochromes, peroxidase, catalase. These proteins contain as a prosthetic group heme.
According to its chemical structure, heme is protoporphyrin IX, associated with ferrous iron. Protoporphyrin IX is an organic compound belonging to the class of porphyrins. Protoporphyrin IX contains four substituted pyrrole rings connected by methine bridges =CH—. The substituents on the pyrrole rings are: four methyl groups CH3—, two vinyl groups CH2=CH— and two propionic acid residues — CH2—CH2—COOH. Heme is connected to the protein part as follows. Non-polar groups. Protoporphyrin IX interacts with hydrophobic regions of amino acids using hydrophobic bonds. In addition, there is a coordination bond between the iron atom and the imidazole histidine radical in the protein chain. Another coordination bond of the iron atom can be used to bind oxygen and other ligands.
The presence of heme-containing proteins in biological material is detected using a benzidine test (when benzidine and hydrogen peroxide are added, the test solution turns blue-green).
compare the structure and function of myoglobin and hemoglobin, remember the characteristic features of each of these proteins.
Myoglobin- a chromoprotein present in muscle tissue and having a high affinity for oxygen. The molecular weight of this protein is about 16,000 Da. The myoglobin molecule has a tertiary structure and represents one polypeptide chain connected to heme. Myoglobin does not have allosteric properties (see 2.4.), its oxygen saturation curve has the shape of a hyperbola (Figure 4). The function of myoglobin is to create an oxygen reserve in the muscles, which is consumed as needed, replenishing the temporary lack of oxygen.
Hemoglobin (Hb)- a chromoprotein present in red blood cells and involved in the transport of oxygen to tissues. Hemoglobin in adults is called hemoglobin A (Hb A). Its molecular weight is about 65,000 Da. The Hb A molecule has a quaternary structure and includes four subunits - polypeptide chains (designated α1, α2, β1 and β2, each of which is associated with heme.
Remember that hemoglobin is an allosteric protein; its molecules can reversibly change from one conformation to another. This changes the affinity of the protein for ligands. The conformation with the least affinity for the ligand is called the tense, or T-conformation. The conformation with the greatest affinity for the ligand is called the relaxed, or R-conformation.
Various environmental factors can shift this balance in one direction or another. Allosteric regulators affecting the affinity of Hb for O2 are: 1) oxygen; 2) H+ concentration (medium pH); 3) carbon dioxide (CO2); 4) 2,3-diphosphoglycerate (DPG). The attachment of an oxygen molecule to one of the hemoglobin subunits promotes the transition of a tense conformation to a relaxed one and increases the affinity for oxygen of other subunits of the same hemoglobin molecule. This phenomenon is called the cooperative effect. The complex nature of the binding of hemoglobin to oxygen is reflected by the hemoglobin O2 saturation curve, which has an S-shape (Figure 3.1).
Figure 3.1. Curves of myoglobin (1) and hemoglobin (2) oxygen saturation.
4. Biological functions of proteins. The role of the spatial organization of the polypeptide chain in the formation of active centers. Interaction of proteins with ligands. Denaturation of proteins.
Proteins play a vital role in the body, performing a variety of biological functions. Remember the most important ones and examples of corresponding proteins by studying Table 2.2.
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Table 2.2 |
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2.2.2. Please note that the basis for the functioning of any protein is its ability to selectively interact with strictly defined molecules or ions (ligands). For example, for enzymes that catalyze chemical reactions, the ligands will be the substances participating in these reactions (substrates), for transport proteins - the substances being transported, etc. 2.2.3. The ligand is able to interact not with the entire surface of the protein molecule, but only with a certain part of it, which is the binding center or active center. This center is formed by spatially close amino acid radicals at the level of the secondary or tertiary structure of the protein. The ability of a ligand to interact with the binding site is determined by its complementarity, that is, the mutual correspondence of their spatial structure (similar to the “key-lock” interaction). Non-covalent (hydrogen, ionic, hydrophobic) as well as covalent bonds are formed between the functional groups of the ligand and the binding center. The complementarity of the ligand and the binding site can explain the high specificity (selectivity) of the protein-ligand interaction. It is important to note that a change in the spatial structure of the protein during denaturation (see 2.4) leads to the destruction of binding centers and loss of the biological function of the protein. |
Denaturation of proteins This is called a change in the native (natural) physicochemical and, most importantly, biological properties of a protein due to a violation of its quaternary, tertiary and even secondary structure. Protein denaturation can be caused by:
- temperature above 60° C;
- ionizing radiation;
- concentrated acids and alkalis;
- salts of heavy metals (mercury, lead, cadmium);
- organic compounds (alcohols, phenols, ketones).
Denatured proteins are characterized by:
- change in the conformation of the molecule;
- decreased solubility in water;
- change in molecular charge;
- less resistance to the action of proteolytic enzymes;
- loss of biological activity.
Please note that under certain conditions it is possible to restore the original (native) conformation of the protein after removing the factor that caused the denaturation. This process is called regeneration.
Remember some examples of the use of protein denaturation in medicine:
- for sedimentation of blood plasma proteins when determining the content of non-protein substances in the blood;
- when carrying out disinfection and sanitary treatment;
- in the treatment and prevention of poisoning with salts of heavy metals (milk or egg white is used as an antidote);
- to obtain medicinal substances of protein nature (denaturation under mild conditions followed by renativation is used).
5. Structure and biological role of nucleotides.
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Nucleic acids or polynucleotides are high-molecular substances consisting of nucleotides connected in a chain by 3", 5" phosphodiester bonds. Each nucleotide consists of a nitrogenous base, a carbohydrate (pentose) and a phosphoric acid residue. The nitrogenous bases that make up nucleotides have the following structure:
Carbohydrates are represented by ribose and deoxyribose:
4.1.2. The nitrogenous base and pentose, connected by an N-glycosidic bond, form nucleoside. If ribose is present as a pentose in a nucleoside, then it is a ribonucleoside, and if deoxyribose is present, then it is a deoxyribonucleoside. |
4.1.3. Nucleotides are phosphorylated nucleosides. The phosphoric acid residue is usually attached to the hydroxyl group of the pentose at the 5" position via an ester bond. Examples:
Nucleoside diphosphates and nucleoside triphosphates, containing two and three phosphoric acid residues, respectively, are also found in cells. The biological role of these compounds will be discussed further.
6. Primary and secondary structures of DNA. Chargaff's rules. Principle of complementarity . Types of bonds in a DNA molecule. Biological role of DNA. Molecular diseases are a consequence of gene mutations.
Primary structure of nucleic acids called sequence of arrangement of mononucleotides in a DNA or RNA chain . The primary structure of nucleic acids is stabilized by 3",5" phosphodiester bonds. These bonds are formed by the interaction of the hydroxyl group in the 3" position of the pentose residue of each nucleotide with the phosphate group of the neighboring nucleotide (Figure 3.2),
Thus, at one end of the polynucleotide chain there is a free 5"-phosphate group (5"-end), and at the other there is a free hydroxyl group in the 3" position (3"-end). Nucleotide sequences are usually written in the direction from the 5" end to the 3" end.
DNA (deoxyribonucleic acid) found in the cell nucleus and has a molecular weight of about 1011 Da. Its nucleotides contain nitrogenous bases adenine, guanine, cytosine, thymine , carbohydrate deoxyribose and phosphoric acid residues. The content of nitrogenous bases in a DNA molecule is determined by Chargaff’s rules:
1) the number of purine bases is equal to the number of pyrimidine bases (A + G = C + T);
2) the amount of adenine and cytosine is equal to the amount of thymine and guanine, respectively (A = T; C = G);
3) DNA isolated from cells of different biological species differ from each other in the specificity coefficient:
(G + C) / (A + T)
These patterns in the structure of DNA are explained by the following features of its secondary structure:
1) a DNA molecule is built from two polynucleotide chains connected to each other by hydrogen bonds and oriented antiparallel (that is, the 3" end of one chain is located opposite the 5" end of the other chain and vice versa);
2) hydrogen bonds are formed between complementary pairs of nitrogenous bases. Thymine is complementary to adenine; this pair is stabilized by two hydrogen bonds. Cytosine is complementary to guanine; this pair is stabilized by three hydrogen bonds (see figure b). The more G-C pairs in a DNA molecule, the greater its resistance to high temperatures and ionizing radiation;
3) both DNA strands are twisted into a helix that has a common axis. The nitrogenous bases face the inside of the helix; In addition to hydrogen interactions, hydrophobic interactions also arise between them. The ribose phosphate moieties are located along the periphery, forming the core of the helix (see Figure 3.4).
Figure 3.4. DNA structure diagram. 
7. Primary and secondary structures of RNA. Types of RNA: structural features. The main components of the protein synthesizing system. Function of ribosomes. Adapter function of tRNA and the role of mRNA in protein synthesis.
RNA (ribonucleic acid) is found predominantly in the cytoplasm of the cell and has a molecular weight in the range of 104 - 106 Da. Its nucleotides contain nitrogenous bases adenine, guanine, cytosine, uracil , carbohydrate ribose and phosphoric acid residues. Unlike DNA, RNA molecules are built from a single polynucleotide chain, which can contain sections that are complementary to each other (Figure 3.5). These regions can interact with each other, forming double helices alternating with non-helical regions.
Figure 3.5. Scheme of the structure of transfer RNA.
Based on their structure and function, there are three main types of RNA:
1) messenger RNA (mRNA) transmit information about the structure of the protein from the cell nucleus to ribosomes;
2) transfer RNAs (tRNAs) transport amino acids to the site of protein synthesis;
3) ribosomal RNA (rRNA) are part of ribosomes and participate in protein synthesis.
8. Biosynthesis of DNA (replication) and mRNA (transcription). Processes of “maturation” of the primary transcript during the formation of mRNA.
Matrix biosynthesis- the process of assembling new macromolecules from monomers, the sequence of which is programmed using nucleic acids. Molecules used as programs in template biosynthesis are called matrices.
The three main template biosyntheses inherent in all living organisms without exception are DNA replication, transcription and translation.
- DNA replication occurs in the nucleus, precedes cell division, resulting in daughter cells receiving a full set of genes;
- transcription also occurs in the nucleus, during which matrix, transport and ribosomal RNAs are formed, which are involved in protein synthesis in the cell;
- broadcast occurs on ribosomes and leads to the formation of specific cellular proteins.
The connection between these processes is reflected in basic postulate of molecular biology: direction of information flow from genotype to phenotype: DNA → RNA → protein(arrows indicate the direction of information transfer).
4.3.2. In addition, some types of viruses are characterized by two more types of matrix syntheses:
- RNA replication - RNA synthesis on an RNA template;
- reverse transcription - DNA synthesis using RNA molecules as a template.
4.3.3. Let us try to formulate general patterns that are characteristic of all matrix biosyntheses.
- Monomers (nucleotides, amino acids) cannot directly participate in the synthesis of polymers; they must be in active form - nucleotides - in the form of nucleoside triphosphates, amino acids - in the form of compounds with tRNA.
- The synthesis of all polynucleotide and polypeptide chains consists of three main stages- initiation, elongation and termination.
- The matrix has special signal or a group of signals allowing identification of a coding element, where does it start information about the synthesized biopolymer chain. This signal, as a rule, does not coincide with the physical start point of the polymer chain of the matrix. Initiation is the process in which the first monomer unit is attached to the matrix molecule.
- For each act of initiation of biosynthesis there is a large number of elongation events, i.e. connection of the next monomer with the growing chain. Three components are involved in elongation: a) the terminal group of the synthesized polymer, b) the coding element of the matrix, c) the next molecule of the active monomer. All of them must be fixed in a certain way in the active center of the enzyme or ribosome.
- Each act of elongation begins with substrate selection by searching through all the substrates present in the system. The entry of the desired substrate into the active center is a signal for the enzymatic reaction to occur. connection of the monomer fragment with the end of the synthesized polymer chain. The addition of a monomer to a growing chain serves as a signal for moving the active center to one coding element of the matrix.
- The end of the product most often does not correspond to the end of the matrix; there should be a special signal that ensures that the chain stops growing, i.e. termination.
- The synthesis of a biologically active molecule is usually does not end with termination. The resulting polymer undergoes a number of transformations, such as partial hydrolysis and combination of several chains into one, modification of monomers in the polymer, attachment of a prosthetic part (to a polypeptide) or an apoprotein (to a polynucleotide).
Replication- the process of DNA self-duplication, or the biosynthesis of a daughter DNA molecule that is completely identical to the original molecule (matrix). Localization of the process is the cell nucleus. Basic principles of DNA replication:
- complementarity
- antiparallelism
- unipolarity
- need for priming- enzymes that synthesize DNA are only capable of extending the existing polynucleotide chain, so first a short RNA chain (seed or primer) is synthesized, to which deoxyribonucleotides are attached; the RNA primer that has fulfilled its role is removed;
- intermittency- one of the daughter strands (leading) grows continuously during the replication process, and the other (lagging) grows in the form of fragments several hundred nucleotides long (Okazaki fragments);
- semi-conservative- as a result of replication, two double daughter DNAs are formed, each of which retains (preserves) unchanged one of the halves of the maternal DNA.
4.4.2. Conditions required for DNA replication:
1) Matrix - DNA molecule (Figure 26.1, a);
2) Unraveling squirrels - break hydrogen bonds between the complementary bases of the DNA double helix, resulting in the formation of a replication fork (Figure 26.1, b);
3) DNA binding proteins- attach to separated DNA strands and prevent their reunification;
4) Primase (RNA polymerase)- an enzyme that synthesizes seed RNA.
5) - deoxyribonucleoside triphosphates (dATP, dGTP, dTTP, dCTP). They attach to the nitrogenous bases of polynucleotide chains using hydrogen bonds according to the principle of complementarity;
6) DNA polymerase - an enzyme that forms new polynucleotide chains from nucleoside triphosphates due to the formation of 3’,5’-phosphodiester bonds. The source of energy is the high-energy bonds of nucleoside triphosphates. On one branch of the replication fork, a continuous chain is synthesized, on the other - Okazaki fragments (Figure 26.1, c);
7) DNA ligase - an enzyme that connects Okazaki fragments into a single chain (Figure 26.1, d).
As a result, two identical DNA molecules are formed (Figure 26.1, e).
Transcription- RNA biosynthesis on a DNA matrix. The process of transcription also occurs in the cell nucleus. Basic principles of transcription:
- complementarity- the synthesized chains are complementary to the matrix;
- antiparallelism- the 5" end of the synthesized polynucleotide chain is opposite the 3" end of the template and vice versa;
- unipolarity- the synthesis of polynucleotide chains always occurs in the direction 5" → 3";
- bareness- RNA biosynthesis does not require a primer;
- asymmetry- the synthesis of the daughter chain occurs only on one strand of the DNA template, while the second is blocked.
4.5.2. Conditions required for transcription:
- Matrix- a section of one of the DNA chains (Figure 8.2, a);
- DNA-dependent RNA polymerase- the main enzyme involved in transcription. The place where the enzyme attaches to DNA is the promoter;
- Substrates and energy sources- ribonucleoside triphosphates (ATP, GTP, UTP, CTP). They bind to the nitrogenous bases of the transcribed DNA chain by hydrogen bonds according to the principle of complementarity.
9. Biosynthesis of proteins. Genetic code. The sequence of reactions during the synthesis of a polypeptide chain (initiation, elongation, termination) during translation on ribosomes. Post-translational modification of protein molecules. Protein synthesis disorders in childhood (kwashiorkor) .
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Broadcast(from English translation- translation) - translation of genetic information contained in mRNA into a linear sequence of amino acids in a polypeptide chain. This translation is carried out through the genetic (biological) code. 5.1.2.Genetic code - a sequence of nucleotides corresponding to certain amino acids. The genetic code is characterized by the following properties:
5.1.3. Amino acids and the nucleotide triplets that encode them are not complementary to each other. Therefore, there must be adapter molecules, each of which can interact with both a specific codon and the corresponding amino acid. Such molecules are transfer RNAs(Figure 8.3). Each tRNA contains a triplet of nucleotides - anticodon , which is complementary to a strictly defined mRNA codon. The 3' end of tRNA (acceptor region) is the site of attachment of the amino acid corresponding to the codon of the mRNA. |
Amino acid activation - preparatory stage of protein biosynthesis - includes their binding to specific tRNAs with the participation of an enzyme aminoacyl-tRNA synthetases. The reaction occurs in the cytoplasm of cells.
The translation process itself includes 3 stages - initiation, elongation, termination and occurs on ribosomes.
Each ribosome consists of a large and small subunit (40S and 60S) and contains an aminoacyl (A) and peptidyl (P) region. The peptidyl site binds the initiating aminoacyl-tRNA, all other aminoacyl-tRNAs are attached to the aminoacyl site.
1) Initiation stage - start of the broadcast. Conditions required for initiation:
- mRNA start codon (AUG);
- protein initiation factors;
- small and large ribosomal subunits;
- GTP (energy source for the closure of ribosomal subparticles);
- magnesium ions;
- initiating aminoacyl-tRNA (methionyl-tRNA) - binds with its anticodon to the initiating codon of mRNA in the peptidyl region of the ribosome.
As a result, initiation complex : mRNA - ribosome - methionyl-tRNA (Figure 5.3, a).
2) Elongation stage - lengthening of the polypeptide chain by 1 amino acid residue occurs in three steps:
- attachment to the initiating complex of aminoacyl-tRNA corresponding to the codon located in the aminoacyl region of the ribosome (Figure 5.3, b);
- transpeptidation - the formation of a peptide bond between amino acid residues (Figure 5.3, c). Energy source - GTP;
- translocation - movement of the ribosome relative to the mRNA by 1 triplet (Figure 5.3, d). The energy source is GTP. Protein factors take part in elongation.
The described process is repeated many times (according to the number of amino acids in the chain).
3) Termination stage - end of broadcast. It is ensured by the presence in the mRNA chain of one of the termination (nonsense) codons - UAA, UGA or UAG. Protein termination factors are involved in the release of the polypeptide (Figure 5.3e). When one of the nonsense codons appears in the aminoacyl region, termination factors stimulate the hydrolase activity of peptidyl transferase. Due to this, the bond between tRNA and peptide is hydrolyzed. GTP is not required for this reaction. After this, the peptide chain, tRNA and mRNA leave the ribosome, and its subparticles dissociate.
Thus, translation of mRNA leads to the formation of a peptide chain with a strictly defined sequence of amino acid residues. The next stage of protein formation is folding, i.e. folding of the peptide chain into a regular three-dimensional structure. If a protein consists of several subunits, then folding also includes combining them into a single macromolecule.
It is believed that small protein molecules containing about 100 aminoacyl residues can independently take on a three-dimensional structure; folding of larger polypeptide chains requires the participation of special proteins - chaperones.
Chaperones are called differently heat shock proteins since they not only ensure the correct folding of newly formed proteins, but also the renaturation of previously synthesized proteins that have undergone partial denaturation in the cell under the influence of various factors (overheating, irradiation, the action of free radicals, etc.).
5.2.2. Post-translational modifications protein molecules may include:
- partial proteolysis (for example, the conversion of a proenzyme to an enzyme);
- addition of a prosthetic group (phosphoric acid residues, carbohydrate residues, heme groups, etc.);
- modifications of side chains of amino acid residues:
- hydroxylation of proline to hydroxyproline in collagen,
- arginine methylation in histone,
- iodination of tyrosine in thyroglobulin).
5.2.3. The effect of toxic and medicinal substances on protein biosynthesis. Protein biosynthesis is one of the most complex processes occurring in cells. Its interruption or perversion is possible as a result of a violation of any of the three matrix syntheses.
Thus, mutagens (benzo(a)pyrene, lindane) disrupt DNA replication and thus interrupt protein synthesizing processes.
Some toxic substances (gossypol) can change the rate of transcription.
Drugs that affect protein biosynthesis include antibiotics and interferons.
Antibiotics that block matrix biosynthesis are used in the treatment of infectious diseases and malignant tumors. (see table 5.1).
Table 5.1
Antibiotics that inhibit matrix biosynthesis
10. Regulation of protein synthesis. Introduction to the operon. Induction and repression of synthesis in the human body. The role of hormones in the regulation of gene action. Inhibitors of matrix synthesis - antibiotics, interferons.
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5.3.1. Operon (transcripton)- a set of genes that can be turned on and off depending on the metabolic needs of the cell. The composition of the operon, along with structural genes (SG) , encoding the structure of certain proteins, includes DNA sections that perform regulatory functions (Figure 5.4). A group of structural genes responsible for the synthesis of enzymes of one metabolic pathway is under the control gene operator (GO) located nearby. The function of the operator gene is controlled by something spatially distant from it gene regulator (GR) , which produces repressor protein , in active or inactive form. An active repressor protein is able to bind to the operator gene and inhibit the transcription of structural genes, therefore, suppress protein synthesis. Substances that cause inactivation of the repressor protein are inductors protein synthesis, which have the opposite effect - corepressors. The initial substrates of metabolic pathways can act as inducers, and the end products of these pathways can act as corepressors. 5.3.2. There are two mechanisms for regulating protein synthesis - induction and repression . An example of an operon that is regulated by an inductive mechanism is lactose operon , which, along with the operator gene, includes 3 structural genes encoding enzymes for lactose catabolism (see Figure 5.4). Lactose is the inducer of this operon. At a high concentration of lactose in the medium, enzymes are synthesized, but at a low concentration - not. |
5.3.3. According to the mechanism of repression it is regulated histidine operon , containing an operator gene and 10 structural genes encoding enzymes necessary for the biosynthesis of histidine (see Figure 5.5). Histidine is a corepressor of this operon. At a high concentration of histidine in the medium, the synthesis of enzymes stops; in the absence of histidine, they are synthesized.
11. The role of enzymes in metabolism. Hereditary enzymopathies V early childhood . Variety of enzymes. Specificity of enzyme action. Classification of enzymes. Isoenzymes, multienzymes.
The course of metabolic processes in the body is determined by the action of numerous enzymes - biological catalysts of protein nature. They speed up chemical reactions without being consumed. Term "enzyme" comes from the Latin word fermentum - sourdough. Along with this concept, the equivalent term is used in the literature "enzyme" (en zyme - in yeast) of Greek origin. Hence the branch of biochemistry that studies enzymes is called “enzymology”.
Enzymology forms the basis for knowledge at the molecular level of the most important problems of human physiology and pathology. The digestion of nutrients and their use for energy production, the formation of structural and functional components of tissues, muscle contraction, the transmission of electrical signals along nerve fibers, the perception of light by the eye, blood clotting - each of these physiological mechanisms is based on the catalytic action of certain enzymes. Numerous diseases have been shown to directly impair enzymatic catalysis; determination of enzyme activity in blood and other tissues provides valuable information for medical diagnostics; enzymes or their inhibitors can be used as medicinal substances. Thus, knowledge of the most important features of enzymes and the reactions they catalyze is necessary for a rational approach to the study of human diseases, their diagnosis and treatment.
The classification is based on the most important feature by which one enzyme differs from another - this is the reaction it catalyzes. The number of types of chemical reactions is relatively small, which made it possible to divide all currently known enzymes into 6 most important classes, depending on the type of reaction being catalyzed. These classes are:
- oxidoreductases (redox reactions);
- transferases (transfer of functional groups);
- hydrolases (cleavage reactions involving water);
- lyases (breaking bonds without the participation of water);
- isomerases (isomeric transformations);
- ligases (synthesis with the consumption of ATP molecules).
7.4.3.
Enzymes of each class are divided into subclasses,
guided by the structure of the substrates. Subclasses combine enzymes that act on similarly constructed substrates. Subclasses are divided into subclasses, V which further refine the structure of chemical groups that distinguish substrates from each other. Within the sub-subclasses they list individual enzymes.
All classification divisions have their own numbers. Thus, any enzyme receives its own unique code number, consisting of four numbers separated by dots. The first number denotes the class, the second - the subclass, the third - the subsubclass, the fourth - the number of the enzyme within the subclass. For example, the enzyme α-amylase, which breaks down starch, is designated as 3.2.1.1, where:
3 — type of reaction (hydrolysis);
2 - type of bond in the substrate (glycosidic);
1 - type of bond (O-glycosidic);
1 - enzyme number in the subclass
The above-described decimal numbering method has one important advantage: it allows one to bypass the main inconvenience of continuous numbering of enzymes, namely: the need to change the numbers of all subsequent ones when including a newly discovered enzyme in the list. A new enzyme may be placed at the end of the corresponding subclass without disturbing the rest of the numbering. Likewise, when new classes, subclasses and sub-subclasses are identified, they can be added without disturbing the numbering order of previously established divisions. If, after receiving new information, it becomes necessary to change the numbers of some enzymes, the previous numbers are not assigned to new enzymes in order to avoid misunderstandings.
Speaking about the classification of enzymes, it should also be noted that enzymes are classified not as individual substances, but as catalysts for certain chemical transformations. Enzymes isolated from different biological sources and catalyzing identical reactions can differ significantly in their primary structure. However, in the classification list they all appear under the same code number.
So, knowing the enzyme code number allows you to:
- eliminate ambiguities if different researchers use the same name for different enzymes;
- make searching for information in literary databases more efficient;
- obtain additional information about the amino acid sequence, spatial structure of the enzyme, and genes encoding enzyme proteins from other databases.
Isoenzymes - these are multiple forms of one enzyme that catalyze the same reaction, but differ in physical and chemical properties (affinity for the substrate, maximum speed of the catalyzed reaction, electrophoretic mobility, different sensitivity to inhibitors and activators, pH optimum and thermal stability). Isoenzymes have a quaternary structure, which is formed by an even number of subunits (2, 4, 6, etc.). Enzyme isoforms are formed by different combinations of subunits.
As an example, consider lactate dehydrogenase (LDH), an enzyme that catalyzes a reversible reaction:
NADH 2 NAD +
pyruvate ← LDH → lactate
LDH exists in the form of 5 isoforms, each of which consists of 4 protomers (subunits) of 2 types M (muscle) and H (heart). The synthesis of M and H type protomers is encoded by two different genetic loci. LDH isoenzymes differ at the level of quaternary structure: LDH 1 (NNNN), LDH 2 (NNMM), LDH 3 (NNMM), LDH 4 (NMMM), LDH 5 (MMMM).
Polypeptide chains of the H and M types have the same molecular weight, but the former are dominated by carboxylic amino acids, the latter by diamino acids, so they carry different charges and can be separated by electrophoresis.
Oxygen metabolism in tissues affects the isoenzyme composition of LDH. Where aerobic metabolism dominates, LDH 1, LDH 2 predominate (myocardium, adrenal glands), where anaerobic metabolism - LDH 4, LDH 5 (skeletal muscles, liver). During the individual development of the organism, changes in oxygen content and LDH isoforms occur in tissues. In the embryo, LDH 4 and LDH 5 predominate. After birth, the content of LDH 1 and LDH 2 increases in some tissues.
The existence of isoforms increases the adaptive capacity of tissues, organs, and the body as a whole to changing conditions. The metabolic state of organs and tissues is assessed by changes in isoenzyme composition.
12. Properties of enzymes. Dependence of the rate of enzymatic reaction on the concentration of enzyme and substrate, temperature and pH of the environment.
The protein nature of enzymes determines the appearance of a number of properties in them that are generally uncharacteristic of inorganic catalysts: oligodynamicity, specificity, dependence of the reaction rate on temperature, pH of the medium, concentration of the enzyme and substrate, the presence of activators and inhibitors.
Under oligodynamism Enzymes are highly effective in very small quantities. This high efficiency is explained by the fact that enzyme molecules continuously regenerate during their catalytic activity. A typical enzyme molecule can regenerate millions of times per minute. It must be said that inorganic catalysts are also capable of accelerating the transformation of a quantity of substances that is many times greater than their own mass. But no inorganic catalyst can compare with enzymes in terms of efficiency.
An example is the enzyme rennin, produced by the gastric mucosa of ruminants. One molecule of it in 10 minutes at 37°C is capable of causing coagulation (curdling) of about a million molecules of milk caseinogen.
Another example of the high efficiency of enzymes is provided by catalase. One molecule of this enzyme at 0°C breaks down about 50,000 molecules of hydrogen peroxide per second:
2 H2O2 2 H2O + O2
The effect of catalase on hydrogen peroxide is to change the activation energy of this reaction from approximately 75 kJ/mol without a catalyst to 21 kJ/mol in the presence of the enzyme. If colloidal platinum is used as a catalyst for this reaction, then the activation energy is only 50 kJ/mol.
7.2.2. When studying the influence of any factor on the rate of an enzymatic reaction, all other factors should remain unchanged and, if possible, have an optimal value.
The rate of enzymatic reactions is measured by the amount of substrate converted per unit of time, or the amount of product formed. The speed change is carried out at the initial stage of the reaction, when the product is still practically absent and the reverse reaction does not occur. In addition, at the initial stage of the reaction, the concentration of the substrate corresponds to its original amount.
7.2.3. Dependence of the rate of enzymatic reaction (V) on enzyme concentration [E](Figure 7.3). At a high substrate concentration (multiple times the enzyme concentration) and other factors remaining constant, the rate of the enzymatic reaction is proportional to the enzyme concentration. Therefore, knowing the rate of the reaction catalyzed by the enzyme, we can draw a conclusion about its amount in the material under study.
Figure 7.3.Dependence of the rate of enzymatic reaction on enzyme concentration
7.2.4. Dependence of reaction rate on substrate concentration [S]. The dependence graph looks like a hyperbola (Figure 7.4). At a constant enzyme concentration, the rate of the catalyzed reaction increases with increasing substrate concentration up to the maximum value Vmax, after which it remains constant. This should be explained by the fact that at high substrate concentrations, all active centers of enzyme molecules are associated with substrate molecules. Any excess substrate can combine with the enzyme only after the reaction product is formed and the active site is freed.
Figure 7.4.Dependence of the rate of enzymatic reaction on the concentration of the substrate.
The dependence of the reaction rate on the substrate concentration can be expressed by the Michaelis-Menten equation:
,
where V is the reaction rate at the substrate concentration [S], Vmax is the maximum speed and KM is the Michaelis constant.
The Michaelis constant is equal to the substrate concentration at which the reaction rate is half the maximum. The determination of KM and Vmax is of great practical importance, as it allows one to quantitatively describe most enzymatic reactions, including reactions involving two or more substrates. Different chemicals that alter enzyme activity have different effects on Vmax and KM values.
7.2.5. Dependence of the reaction rate on t - the temperature at which the reaction occurs (Figure 7.5) is complex. The temperature value at which the reaction rate is maximum represents the temperature optimum of the enzyme. The temperature optimum for most enzymes in the human body is approximately 40°C. For most enzymes, the optimal temperature is equal to or higher than the temperature at which the cells are located.
Figure 7.5. Dependence of the rate of enzymatic reaction on temperature.
At lower temperatures (0° - 40°C), the reaction rate increases with increasing temperature. When the temperature rises by 10°C, the rate of the enzymatic reaction doubles (temperature coefficient Q10 is 2). The increase in reaction rate is explained by an increase in the kinetic energy of the molecules. With a further increase in temperature, the bonds that support the secondary and tertiary structure of the enzyme are broken, that is, thermal denaturation. This is accompanied by a gradual loss of catalytic activity.
7.2.6. Dependence of the reaction rate on the pH of the medium (Figure 7.6). At a constant temperature, the enzyme works most efficiently within a narrow pH range. The pH value at which the reaction rate is maximum represents the optimum pH of the enzyme. Most enzymes in the human body have an optimum pH within the range of pH 6 - 8, but there are enzymes that are active at pH values outside this range (for example, pepsin, which is most active at pH 1.5 - 2.5).
A change in pH, either in the acidic or alkaline direction from the optimum, leads to a change in the degree of ionization of the acidic and basic groups of amino acids that make up the enzyme (for example, COOH groups of aspartate and glutamate, NH2 groups of lysine, etc.). This causes a change in the conformation of the enzyme, resulting in a change in the spatial structure of the active center and a decrease in its affinity for the substrate. In addition, at extreme pH values, the enzyme denatures and is inactivated.
Figure 7.6. Dependence of the rate of enzymatic reaction on the pH of the medium.
It should be noted that the pH optimum characteristic of an enzyme does not always coincide with the pH of its immediate intracellular environment. This suggests that the environment in which the enzyme is located regulates its activity to some extent.
7.2.7. Dependence of the reaction rate on the presence of activators and inhibitors . Activators increase the rate of the enzymatic reaction. Inhibitors reduce the rate of enzymatic reactions.
Inorganic ions can act as enzyme activators. It is believed that these ions cause the enzyme or substrate molecules to adopt a conformation that promotes the formation of an enzyme-substrate complex. This increases the probability of interaction between the enzyme and the substrate, and consequently the rate of the reaction catalyzed by the enzyme. For example, salivary amylase activity increases in the presence of chloride ions.
13. Mechanism of action of enzymes. Catalytic (active) center. Coenzymes and cofactors. Competitive and non-competitive inhibition. Use of competitive inhibitors as drugs.
Active center (Ac) is a part of the enzyme molecule that specifically interacts with the substrate and is directly involved in catalysis. Ats, as a rule, is located in a niche (pocket). Two regions can be distinguished in Ac: the substrate binding site - substrate area (contact pad) and actually catalytic center .
Most substrates form at least three bonds with the enzyme, due to which the substrate molecule attaches to the active site in the only possible way, which ensures the substrate specificity of the enzyme. The catalytic center provides the choice of the chemical transformation path and the catalytic specificity of the enzyme.
A group of regulatory enzymes has allosteric centers , which are located outside the active center. “+” or “-” modulators that regulate enzyme activity can be attached to the allosteric center.
There are simple enzymes, consisting only of amino acids, and complex enzymes, which also include low molecular weight organic compounds of a non-protein nature (coenzymes) and (or) metal ions (cofactors).
Coenzymes - these are organic substances of a non-protein nature that take part in catalysis as part of the catalytic site of the active center. In this case, the protein component is called apoenzyme , and the catalytically active form of a complex protein is holoenzyme . Thus: holoenzyme = apoenzyme + coenzyme.
The following function as coenzymes:
- hemes,
- nucleotides,
- coenzyme Q,
- FAFS,
- Glutathione
- derivatives of water-soluble vitamins:
A coenzyme that is attached to the protein part by covalent bonds is called prosthetic group . These are, for example, FAD, FMN, biotin, lipoic acid. The prosthetic group is not separated from the protein part. A coenzyme that is attached to the protein part by non-covalent bonds is called cosubstrate . These are, for example, NAD +, NADP +. The cosubstrate attaches to the enzyme at the time of reaction.
Enzyme cofactors are metal ions necessary for the catalytic activity of many enzymes. Potassium, magnesium, calcium, zinc, copper, iron, etc. ions act as cofactors. Their role is diverse; they stabilize substrate molecules, the active center of the enzyme, its tertiary and quaternary structure, and ensure substrate binding and catalysis. For example, ATP binds to kinases only in conjunction with Mg 2+ .
14. Basic mechanisms for regulating the action of enzymes and their role in the regulation of metabolism. Proenzymes.
8.4.1. As already noted, enzymes are catalysts whose activity can be regulated. Therefore, through enzymes it is possible to control the speed of chemical reactions in the body. Regulation of enzyme activity can be carried out by interaction with them of various biological components or foreign compounds (for example, drugs and poisons), which are commonly called modifiers or enzyme regulators. Under the influence of modifiers on the enzyme, the reaction can be accelerated (in this case they are called activators) or slow down (in this case they are called inhibitors).
8.4.2. Enzyme activation is determined by the acceleration of biochemical reactions that occurs after the action of the modifier. One group of activators consists of substances that affect the region of the active center of the enzyme. These include enzyme cofactors and substrates. Cofactors (metal ions and coenzymes) are not only obligatory structural elements of complex enzymes, but also essentially their activators.
Of the metal ions, the activity of many enzymes is affected by: NH4+, Na+, Mg2+, K+, Ca2+, Mn2+, Zn2+, Fe2+, Fe3+, Co2+. Heavy metal ions, as a rule, have an inhibitory effect. The actions of cations are generally quite specific, but in most cases the enzyme is activated by more than one cation. The phenomenon of antagonism between ions is also observed. The best known is the antagonism between Na+ and K+ and between Mg2+ and Ca2+.
Magnesium is a natural activator of enzymes acting on phosphorylated substrates (phosphatases, kinases, synthetases), but under in vitro conditions it can be replaced by manganese.
Anions generally have little effect on enzyme activity, and their effects lack specificity. The exception is amylase, which is activated by chlorides and, to a lesser extent, by other halogens. The effect of the activating ion also varies depending on pH. The degree of enzyme purification also affects the activating ion concentration and the specificity of activation. Highly purified enzymes are characterized by greater selectivity towards activating ions.
8.4.3. The activating effect of metal ions is realized in various ways. The most typical mechanism is the inclusion of an ion in the structure of the catalytic center of the enzyme, which is inactive without it. This is a typical function of a metal as a coenzyme. Another fairly common function of an activating metal is to form a bond between an enzyme and a substrate, or between an enzyme, a coenzyme and a substrate. For example, Zn2+ ions in the alcohol dehydrogenase enzyme form 2 coordination bonds with the NAD+ coenzyme molecule, 3 coordination bonds with the apoenzyme molecule, and the sixth coordination bond attaches the substrate.
Metal ions, as well as substrates, coenzymes, their precursors and structural analogs, can be used in practice as drugs that regulate enzyme activity.
While the catalytically active protein is called an enzyme (or enzyme), the inactive enzyme precursor is called a proenzyme (or zymogen).
Activation of proteins by partial proteolysis is a process widespread in biological systems. Here are some examples.
- digestive enzymes that hydrolyze proteins are synthesized in the stomach and pancreas in the form of proenzymes: pepsin - in the form of pepsinogen, trypsin - in the form of trypsinogen, etc.
- Blood coagulation is a cascade of reactions of proteolytic activation of proenzymes. This provides a quick response to blood vessel damage.
- some protein hormones are synthesized as inactive precursors. For example, insulin is formed from proinsulin.
- fibrillar connective tissue protein collagen is also formed from a precursor - procollagen.
15. Principles of quantitative determination of enzymes. Units of enzyme activity. Main directions of use of enzymes in medicine. Enzymodiagnostics, enzyme therapy, use of enzymes as reagents.
The unique property of enzymes to accelerate chemical reactions can be used to quantify the content of these biocatalysts in biological material (tissue extract, blood serum, etc.). Under correctly selected experimental conditions, there is almost always a proportionality between the amount of enzyme and the rate of the catalyzed reaction, therefore, by the activity of the enzyme, one can judge its quantitative content in the test sample.
Measurement of enzyme activity is based on comparing the rate of a chemical reaction in the presence of an active biocatalyst with the rate of reaction in a control solution in which the enzyme is absent or inactivated.
The material under study is placed in an incubation environment where optimal temperature, pH, concentrations of activators and substrates are created. At the same time, a control sample is carried out, to which the enzyme is not added. After some time, the reaction is stopped by adding various reagents (changing the pH of the medium, causing denaturation of proteins, etc.) and analyzing the samples.
In order to determine the rate of an enzymatic reaction, you need to know:
- the difference in concentrations of the substrate or reaction product before and after incubation;
- incubation time;
- amount of material taken for analysis.
Most often, enzyme activity is assessed by the amount of reaction product formed. This is done, for example, when determining the activity of alanine aminotransferase, which catalyzes the following reaction:
Enzyme activity can also be calculated based on the amount of substrate consumed. An example is a method for determining the activity of α-amylase, an enzyme that breaks down starch. By measuring the starch content in the sample before and after incubation and calculating the difference, the amount of substrate broken down during incubation is found.
There are a large number of methods for measuring enzyme activity, differing in technique, specificity, and sensitivity.
Most often used to determine photoelectrocolorimetric methods . These methods are based on color reactions with one of the products of enzyme action. In this case, the color intensity of the resulting solutions (measured on a photoelectrocolorimeter) is proportional to the amount of the product formed. For example, during reactions catalyzed by aminotransferases, α-keto acids accumulate, which give red-brown compounds with 2,4-dinitrophenylhydrazine:
If the biocatalyst under study has low specificity of action, then it is possible to select a substrate whose reaction results in the formation of a colored product. An example is the determination of alkaline phosphatase, an enzyme widely distributed in human tissues; its activity in blood plasma changes significantly in diseases of the liver and skeletal system. This enzyme, in an alkaline environment, hydrolyzes a large group of phosphate esters, both natural and synthetic. One of the synthetic substrates is paranitrophenyl phosphate (colorless), which in an alkaline environment breaks down into orthophosphate and paranitrophenol (yellow).
The progress of the reaction can be monitored by measuring the gradually increasing color intensity of the solution:
For enzymes with high specificity of action, such a selection of substrates is usually impossible.
Spectrophotometric methods are based on changes in the ultraviolet spectrum of chemicals taking part in the reaction. Most compounds absorb ultraviolet rays, and the absorbed wavelengths are characteristic of certain groups of atoms present in the molecules of these substances. Enzymatic reactions cause intramolecular rearrangements, as a result of which the ultraviolet spectrum changes. These changes can be recorded on a spectrophotometer.
Spectrophotometric methods, for example, determine the activity of redox enzymes containing NAD or NADP as coenzymes. These coenzymes act as acceptors or donors of hydrogen atoms and are thus either reduced or oxidized during metabolic processes. Reduced forms of these coenzymes have an ultraviolet spectrum with an absorption maximum at 340 nm; oxidized forms do not have this maximum. Thus, when lactate dehydrogenase acts on lactic acid, hydrogen is transferred to NAD, which leads to an increase in the absorption of NADH at 340 nm. The magnitude of this absorption in optical units is proportional to the amount of reduced form of the coenzyme formed.
By changing the content of the reduced form of the coenzyme, the activity of the enzyme can be determined.
Fluorimetric methods. These methods are based on the phenomenon of fluorescence, which consists in the fact that the object under study, under the influence of radiation, emits light with a shorter wavelength. Fluorimetric methods for determining enzyme activity are more sensitive than spectrophotometric methods. Relatively new and even more sensitive are chemiluminescent methods using the luciferin-luciferase system. Such methods make it possible to determine the rate of reactions that occur with the formation of ATP. When luciferin (a carboxylic acid of complex structure) interacts with ATP, luciferyl adenylate is formed. This compound is oxidized with the participation of the enzyme luciferase, which is accompanied by a flash of light. By measuring the intensity of light flashes, it is possible to determine amounts of ATP on the order of several picomoles (10-12 mol).
Titrometric methods . A number of enzymatic reactions are accompanied by a change in the pH of the incubation mixture. An example of such an enzyme is pancreatic lipase. Lipase catalyzes the reaction:
The resulting fatty acids can be titrated, and the amount of alkali used for titration will be proportional to the amount of fatty acids released and, therefore, to the lipase activity. Determination of the activity of this enzyme is of clinical importance.
Manometric methods are based on measuring in a closed reaction vessel the volume of gas released (or absorbed) during an enzymatic reaction. Using such methods, the oxidative decarboxylation reactions of pyruvic and α-ketoglutaric acids, which proceed with the release of CO2, were discovered and studied. Currently, these methods are rarely used.
The International Enzyme Commission has proposed unit of activity of any enzyme, take such an amount of enzyme that, under given conditions, catalyzes the conversion of one micromole (10-6 mol) of substrate per unit time (1 min, 1 hour) or one microequivalent of the affected group in cases where more than one group in each substrate molecule is attacked (proteins, polysaccharides and others). The temperature at which the reaction is carried out must be indicated. Enzyme activity measurements can be expressed in units general, specific and molecular activity.
For a unit total enzyme activity based on the amount of material taken for research. Thus, the activity of alanine aminotransferase in the liver of rats is 1670 μmol of pyruvate per hour per 1 g of tissue; Cholinesterase activity in human serum is 250 µmol acetic acid per hour per 1 ml of serum at 37°C.
High values of enzyme activity both in normal and pathological conditions require special attention of the researcher. It is recommended to work with low levels of enzyme activity. To do this, the enzyme source is taken in smaller quantities (the serum is diluted several times with physiological solution, and a smaller percentage homogenate is prepared for the tissue). In this case, conditions for saturation with the substrate are created in relation to the enzyme, which contributes to the manifestation of its true activity.
Total enzyme activity is calculated using the formula:
Where A- enzyme activity (total), ΔС- difference in substrate concentrations before and after incubation; IN- amount of material taken for analysis, t- incubation time; n- breeding.
It should be borne in mind that indicators of the activity of enzymes in blood serum and urine, studied for diagnostic purposes, are expressed in units of total activity.
Since enzymes are proteins, it is important to know not only the overall enzyme activity in the material being tested, but also the enzymatic activity of the protein present in the sample. For a unit specific activity take an amount of enzyme that catalyzes the conversion of 1 µmol of substrate per unit time per 1 mg of sample protein. To calculate the specific activity of the enzyme, it is necessary to divide the total activity by the protein content in the sample:
The worse the enzyme is purified, the more extraneous ballast proteins are in the sample, the lower the specific activity. During purification, the amount of such proteins decreases, and accordingly, the specific activity of the enzyme increases. Suppose that in the original biological material that is the source of the enzyme (chopped liver, pulp from plant tissue), the specific activity was equal to 0.5 µmol/(mg protein × min). After fractional precipitation with ammonium sulfate and gel filtration through Sephadex, it increased to 25 µmol/(mg protein x min), i.e. increased 50 times. Evaluating the efficiency of purification of enzyme preparations is used in the production of medicines of an enzymatic nature.
Specific activity is determined when it is necessary to compare the activity of different preparations of the same enzyme. If it is necessary to compare the activity of different enzymes, molecular activity is calculated.
Molecular activity (or enzyme turnover number) is the number of moles of substrate that are converted by 1 mole of enzyme per unit of time (usually 1 minute). Different enzymes have different molecular activities. A decrease in the number of enzyme turnover occurs under the influence of non-competitive inhibitors. By changing the conformation of the catalytic center of the enzyme, these substances reduce the affinity of the enzyme for the substrate, which leads to a decrease in the number of substrate molecules reacting with one enzyme molecule per unit time.
16. Nutrition is an integral part of metabolism. The main components of the diet and their role. Replaceable and irreplaceable components of the diet. Balanced diet. Consequences of unbalanced nutrition in children .
Full-fledged is a diet that meets a person’s energy needs and contains the required amount of essential nutrients to ensure normal growth and development of the body.
Factors influencing the body's need for energy and nutrients: gender, age and body weight of a person, his physical activity, climatic conditions, biochemical, immunological and morphological characteristics of the body.
All nutrients can be divided into five classes:
1. proteins; 2. fats; 3. carbohydrates; 4. vitamins; 5. minerals.
In addition, any diet must contain water, as a universal solvent.
The essential components of the diet are:
- essential amino acids - valine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan;
- essential fatty acids - linoleic, linolenic, arachidonic;
- water- and fat-soluble vitamins;
- inorganic (mineral) elements - calcium, potassium, sodium, chlorine, copper, iron, chromium, fluorine, iodine and others.
11.1.2. Balanced diet. A diet containing nutrients in a ratio optimal for maximum satisfaction of the plastic and energy needs of the human body is called balanced diet. It is believed that the most favorable ratio of proteins, fats and carbohydrates is close to 1:1:4, provided that the total calorie content of the diet corresponds to the energy expenditure of a given person. So, for a male student weighing 60 kg, energy consumption is on average 2900 kcal per day and the diet should contain: 80-100 g of proteins, 90 g of fats, 300 - 400 g of carbohydrates.
17. Biological value of food proteins. Quantity and quality of proteins in human nutrition. Replaceable and essential amino acids. Combination of food products that are mutually complementary in amino acid composition. Characteristics of the protein diet of children. Consequences of insufficient protein nutrition in children.
Biological role of food proteins is that they serve as a source of irreplaceable and replaceable amino acids. Amino acids are used by the body to synthesize its own proteins; as precursors of non-protein nitrogenous substances (hormones, purines, porphyrins, etc.); as a source of energy (oxidation of 1 g of proteins provides approximately 4 kcal of energy).
Food proteins are divided into complete and incomplete.
Complete food proteins - of animal origin, contain all amino acids in the required proportions and are well absorbed by the body.
Incomplete proteins - of plant origin, do not contain, or contain insufficient amounts of one or more essential amino acids. Thus, grain crops are deficient in lysine, methionine, and threonine; Potato protein contains little methionine and cysteine. To obtain protein-rich diets, you should combine plant proteins that complement each other in amino acid composition, for example, corn and beans.
Daily requirement: at least 50 g per day, on average 80-100 g.
11.2.2. Protein deficiency in childhood causes: 1. decreased body resistance to infections; 2. growth arrest due to impaired synthesis of growth factors; 3. energy deficiency of the body (depletion of carbohydrate and fat depots, catabolism of tissue proteins); 4. weight loss - malnutrition. During protein starvation, edema is observed, which occurs due to a decrease in protein content in the blood ( hypoalbuminemia) and disturbances in the distribution of water between blood and tissues.
18. Digestion of proteins. Proteinases. Mechanism of activation of gastrointestinal proteinases . Specificity (selectivity) of hydrolysis of peptide bonds. Features of protein digestion in infants, protein digestion disorders in children . Rotting of proteins (amino acids) in the large intestine.
The digestion of proteins, that is, their breakdown into individual amino acids, begins in the stomach and ends in the small intestine. Digestion occurs under the action of gastric, pancreatic and intestinal juices, which contain proteolytic enzymes (proteases or peptidases). Proteolytic enzymes belong to the class of hydrolases. They catalyze the hydrolysis of peptide bonds CO—NH protein molecule.
All proteolytic enzymes can be divided into two groups:
- exopeptidases- catalyze the rupture of the terminal peptide bond with the release of the N- or C-terminal amino acid;
- endopeptidases- hydrolyze peptide bonds within the polypeptide chain, the reaction products are peptides with a lower molecular weight.
10.1.3. Most proteolytic enzymes involved in the digestion of proteins and peptides are synthesized and secreted into the cavity of the digestive tract in the form of inactive precursors - proenzymes (zymogens). Therefore, the proteins of the cells producing proenzymes are not digested. Activation of proenzymes occurs in the lumen of the gastrointestinal tract through partial proteolysis - the cleavage of part of the zymogen peptide chain.
The bulk of amino acids formed in the digestive tract as a result of protein digestion are absorbed into the blood and replenish the body’s amino acid pool. A certain amount of unabsorbed amino acids undergo decay in the large intestine.
Rotting - transformations of amino acids caused by the activity of microorganisms in the large intestine. Strengthening the processes of decay of amino acids can be facilitated by:
- excess intake of proteins from food;
- congenital and acquired disorders of the absorption of amino acids in the intestines;
- decreased intestinal motor function.
As a result of the decay of amino acids, various substances are formed, many of which are toxic to the body. Some examples of decay products are given in Table 10.2.
Table 10.2
Products of decay of amino acids in the intestines.
10.2.2. The products of amino acid decay are xenobiotics- substances that are foreign to the human body and must be neutralized (inactivated).
19. The role of lipids in the body. Dietary lipids, daily requirements for children of different ages. Features of the use of lipids in various tissues. Brown adipose tissue. Deposition and mobilization of fats in adipose tissue. Obesity.
The composition of dietary fats consists mainly of triacylglycerols (98%), phospholipids and cholesterol. Triacylglycerols of animal origin contain many saturated fatty acids and have a solid consistency. Vegetable fats contain more unsaturated fatty acids and have a liquid consistency (oils).
Biological role: 1. are one of the main sources of energy; 2. serve as a source of essential polyunsaturated fatty acids; 3. promote the absorption of fat-soluble vitamins from the intestines. Polyunsaturated fatty acids necessary for the body to build phospholipids, which form the basis of all cell membrane structures and blood lipoproteins. In addition, linoleic acid is used for the synthesis of arachidonic acid, which serves as a precursor to prostaglandins, prostacyclins, thromboxanes and leukotrienes.
Daily requirement: 90-100 g, of which 30% should be vegetable oils. The nutritional value of vegetable fats is higher than that of animal fats, since with the same energy effect - 9 kcal per 1 g, they contain more essential fatty acids.
11.3.2. Violation of the ratio of the proportion of plant and animal fats in the diet leads to a change in the ratio of various classes of lipoproteins in the blood and, as a consequence, to coronary heart disease and atherosclerosis.
20. Digestion of fats. Lipases and phospholipases. Bile acids and paired bile acids: structure, formation, biological role. Features of lipid digestion in children . Lipid digestion disorders.
The main site of lipid digestion is the upper small intestine. The following conditions are necessary for the digestion of lipids:
- presence of lipolytic enzymes;
- conditions for lipid emulsification;
- optimal pH values of the environment (within 5.5 - 7.5).
10.3.2. Various enzymes are involved in the breakdown of lipids. Dietary fats in an adult are broken down mainly by pancreatic lipase; Lipase is also found in intestinal juice and saliva; in infants, lipase is active in the stomach. Lipases belong to the class of hydrolases; they hydrolyze ester bonds -O-SO- with the formation of free fatty acids, diacylglycerols, monoacylglycerols, glycerol
Glycerophospholipids supplied with food are exposed to specific hydrolases - phospholipases, which cleave ester bonds between the components of phospholipids. The specificity of the action of phospholipases is shown in Figure 10.4.
Figure 10.4. Specificity of the action of enzymes that break down phospholipids.
The products of phospholipid hydrolysis are fatty acids, glycerol, inorganic phosphate, nitrogenous bases (choline, ethanolamine, serine).
Dietary cholesterol esters are hydrolyzed by pancreatic cholesterol esterase to form cholesterol and fatty acids.
10.3.3. Understand the structure of bile acids and their role in the digestion of fats. Bile acids are the end product of cholesterol metabolism and are formed in the liver. These include: cholic (3,7,12-trioxycholanic), chenodeoxycholic (3,7-dioxycholanic) and deoxycholic (3, 12-dioxycholanic) acids (Figure 10.5, a). The first two are primary bile acids (formed directly in hepatocytes), deoxycholic acid is secondary (as it is formed from primary bile acids under the influence of intestinal microflora).
In bile, these acids are present in conjugated form, i.e. in the form of compounds with glycine H2N-CH2-COOH or taurine H2N-CH2-CH2-SO3H(Figure 10.5, b).
Figure 10.5. The structure of unconjugated (a) and conjugated (b) bile acids.
15.1.4. Bile acids have amphiphilic properties: hydroxyl groups and side chain are hydrophilic, cyclic structure is hydrophobic. These properties determine the participation of bile acids in the digestion of lipids:
1) bile acids are capable emulsify fats, their molecules with their non-polar part are adsorbed on the surface of fat droplets, at the same time hydrophilic groups interact with the surrounding aqueous environment. As a result, the surface tension at the interface between the lipid and aqueous phases decreases, as a result of which large fat droplets are broken into smaller ones;
2) bile acids, along with bile colipase, are involved in activation of pancreatic lipase, shifting its pH optimum to the acidic side;
3) bile acids form water-soluble complexes with hydrophobic products of fat digestion, which contributes to their absorption into the wall of the small intestine.
Bile acids, which penetrate into the enterocytes during absorption along with hydrolysis products, enter the liver through the portal system. These acids can be re-secreted with bile into the intestines and participate in the processes of digestion and absorption. Such enterohepatic circulation bile acids can be carried out up to 10 or more times a day.
In many cases, proteins are synthesized as predecessors– biologically inactive molecules. Their functional activity manifests itself as a result of transformations called postsynthetic or post-translational modification (processing).
Examples of post-translational modification of proteins:
Proteolytic cleavage of N-terminal formylmethionine or methionine;
Cleavage of signal peptides;
Partial proteolysis;
- post-translational modification of proteins by amino acid radicals: covalent addition of a prosthetic group, methylation of lysine and arginine radicals, acetylation of the N-terminal amino acid, phosphorylation of histones and non-histone chromatin proteins, hydroxylation of the proline radical; addition of oligosaccharide fragments (glycosylation) to asparagine, serine and threonine radicals, etc.
The selection of the correct protein structure occurs with the participation of proteins chaperones. Hydrophobic regions on the surface of the chaperone-70 globule interact with hydrophobic regions of the synthesized chain, protecting it from improper interactions with other cytosolic proteins. Chaperone-60s are involved in correcting the spatial structure of misfolded or damaged chains.
Mutations in a chaperone that is part of the eye lens lead to clouding of the lens due to protein aggregation and the development of cataracts.
Transport of synthesized proteins across membranes
The synthesized protein enters the cytosol from the ribosome. If it is not used for the needs of the cell itself, i.e. refers to exported (secreted) proteins, then it is transported through the membrane using low molecular weight peptides (15-30 amino acid residues) containing hydrophobic radicals. This signal peptides. A channel is formed in the membrane through which the signal peptide penetrates into the endoplasmic reticulum tank and drags along the synthesized protein molecule. Under the influence signal peptidase The N-terminal signal sequence is cleaved off, and the protein exits the cell through the Golgi apparatus in the form of a secretory vesicle.
REGULATION OF PROTEIN SYNTHESIS
The concentration of many proteins in the cell is not constant and changes depending on the state of the cell and external conditions. This occurs as a result of regulation of the rates of protein synthesis and breakdown.
In mammalian cells exist two types of regulation of protein biosynthesis:
- short-term, ensuring the body’s adaptation to environmental changes;
- long-term, stable, which determines cell differentiation and different protein composition of organs and tissues.
The most common mechanism for regulating protein synthesis is regulation at the transcription level(formation of the primary transcript).
Synthesis in the basal state is called constitutive synthesis.
Distinguish two forms of regulation – induction of synthesis(positive regulation) and repression of synthesis(negative regulation). Concepts of induction and repression suggest a change in the rate of protein synthesis relative to initial (basal) level. If the rate of constitutive protein synthesis is high, then the protein is regulated by the mechanism of synthesis repression, and, conversely, at a low basal rate, induction of synthesis is observed.
According to the theory of F. Jacob and J. Monet, in the genetic apparatus of a bacterial cell there are operons– DNA segments containing structural genes for certain proteins ( cistrons), and regulatory regions.
Reading the genetic code begins with promoter located next to operator genome. The operator gene is located on the extreme segment of the structural gene. It either prohibits or allows the replication of mRNA on DNA.
The activity of the operon controls gene regulator. Repressor protein communicates between the operon and the gene-regulator. The repressor is formed in nuclear ribosomes on mRNA synthesized at the regulator gene. It forms a complex with the operator gene and blocks the synthesis of mRNA, and, consequently, protein. The repressor can bind to low molecular weight substances - inducers or effectors. After this, it loses the ability to bind to the operator gene, the operator gene leaves the control of the regulator gene, and mRNA synthesis begins. This is the induction of synthesis (Fig. 14).
U eukaryotes the mechanisms regulating protein synthesis are more complex. Positive regulatory mechanisms predominate. The main regulatory point is the transcription initiation stage. Regulatory elements that stimulate transcription are called enhancers, and those suppressing it - silencers. They can be located either close to the promoter or at a distance from it, and selectively bind to protein regulators: enhancers – with inducer proteins, silencers – with repressor proteins. The process of interaction of regulatory elements with regulatory proteins is regulated by signaling molecules - hormones, some metabolites.
Knowledge of the structure and functioning of ribosomes in prokaryotes and eukaryotes has made it possible to develop new types of antibiotics. The synthesis of nucleic acids and proteins is a key process necessary to maintain cell life. If it is turned off in any way, the cell will die. There are drugs that disrupt the synthesis of purine bases and amino acids, the synthesis of nucleic acids (Fig. 16), and protein synthesis at various levels only in bacterial cells (Table 4).
Figure 16. Drugs that disrupt the synthesis of nucleic acids
Table 4
Agents that inhibit the synthesis of bacterial cell proteins
GENETIC ENGINEERING
Genetic Engineering- a system of experimental techniques that make it possible to construct artificial genetic structures in the laboratory (in vitro) in the form of so-called recombinant or hybrid DNA molecules.
Recombinant DNA Technology uses the following methods:
1. Specific cleavage of DNA with restriction enzymes. The targets of restriction enzymes are often palindromes of 4-6 base pairs - restriction sites. Some restriction enzymes introduce breaks along the axis of symmetry, forming so-called “blunt” ends. Others are shifted; “sticky” ends are formed, that is, the fragments have single-stranded mutually complementary sections four nucleotides long at their ends. Such fragments are especially convenient for creating recombinant DNA.
2. Nucleotide sequencing. Using electrophoresis, DNA fragments that vary in size can be separated, and then each fragment can be examined separately. This allows you to build restriction map, which indicates the position of each restriction site relative to other sites.
3. Construction of recombinant DNA:
To obtain recombinant DNA, plasmids are isolated from E. coli and part of the circular DNA molecule is removed from them using a restriction enzyme. The complementary strands of the DNA molecule are cut in different places, resulting in the formation of “sticky” ends. Sticky ends are created on the DNA fragment selected for transplantation using the same restriction enzyme. If you mix a DNA fragment (gene) and a plasmid, they are connected by “sticky” ends. Then, using a ligase, a circular DNA molecule is again obtained, but now it, together with the plasmid DNA, contains the gene selected for transplantation. That's what it is recombinant DNA.
Possible:
- cross-linking at the same “sticky” ends (restriction enzyme ligase method). Regions that are complementary to each other tend to associate through base pairing. To repair breaks, the enzyme DNA ligase is used.
- stitching along “blunt” ends (connector method). Blunt ends can be joined by DNA ligase. The reaction efficiency is lower than when cross-linking at sticky ends.
- stitching together fragments with unlike sticky ends. Apply linkers- chemically synthesized oligonucleotides that represent restriction sites or a combination thereof. There are blunt end-sticky end linkers.
The procedure described above is complex and produces only very small amounts of recombinant DNA.
4. Cloning (reproduction) of recombinant DNA:
- in vivo DNA cloning.
If recombinant plasmids are added to an E. coli culture, they can be incorporated into bacterial cells - recombinant bacteria are obtained. Plasmids in the cell begin to replicate. When bacteria multiply, the newly formed bacterial cells also contain these plasmids. Cloned recombinant plasmids can be isolated from recombinant bacteria, and from them the DNA fragment under study can be isolated. In this way, a gene or any other DNA fragment can be isolated in quantities sufficient for research purposes.
- amplification(increase number of copies) DNA in vitro.
In 1985, K. Mullis and his colleagues developed a method for cloning DNA sequences in vitro, which was called polymerase chain reaction (PCR). An excess of 2 synthetic primers is added to the DNA sample being analyzed. The primers are oriented in such a way that synthesis by polymerase occurs only between them, doubling the number of copies of that DNA section. The amplified region is called amplicon. Amplification consists of repeated cycles, which are a three-step process: I- DNA denaturation at 95 °C; II- annealing of primers with complementary sequences (40-60 °C); III- subsequent completion of polynucleotide chains from primers using DNA polymerase at a temperature of 70-75 ° C (Fig. 17).
The duration of one cycle is less than 3 minutes. Thus, in 2 hours, about a billion copies of the detectable DNA sequence can be obtained. The fragment propagated in vitro is obtained in quantities sufficient for its direct sequencing. PCR is called cell-free molecular cloning.
New diagnostic tests for genetic and infectious diseases have been developed using PCR. The method is used for early diagnosis of the presence of human immunodeficiency virus (HIV) in the body. The method of analyzing individual spermatozoa has found practical application in forensic medicine. Using PCR, it was possible to amplify and clone fragments of mitochondrial DNA from the fossil remains of a 7,000-year-old human brain.
5. Introduction of a gene into a cell. The gene must be introduced into the cell in such a way that it is not destroyed by cellular nucleases, but is integrated with the cell genome. Use 2 ways.
Transduction.
Vector- a DNA or RNA molecule consisting of two components: a vector part (carrier) and a cloned foreign gene. The task of the vector is to deliver the selected DNA to the recipient cell and integrate it into the genome.
The vector must include marker gene, allowing the selection of altered cells. Two groups of marker genes can be distinguished:
- selective genes, responsible for resistance to antibiotics or herbicides;
- reporter genes, encoding cell-neutral proteins, the presence of which in tissues can be easily tested.
Responsible for the ability of a gene to express regulatory sequences, which also need to be integrated into the vector molecule.
Vector types:
- bacterial plasmids. One of the most commonly used cloning plasmids, pBR 322, is based on plasmids isolated from E. coli;
- viruses. There are viruses that do not lead to cell death, but are integrated into the genome of the host cell and multiply together with it, or cause its uncontrolled growth, i.e. turns into cancer;
- hybrid vectors containing phage DNA and plasmids - cosmids and phasmids.
2. Direct introduction of a gene into a cell – transformation. Its types:
Transfection. DNA is adsorbed on calcium phosphate crystals. They are taken up by the cell by phagocytosis.
DNA microinjection using micropipettes with a diameter of 0.1-0.5 microns and a micromanipulator.
Electroporation is based on the fact that high voltage pulses reversibly increase the permeability of biomembranes.
"Mini cells" obtained by blocking donor cells in mitosis colcemid. A new nuclear membrane forms around each chromosome. They are then processed cytochalasin B and centrifuge. Mini-cells are formed - micronuclei, encapsulated in the cytoplasmic membrane. For their fusion, special mild conditions are selected.
Packaging in liposomes used to protect exogenous genetic material from the destructive action of restriction enzymes.
Biological ballistics method is one of the most effective methods of plant transformation today. Vector DNA is sprayed onto tiny particles of tungsten. They are placed inside a biolistic gun, from where they are ejected at tremendous speed and, tearing the cell walls, enter the cytoplasm and nucleus of the cells.
With the development of genetic engineering, it became possible to force microorganisms to synthesize substances that are difficult to obtain by other methods - interferon, insulin, somatostatin, somatotropin, the enzyme urokinase, some blood clotting factors, etc. All these proteins are used to treat diseases.
Plasmids can also be introduced into eukaryotic cells. Genetic transformation of somatic cells of mammals makes it possible to study the subtle mechanisms of regulation of gene expression and purposefully modify the genetic apparatus of the cell, which is of great importance for medical genetics.