What are enzymes in chemistry definition. Enzymes. Structural and functional organization of enzymes

Chemical nature of enzymes. The importance of enzymes for the life of the body.

7.1.1. 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) 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 gives valuable information for medical diagnostics; enzymes or their inhibitors can be used as medicinal substances. Thus, knowledge the most important features enzymes and the reactions they catalyze are necessary for a rational approach to the study of human diseases, their diagnosis and treatment.

7.1.2. Substances whose transformations are catalyzed by enzymes are called substrates . The enzyme combines with the substrate to form enzyme-substrate complex (Figure 7.1).

Figure 7.1. Formation of an enzyme-substrate complex during a catalyzed reaction.

The formation of this complex helps to reduce the energy barrier that the substrate molecule must overcome to enter into a reaction (Figure 7.2). Upon completion of the reaction, the enzyme-substrate complex breaks down into product(s) and enzyme. At the end of the reaction, the enzyme returns to its original state and can interact with a new substrate molecule.

Figure 7.2. The influence of the enzyme on the energy barrier of the reaction. Enzymes, by acting as catalysts, lower the activation energy required for a reaction to occur.

7.1.3. Enzymes are characterized by properties common to all proteins. In particular, enzyme molecules, like other proteins, are built from α-amino acids connected by peptide bonds. Therefore, enzyme solutions give positive biuret reaction, and their hydrolysates - positive ninhydrin reaction. The native properties and functions of enzymes are determined by the presence of a certain spatial structure (conformation) of their polypeptide chain. Changing this structure as a result thermal denaturation leads to loss of catalytic properties. The presence of high molecular weight in enzymes determines their inability to dialysis, and the presence of charged functional groups in molecules is mobility in an electric field. Like other proteins, enzymes form colloidal solutions, from which can be precipitated by acetone, alcohol, ammonium sulfate- substances that contribute to the destruction of the hydration shell and neutralization of the electrical charge.

Basic properties of enzymes. Oligodynamicity and reversibility of enzyme action. The influence of the concentration of enzyme and substrate, temperature and pH of the environment on the rate of the enzymatic reaction.

7.2.1. 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 N 2 O2 2 H2 O + 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.

With more low 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, both in the acidic and alkaline directions 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 is denatured and 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.

Specificity of enzyme action (absolute, relative and stereochemical).

7.3.1. Important property What distinguishes enzymes from inorganic catalysts is specificity of action. As is known, the structure of the active center of an enzyme is complementary to the structure of its substrate. Therefore, the enzyme selects and attaches only its substrate from all substances present in the cell. Enzymes are characterized by specificity not only with respect to the substrate, but also with respect to the pathway of substrate conversion.

Enzymes have absolute, relative, and stereochemical specificity.

7.3.2. Absolute specificity- the selective ability of an enzyme to catalyze only one of the possible transformations of one substrate. This can be explained by the conformational and electrostatic complementarity of the substrate and enzyme molecules.

For example, the arginase enzyme catalyzes only the hydrolysis of the amino acid arginine, the urease enzyme only catalyzes the breakdown of urea and does not act on other substrates.

7.3.3. Relative specificity- the selective ability of the enzyme to catalyze similar transformations of substrates of similar structure.

Such enzymes act on the same functional groups or on the same type of bonds in substrate molecules. For example, different hydrolytic enzymes act on a certain type of bonds:

• amylase - on glycosidic bonds;
• pepsin and trypsin - for peptide bonds;
• lipase and phospholipase - into ester bonds.

The action of these enzymes extends to a large number of substrates, which allows the body to get by with a small amount of digestive enzymes - otherwise they would need much more.

7.3.4. Stereochemical (optical) specificity- the selective ability of the enzyme to catalyze the transformation of only one of the possible spatial isomers of the substrate.

Thus, most mammalian enzymes catalyze the conversion of only L-isomers of amino acids, but not D-isomers. enzymes involved in monosaccharide metabolism, on the contrary, catalyze the conversion of only D-, but not L-phosphosaccharides. Glycosidases are specific not only to the monosaccharide fragment, but also to the nature of the glycosidic bond. For example, α-amylase cleaves α-1,4-glycosidic bonds in the starch molecule, but does not act on α-1,2-glycosidic bonds in the sucrose molecule.

Basic principles underlying the modern classification and nomenclature of enzymes.

7.4.1. Currently, more than two thousand chemical reactions catalyzed by enzymes are known, and this number is constantly increasing. To navigate so many transformations. There was an urgent need for a systematic classification and nomenclature by which any enzyme could be accurately identified. The nomenclature that was used until the middle of the 20th century was very far from perfect. When researchers discovered a new enzyme, they gave it a name at their own discretion, which inevitably led to confusion and all sorts of contradictions. Some names turned out to be erroneous, others said nothing about the nature of the catalyzed reaction. Scientists from different schools often used different names for the same enzyme or, conversely, the same name for several different enzymes.

It was decided to develop a rational international classification and a nomenclature of enzymes that could be used by biochemists in all countries. For this purpose, the Commission on Enzymes was created under the International Union of Biochemistry and Molecular Biology (IUВMB), which proposed the basic principles of such classification and nomenclature in 1964. It is constantly being improved and supplemented; currently the sixth edition of this nomenclature is in effect (1992), to which additions are published annually.

OXIDOREDUCTASES.

To the class oxidoreductases include enzymes that catalyze redox reactions. General scheme they can be represented as follows:

where AH2 is a hydrogen donor, B is a hydrogen acceptor. In living organisms, oxidation occurs primarily through the abstraction of hydrogen atoms or electrons from donor substrates. Various substances can be acceptors of hydrogen atoms or electrons - coenzymes (NAD, NADP, FAD, FMN, glutathione, lipoic acid, ubiquinone), cytochromes. iron sulfur proteins and oxygen.

Subclasses of oxidoreductases are formed depending on the nature of the functional group of the hydrogen donor (electrons). In total there are 19 subclasses. The main ones are the following:

Oxidoreductases acting on the CH-OH group of donors. Enzymes belonging to this subclass oxidize alcohol groups to aldehyde or ketone groups. An example is the enzyme alcohol dehydrogenase (alcohol:NAD oxidoreductase; EC 1.1.1.1). involved in the metabolism of ethanol in tissues:

In addition to the oxidation of alcohols, enzymes of this subclass are involved in the dehydrogenation of hydroxy acids (lactic, malic, isocitric), monosaccharides and other compounds containing hydroxyl groups.

Oxidoreductases acting on the aldehyde or ketone group of donors. These enzymes oxidize aldehydes and ketones to carboxylic acids. For example, a representative of this subclass - glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD-oxidoreductase (phosphorylating), EC 1.2.1.12) - catalyzes one of the intermediate reactions of glucose breakdown:

It is important to note that the product of this reaction contains an energy-rich phosphate bond at the 1-position. The phosphoric acid residue forming this bond can be transferred from 1,3-diphosphoglycerate to ADP to form ATP (see below).

Oxidoreductases acting on the CH-CH group of donors. As a result of the reactions they catalyze, CH-CH groups are converted into C=C groups. that is, the formation of unsaturated compounds from saturated ones occurs. For example, an enzyme in the tricarboxylic acid cycle succinate dehydrogenase (succinate: acceptor - oxidoreductase, EC 1.3.99.1) accelerates the oxidation of succinic acid with the formation of unsaturated fumaric acid:

Oxidoreductases acting on CH-NH2 - donor group. These enzymes catalyze the oxidative deamination of amino acids and biogenic amines. Amines are converted into aldehydes or ketones, amino acids into keto acids, and ammonia is released. So, glutamate dehydrogenase (L-glutamate:NAD(P) - oxidoreductase (deamination), EC 1.4.1.3) takes part in the following transformation of glutamate:

Oxidoreductases acting on sulfur-containing groups of donors catalyze the oxidation of thiol (sulfhydryl) groups to disulfide groups, and sulfites to sulfates. An example of an enzyme is dihydrolipoyl dehydrogenase (EC 1.8.1.4), catalyzing one of the intermediate reactions of oxidative decarboxylation of pyruvate:

Oxidoreductases, which act on hydrogen peroxide as an acceptor, are relatively few in number and are combined into a separate subclass, also known under the trivial name peroxidases. An example of an enzyme is glutathione peroxidase (glutathione: H2 O2 - oxidoreductase. EC 1.11.1.9), involved in the inactivation of hydrogen peroxide in erythrocytes, liver and some other tissues:

Oxidoreductases acting on a pair of donors with the inclusion of molecular oxygen, or monooxygenases - enzymes that catalyze the oxidation of organic compounds by molecular oxygen, leading to the inclusion of one of the oxygen atoms in the molecules of these compounds. In this case, the second oxygen atom is included in the water molecule. This is how the reaction of phenylalanine to tyrosine is catalyzed phenylalanine 4-monooxygenase (KF 1.14.16.1):

In some people, a genetic defect in this enzyme causes a disease called phenylketonuria.

Monooxygenases also include an enzyme known as cytochrome P450 (EC 1.14.14.1) It is found mainly in liver cells and carries out hydroxylation of lipophilic compounds foreign to the body, formed as by-products reactions or entering the body from the outside. For example, indole, formed from tryptophan as a result of the activity of intestinal microorganisms, undergoes hydroxylation in the liver according to the following scheme:

The appearance of a hydroxyl group increases the hydrophilicity of substances and facilitates their subsequent removal from the body. In addition, cytochrome P450 takes part in certain stages of the conversion of cholesterol and steroid hormones. The presence of a highly efficient cytochrome P450 system in living organisms leads in some cases to undesirable practical consequences: it reduces the time spent in the human body medicines and thereby reduces their therapeutic effect.

Oxidoreductases acting on one donor with the inclusion of molecular oxygen, or dioxygenases, catalyze transformations during which both atoms of the O2 molecule are included in the composition of the oxidized substrate. For example, in the process of catabolism of phenylalanine and tyrosine, maleylacetoacetate is formed from homogentisic acid, which includes both oxygen atoms:

The enzyme that catalyzes this reaction is called homogentisate 1,2-dioxygenase(KF 1.13.11.5). In some cases, congenital deficiency of this enzyme occurs, which leads to the development of a disease called alkaptonuria.

TRANSFERASES.

where X is the transferred functional group. AX is the group donor, B is the acceptor. The division into subclasses depends on the nature of the groupings being transferred.

Transferases that transfer one-carbon fragments. This subclass includes enzymes that accelerate the transfer of methyl (-CH3), methylene (-CH2-), methenyl (-CH=), formyl and related groups. Yes, with the participation guanidine acetate methyltransferase (S-adenosylmethionine guanidine acetate methyltransferase, EC 2.1.1.2) synthesis occurs biologically active substance creatine:

Transferases that transfer carboxylic acid residues (acyltransferases). They catalyze a variety of chemical processes associated with the transfer of residues of various acids (acetic, palmitic, etc.) mainly from coenzyme A thioesters to various acceptors. An example of a transacetylation reaction would be the formation of the mediator acetylcholine with the participation choline acetyltransferase (acetyl-CoA:choline-O-acetyltransferase, EC 2.3.1.6):

Transferases that transfer glycosyl residues (glycosyltransferases) catalyze the transport of glycosyl residues from phosphorus ester molecules to molecules of monosaccharides, polysaccharides and other substances. These enzymes, in particular, play a major role in the synthesis of glycogen and starch, as well as in the first phase of their destruction. Another enzyme of this subclass - UDP-glucuronyltransferase (UDP-glucuronate-glucuronyl transferase (non-sink specific), EC 2.4.1.17) - participates in the processes of neutralization of endogenous and foreign toxic substances in the liver:

Transferases that transfer nitrogenous groups. This subclass includes aminotransferases, accelerating the transfer of the α-amino group of amino acids to the α-carbon atom of keto acids. The most important of these enzymes is alanine aminotransferase (L-alanine:2-oxoglutarate aminotransferase, EC 2.6.1.2). catalyzing reaction:

Transferases that transfer phosphate groups (phosphotransferases). This group of enzymes catalyzes biochemical processes, associated with the transport of phosphoric acid residues to various substrates. These processes have important for the life of the body, since they ensure the conversion of a number of substances into organic phosphoesters, which have high chemical activity and easily enter into subsequent reactions. Phosphotransferases that use ATP as a phosphate donor are called kinases . A widely distributed enzyme is hexokinase (ATP:D-hexose-6-phosphotransferase. EC 2.7.1.1.), accelerating the transfer of the phosphate group from ATP to monosaccharides:

In some cases, reverse transfer of the phosphate group from the substrate to ADP with the formation of ATP is also possible. Yes, enzyme phosphoglycerate kinase (ATP:D-3-phosphoglycerate-1-phosphotransferase, EC 2.7.2.3) converts the previously mentioned (see “Oxidoreductase”) 1.3-diphosphoglycerate:

Similar reactions of phosphorylation of ADP with the formation of ATP, coupled with the conversion of the substrate (and not with the transfer of electrons in the respiratory chain), are called substrate phosphorylation reactions. The role of these reactions in the cell increases significantly with a lack of oxygen in the tissues.

HYDROLASES.

Hydrolases are a class of enzymes that catalyze the breakdown of organic compounds with the participation of water (hydrolysis reactions). These reactions proceed according to the following scheme:

where A-B is a complex compound, A-H and B-OH are the products of its hydrolysis. Reactions of this type actively occur in the body; they come with the release of energy and, as a rule, are irreversible.

Subclasses of hydrolases are formed depending on the type of bond being hydrolyzed. The most important are the following subclasses:

Hydrolases acting on esters (or esterase) hydrolyze esters of carboxylic, phosphoric, sulfuric and other acids. A widespread enzyme of this subclass is triacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.3). accelerating the hydrolysis of acylglycerols:

Other representatives of esterases cleave ester bonds in acetylcholine (acetylcholinesterase), phospholipids (phospholipases), nucleic acids (nucleases), and organophosphorus esters (phosphatases).

Hydrolases acting on glycosidic bonds (glycosidases) accelerate the hydrolysis reactions of oligo- and polysaccharides, as well as other compounds containing monosaccharide residues (for example, nucleosides). A typical representative is sucrase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26). catalyzing the breakdown of sucrose:

Hydrolases acting on peptide bonds (peptidases) catalyze reactions of hydrolysis of peptide bonds in proteins and peptides. This group includes pepsin, trypsin, chymotrypsin, cathepsin and other proteolytic enzymes. Hydrolysis of peptide bonds occurs according to the following scheme:

Hydrolases acting on C-N bonds other than peptide ones - enzymes that accelerate the hydrolysis of organic acid amides. Representative of this subclass - glutaminase (L-glutamyl amidohydrolase, EC 3.5.1.2) - participates in maintaining the acid-base state of the body by catalyzing the hydrolysis of glutamine in the kidneys:

LYASES.

Lyases are a class of enzymes that catalyze non-hydrolytic reactions of cleavage of substrates with the formation of double bonds or, conversely, addition at the site of the break of the double bond. The general scheme of these reactions:

where A-B is the substrate, A and B are the reaction products. As a result of such reactions, simple substances are often released, for example, CO2, NH3, H2 O.

Carbon-carbon-lyase catalyze the breaking of a bond between two carbon atoms. Among them highest value have carboxy-lyases (decarboxylases), under the influence of which decarboxylation of a-keto and amino acids occurs, keto acid lyases , which includes citrate synthase, aldehyde lyases (aldolases). The latter includes fructose diphosphate aldolase (fructose-1,6-diphosphate-D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13), catalyzing the reaction:

Carbon-oxygen-lyase catalyze the breaking of bonds between carbon and oxygen atoms. This subclass includes primarily hydrolyases, participating in dehydration and hydration reactions. An example would be serine dehydratase (L-serine hydrolyase (deamination), EC 4.2.1.3), which carries out the transformation:

Sometimes a backlash using the term can be taken as the basis for a working title "hydratase". Thus, for the enzyme of the tricarboxylic acid cycle L-malate hydrolyase (EC 4.2.1.2) the recommended name is "fumarate hydratase":

Carbon-nitrogen lyases participate in the elimination of nitrogen-containing groups. A representative of this subclass is histidine ammonia lyase (L-histidine ammonia lyase, EC 4.3.1.3), involved in the deamination of histidine:

Carbon-sulfur lyase catalyze the elimination of sulfhydryl groups. This subclass includes desulfhydrase sulfur-containing amino acids, e.g. cysteine ​​desulfhydrase (L-cysteine ​​hydrogen sulfide lyase (deamination), EC 4.4.1.1).

ISOMERASES.

Isomerases are a class of enzymes that accelerate the processes of intramolecular transformations with the formation of isomers. Reactions of this type can be represented schematically as follows:

where A and A" are isomer substances.

Isomerases are a relatively small class of enzymes; they are divided into the following subclasses depending on the type of isomerization reaction catalyzed:

Racemases and epimerases catalyze the interconversion of isomers containing asymmetric carbon atoms. Racemases are called enzymes that act on substrates with one asymmetric atom, for example, converting L-amino acids into D-amino acids. One of these enzymes is alanine racemase (alanine racemase. EC 5.1.1.1), catalyzing the reaction:

Epimerases are called enzymes that act on substrates with several asymmetric carbon atoms. These enzymes include UDP-glucose epimerase (UDP-glucose-4-epimerase, EC 5.1.3.2). participating in the processes of interconversion of monosaccharides:

Cis-trans isomerases - enzymes, causing change geometric configuration relative to the double bond. An example of such an enzyme is maleylacetoacetate isomerase (maleylacetoacetate-cis-trans-isomerase, EC 5.2.1.2), involved in the catabolism of phenylalanine and tyrosine and converting maleylacetoacetate (see 4.6.1) into fumarylacetoacetate:

Intramolecular oxidoreductases - isomerases that catalyze the interconversion of aldoses and ketoses. In this case, the CH-OH group is oxidized with simultaneous reduction of the neighboring C=O group. So, triosephosphate isomerase (D-glyceraldehyde-3-phosphate-ketol isomerase, EC 5.3.1.1) catalyzes one of the reactions of carbohydrate metabolism:

Isomerases also include intramolecular transferases, carrying out the transfer of one group from one part of the substrate molecule to another part of the same molecule, and intramolecular lyases, catalyzing decyclization reactions, as well as the transformation of one type of ring into another.

It should be emphasized that not all biochemical processes. which results in isomerization, are catalyzed by isomerases. Thus, the isomerization of citric acid into isopimonic acid occurs with the participation of the enzyme aconitate hydratase (citrate (isocitrate) hydrolyase, EC 4.2.1.3), catalyzing the dehydration-hydration reaction with the intermediate formation of cis-aconitic acid:

LIGASES.

Ligases are a class of enzymes that catalyze the synthesis of organic compounds from starting substances activated due to the breakdown of ATP (or GTP, UTP, CTP). For enzymes of this class, the trivial name is also retained synthetase. IN Therefore, according to IUBMB recommendations, the term “synthetases” should not be used for enzymes whose action does not involve nucleoside triphosphates. Reactions catalyzed by ligases (synthetases) proceed according to the following scheme:

,

where A and B are interacting substances; A-B is a substance formed as a result of interaction.

Since new chemical bonds are formed as a result of the action of these enzymes, subclasses of class VI are formed depending on the nature of the newly formed bonds.

Ligases that form carbon-oxygen bonds. These include a group of enzymes known as amino acid-tRNA ligases (aminoacyl-tRNA synthetases). which catalyze reactions between amino acids and the corresponding transport RNAs. These reactions produce active forms of amino acids that can participate in the process of protein synthesis on ribosomes. An example of an enzyme is tyrosyl-tRNA synthetase (L-tyrosine: tRNA ligase (AMP-forming), EC 6.1.1.1), involved in the reaction:

Ligases that form carbon-sulfur bonds. This subclass is represented primarily by enzymes that catalyze the formation of thioesters fatty acids with coenzyme A. With the participation of these enzymes, acyl-CoA is synthesized - active forms of fatty acids that can enter into various biosynthesis and breakdown reactions. Let us consider one of the reactions of activation of fatty acids that occurs in the presence of the enzyme acyl-CoA synthetase (carboxylic acid: coenzyme A-ligase (AMP-forming). EC 6.2.1.2):

Ligases that form carbon-nitrogen bonds catalyze numerous reactions of introducing nitrogen-containing groups into organic compounds. An example would be glutamine synthetase (L-glutamine: ammonia-γ-ligase (ADP-forming), EC 6.3.1.2). participating in the neutralization of a toxic metabolic product - ammonia - in a reaction with glutamic acid:

Ligases that form carbon-carbon bonds. Of these enzymes, the most studied carboxylase, providing carboxylation of a number of compounds, resulting in elongation of carbon chains. The most important representative of this class is pyruvate carboxylase (pyruvate:CO2 ligase (ADP-forming), EC 6.4.1.1), accelerating the reaction of the formation of oxaloacetate, a key compound in the tricarboxylic acid cycle and carbohydrate biosynthesis:

Let us recall that reactions involving ATP are catalyzed not only by class VI enzymes, but also by some class II enzymes (phosphotransferases or kinases). It is important to be able to distinguish between these types of reactions. Their difference is that in transferase reactions ATP is donor of phosphate groups , therefore, as a result of these reactions, there is no release of H3 PO4 (see examples above). On the contrary, in synthetase reactions ATP serves source of energy , released during its hydrolysis, therefore one of the products of such a reaction will be inorganic ortho- or pyrophosphate.

Rules for working with enzymes

Enzymes, like all proteins, are relatively unstable substances. They are easily denatured and inactivated. Therefore, when working with them, certain conditions must be met.

• When storing the object of study for more than several hours at room temperature the enzyme is almost completely inactivated. Therefore, analysis for determining enzyme activity should be carried out as soon as possible. If necessary long-term storage possible if the enzyme solution is dried from the frozen state under high vacuum (lyophilization). In this case, the enzyme almost completely retains activity upon further storage at room temperature. Some enzymes are well preserved in concentrated salt solutions, for example, in saturated ammonium sulfate (salting out process). If necessary, the enzyme precipitate can be centrifuged and dissolved in saline or an appropriate buffer. If necessary, excess salt can be removed by dialysis.
• It is necessary to remember the sensitivity of enzymes to fluctuations in the pH of the environment. With few exceptions, most enzymes are inactivated in solutions with a pH below 5 or above 9, and the optimum enzyme action appears in the zone of several units or tenths of a unit of pH value. It is recommended to determine the pH of buffer solutions used when working with enzymes very accurately using a pH meter.
• Enzymes are easily destroyed by potent reagents: acids, alkalis, oxidizing agents, salts heavy metals. It is necessary to work with chemically pure reagents and double-distilled water, since even slight contamination of the reagents, especially with metal impurities that can act as modulators, leads to changes in enzyme activity.
• When working with enzymes, more than anywhere else, strict adherence to the standardization of research conditions is necessary: ​​precise maintenance of temperature and time conditions, the use of reagents from the same batch, and when changing reagents, the data obtained must be calibrated again. If the developing color in the color reaction is unstable over time, it is necessary to strictly observe the timing of photometry.
• It is recommended to work under conditions of sufficient saturation of the enzyme with the substrate, since this circumstance significantly affects end result, the lack of substrate eliminates the differences between the options.
• When working with enzymes, it is necessary to take into account the organ-specific isoenzyme spectrum. Often this specificity affects the conditions under which the enzyme acts. The course of the reaction can be affected by different affinities for the substrate, different sensitivity to pH, characteristic of isoenzymes of a particular organ or tissue. Transferring a method for studying enzyme activity from one object to another (for example, from serum to tissue or from one organ to another) must be done with extreme caution, taking into account all known data about the enzyme and its multiple forms, as well as carefully checking the results.

For the widespread implementation of various biochemical (enzymatic) reactions, automation of the most generally accepted and necessary tests is being introduced, as well as the unification and standardization of laboratory tests. This is rational and necessary both to improve the accuracy and quality of samples, and to compare data obtained in different laboratories.

It is also generally accepted to carry out a mandatory parallel study, along with the pathology being studied, of physiological control - a group of practically healthy people to establish normal, physiological fluctuations. Understanding the relativity of the concept of “normal value”, it should be accepted that in order to identify differences in pathology and evaluate a pathological sign, the arithmetic mean M ± 1σ or 2σ (with a normal Gaussian distribution) is usually taken as the “norm”, depending on the degree of fluctuation of the indicator .

Principles for determining enzyme activity in biological material.

5.6.2. Unique property 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.

Enzyme activity measurement is based on speed comparison chemical reaction in the presence of an active biocatalyst with the reaction rate in a control solution in which the enzyme is absent or inactivated.

The material under study is placed in an incubation medium, where optimal temperature, pH of the environment, concentration of activators and substrates. 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.

Methods for measuring enzyme activity

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 liver diseases 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 based on changes in the ultraviolet spectrum chemical substances, 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 (carboxylic acid) interacts complex structure) luciferyl adenylate is formed with ATP. 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.

Enzyme activity units and their applications.

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 source of the enzyme is taken from smaller quantity(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.

Training tasks and standards for their solution.

1. Objectives

1. What enzymes are called racemases?

2. Decipher the systematic name of the enzyme (separately for each of the elements, highlighted in different colors):
S-adenosylmethionine: guanidine acetate methyl transferase?

Define:
a) type of reaction;
b) enzyme class;
c) subclass.

2. Solution standards

1. Racemases - enzymes that catalyze interconversion optical isomers, containing a single asymmetric carbon atom (see section 2.3).

2. The systematic name of the enzyme is read from the end. The enzyme belongs to the class transferases, catalyzes the transfer reaction methyl group on guanidine acetate (methyl group acceptor) with S-adenosylmethionine (methyl group donor) (see sections 2.2 - 2.3).

3. a) In this reaction occurs splitting of a substance without the participation of water molecules

b) Non-hydrolytic cleavage of the substrate with the formation of two products is catalyzed enzymes belonging to the fourth class (lyases)

c) The bond between the first and second carbon atoms is broken, which leads to the elimination of the carboxyl group in the form of CO2. Hence, enzyme subclass - carbon-carbon-lyase(see section 2.3).