Molecular effects of enzyme action. Enzymes. Features of enzymatic catalysis. Structure and structure of enzymes Enzymatic reaction according to the type of “sequential reactions”

Any catalytic reaction involves a change in the rates of both forward and reverse reactions due to a decrease in its energy. If a chemical reaction proceeds with the release of energy, then it must begin spontaneously. However, this does not happen because the components of the reaction must be transferred to the activated (transition) state. The energy required to convert reacting molecules into an activated state is called activation energy.

Transition state characterized by the continuous formation and breaking of chemical bonds, and thermodynamic equilibrium exists between the transition and ground states. The rate of the forward reaction depends on the temperature and the difference in free energy values ​​for the substrate in the transition and ground states. This difference is called free energy of reaction.

Achieving the transition state of the substrate is possible in two ways:

• due to the transfer of excess energy to reacting molecules (for example, due to an increase in temperature),
• by reducing the activation energy of the corresponding chemical reaction.

Ground and transition states of reacting substances.

Eo, Ek - reaction activation energy without and in the presence of a catalyst; DG-

difference in free energy of reaction.

Enzymes “help” substrates adopt a transition state due to the binding energy during formation enzyme-substrate complex. The decrease in activation energy during enzymatic catalysis is due to an increase in the number of stages of the chemical process. The induction of a number of intermediate reactions leads to the fact that the initial activation barrier is split into several lower barriers, which the reacting molecules can overcome much faster than the main one.

The mechanism of the enzymatic reaction can be represented as follows:

1. connection of enzyme (E) and substrate (S) with the formation of an unstable enzyme-substrate complex (ES): E + S → E-S;
2. formation of an activated transition state: E-S → (ES)*;
3. release of reaction products (P) and regeneration of the enzyme (E): (ES)* → P + E.

To explain the high efficiency of enzyme action, several theories of the mechanism of enzymatic catalysis have been proposed. The earliest is theory of E. Fisher (the theory of “template” or “rigid matrix"). According to this theory, the enzyme is a rigid structure, the active center of which is a “cast” of the substrate. If the substrate approaches the active site of the enzyme like a “key to a lock,” a chemical reaction will occur. This theory well explains two types of substrate specificity of enzymes - absolute and stereospecificity, but turns out to be untenable in explaining the group (relative) specificity of enzymes.

The "rack" theory based on the ideas of G. K. Euler, who studied the action of hydrolytic enzymes. According to this theory, the enzyme binds to the substrate molecule at two points, and the chemical bond is stretched, the electron density is redistributed, and the chemical bond is broken, accompanied by the addition of water. Before joining the enzyme, the substrate has a “relaxed” configuration. After binding to the active center, the substrate molecule undergoes stretching and deformation (it is located in the active center as if on a rack). The longer the chemical bonds in the substrate, the easier they are to break and the lower the activation energy of the chemical reaction.

Recently it has become widespread theory of “induced correspondence” by D. Koshland, which allows for high conformational lability of the enzyme molecule, flexibility and mobility of the active center. The substrate induces conformational changes in the enzyme molecule in such a way that the active center takes on the spatial orientation necessary for binding the substrate, i.e., the substrate approaches the active center like a “hand to a glove.”

According to the theory of induced correspondence, the mechanism of interaction between enzyme and substrate is as follows:

1. The enzyme, based on the principle of complementarity, recognizes and “catches” the substrate molecule. In this process, the protein molecule is aided by the thermal movement of its atoms;
2. amino acid residues of the active center are shifted and adjusted in relation to the substrate;
3. chemical groups are covalently added to the active site - covalent catalysis.

The sequence of events in enzymatic catalysis can be described by the following diagram. First, a substrate-enzyme complex is formed. In this case, a change in the conformations of the enzyme molecule and the substrate molecule occurs, the latter is fixed in the active center in a tense configuration. This is how the activated complex is formed, or transition state, is a high-energy intermediate structure that is energetically less stable than the parent compounds and products. The most important contribution to the overall catalytic effect is made by the process of stabilization of the transition state - the interaction between amino acid residues of the protein and the substrate, which is in a tense configuration. The difference between the free energy values ​​for the initial reactants and the transition state corresponds to the free energy of activation (ΔG #). The reaction rate depends on the value (ΔG #): the smaller it is, the greater the reaction rate, and vice versa. Essentially, the DG represents an “energy barrier” that must be overcome for a reaction to occur. Stabilizing the transition state lowers this “barrier” or activation energy. At the next stage, the chemical reaction itself occurs, after which the resulting products are released from the enzyme-product complex.

There are several reasons for the high catalytic activity of enzymes, which reduce the energy barrier to the reaction.

1. An enzyme can bind molecules of reacting substrates in such a way that their reactive groups will be located close to each other and from the catalytic groups of the enzyme (effect rapprochement).

2. With the formation of a substrate-enzyme complex, fixation of the substrate and its optimal orientation for breaking and formation of chemical bonds are achieved (effect orientation).

3. Binding of the substrate leads to the removal of its hydration shell (exists on substances dissolved in water).

4. Effect of induced correspondence between substrate and enzyme.

5. Stabilization of the transition state.

6. Certain groups in the enzyme molecule can provide acid-base catalysis(transfer of protons in the substrate) and nucleophilic catalysis(formation of covalent bonds with the substrate, which leads to the formation of structures that are more reactive than the substrate).

One example of acid-base catalysis is the hydrolysis of glycosidic bonds in the murein molecule by lysozyme. Lysozyme is an enzyme present in the cells of various animals and plants: in tear fluid, saliva, chicken protein, milk. Lysozyme from chicken eggs has a molecular weight of 14,600 Da, consists of one polypeptide chain (129 amino acid residues) and has 4 disulfide bridges, which ensures high stability of the enzyme. X-ray structural analysis of the lysozyme molecule showed that it consists of two domains forming a “gap” in which the active center is located. Along this “gap” the hexosaccharide binds, and the enzyme has its own site for binding each of the six sugar rings of murein (A, B, C, D, E and F) (Fig. 6.4).

The murein molecule is held in the active site of lysozyme mainly due to hydrogen bonds and hydrophobic interactions. In close proximity to the site of hydrolysis of the glycosidic bond, there are 2 amino acid residues of the active center: glutamic acid, occupying the 35th position in the polypeptide, and aspartic acid, the 52nd position in the polypeptide (Fig. 6.5).

The side chains of these residues are located on opposite surfaces of the “cleft” in close proximity to the attacked glycosidic bond—at a distance of approximately 0.3 nm. The glutamate residue is in a non-polar environment and is not ionized, and the aspartate residue is in a polar environment; its carboxyl group is deprotonated and participates as a hydrogen acceptor in a complex network of hydrogen bonds.

The hydrolysis process is carried out as follows. The protonated carboxyl group of the Glu-35 residue provides its proton to the glycosidic oxygen atom, which leads to the rupture of the bond between this oxygen atom and the C 1 atom of the sugar ring located in site D (stage of general acid catalysis). As a result, a product is formed that includes the sugar rings located in regions E and F, which can be released from the complex with the enzyme. The conformation of the sugar ring located in region D is distorted, taking on the conformation half-chairs, in which five of the six atoms forming the sugar ring lie practically in the same plane. This structure corresponds to the transition state conformation. In this case, the C 1 atom turns out to be positively charged and the intermediate product is called a carbonium ion (carbocation). The free energy of the transition state decreases due to the stabilization of the carbonium ion by the deprotonated carboxyl group of the Asp-52 residue (Fig. 6.5).

At the next stage, a water molecule enters the reaction and replaces the disaccharide residue diffusing from the region of the active center. The proton of the water molecule goes to Glu-35, and the hydroxyl ion (OH -) to the C 1 atom of the carbonium ion (stage of general basic catalysis). As a result, the second fragment of the cleaved polysaccharide becomes a reaction product (chair conformation) and leaves the active center region, and the enzyme returns to its original state and is ready to carry out the next disaccharide cleavage reaction (Fig. 6.5).

Properties of enzymes

When characterizing the properties of enzymes, we first use the concept of “activity.” Enzyme activity is understood as the amount of enzyme that catalyzes the conversion of a certain amount of substrate per unit of time. To express the activity of enzyme preparations, two alternative units are used: international (E) and “catal” (kat). The international unit of enzyme activity is taken to be the amount of enzyme that catalyzes the conversion of 1 µmol of substrate into a product in 1 minute under standard conditions (usually optimal). One katal denotes the amount of enzyme that catalyzes the conversion of 1 mole of substrate in 1 s. 1 cat=6*10 7 E.

Often enzyme preparations are characterized by specific activity, which reflects the degree of purification of the enzyme. Specific activity is the number of units of enzyme activity per 1 mg of protein.

The activity of enzymes depends to a very large extent on external conditions, among which the temperature and pH of the environment are of paramount importance. An increase in temperature in the range of 0-50° C usually leads to a smooth increase in enzymatic activity, which is associated with the acceleration of the formation of the substrate-enzyme complex and all subsequent catalytic events. However, a further increase in temperature is usually accompanied by an increase in the amount of inactivated enzyme due to denaturation of its protein part, which is expressed in a decrease in activity. Each enzyme is characterized temperature optimum- the temperature value at which its greatest activity is recorded. More often, for enzymes of plant origin, the temperature optimum lies within 50-60 ° C, and for animal enzymes - between 40 and 50 ° C. Enzymes of thermophilic bacteria are characterized by a very high temperature optimum.

The dependence of enzyme activity on pH values ​​of the environment is also complex. Each enzyme is characterized optimum pH environment in which it exhibits maximum activity. As you move away from this optimum in one direction or the other, enzymatic activity decreases. This is explained by a change in the state of the active center of the enzyme (a decrease or increase in the ionization of functional groups), as well as the tertiary structure of the entire protein molecule, which depends on the ratio of cationic and anionic centers in it. Most enzymes have a pH optimum in the neutral range. However, there are enzymes that exhibit maximum activity at pH 1.5 (pepsin) or 9.5 (arginase).

Enzyme activity is subject to significant fluctuations depending on exposure inhibitors(substances that reduce activity) and activators(substances that increase activity). The role of inhibitors and activators can be played by metal cations, some anions, carriers of phosphate groups, reducing equivalents, specific proteins, intermediate and final products of metabolism, etc. These substances can enter the cell from the outside or be produced within it. In the latter case, they talk about the regulation of enzyme activity - an integral link in the general regulation of metabolism.

Substances that affect enzyme activity can bind to the active and allosteric centers of the enzyme, as well as outside these centers. Particular examples of such phenomena will be discussed in Chapters 7-19. To generalize some patterns of inhibition of enzyme activity, it should be noted that these phenomena in most cases come down to two types - reversible and irreversible. During reversible inhibition no changes are made to the enzyme molecule after its dissociation with the inhibitor. An example is the action substrate analogues, which can bind to the active site of the enzyme, preventing the enzyme from interacting with the true substrate. However, an increase in the substrate concentration leads to the “displacement” of the inhibitor from the active site, and the rate of the catalyzed reaction is restored ( competitive inhibition). Another case of reversible inhibition is the binding of the inhibitor to a prosthetic group of the enzyme, or apoenzyme, outside the active center. For example, the interaction of enzymes with heavy metal ions that attach to the sulfhydryl groups of amino acid residues of the enzyme, protein-protein interactions or covalent modification of the enzyme. This inhibition of activity is called non-competitive.

Irreversible inhibition in most cases it is based on linking the so-called “ suicidal substrates» with active sites of enzymes. In this case, covalent bonds are formed between the substrate and the enzyme, which are broken down very slowly and the enzyme is not able to perform its function for a long time. An example of a “suicidal substrate” is the antibiotic penicillin (Chapter 18, Fig. 18.1).

Because enzymes are characterized by specificity of action, they are classified according to the type of reaction they catalyze. According to the currently accepted classification, enzymes are grouped into 6 classes:

1. Oxidoreductases (redox reactions).

2. Transferases (reactions of transfer of functional groups between substrates).

3. Hydrolases (hydrolysis reactions, the acceptor of the transferred group is a water molecule).

4. Lyases (reactions of elimination of groups in a non-hydrolytic way).

5. Isomerases (isomerization reactions).

6. Ligases, or synthetases (synthesis reactions due to the energy of cleavage of nucleoside triphosphates, most often ATP).

The number of the corresponding enzyme class is fixed in its code numbering (cipher). The enzyme code consists of four numbers separated by dots, indicating the enzyme class, subclass, subsubclass and serial number in the subclass.

STEPS OF ENZYME CATALYSIS

1. Formation of the enzyme-substrate complex

Enzymes have high specificity and this made it possible to put forward a hypothesis according to which the active center of the enzyme is complementary to the substrate, i.e. corresponds to it like a “key to a lock.” After the “key” substrate interacts with the “lock” active center, chemical transformations of the substrate into the product occur.

Later, another version of this hypothesis was proposed - the active center is a flexible structure in relation to the substrate. The substrate, interacting with the active center of the enzyme, causes a change in its conformation, leading to the formation of an enzyme-substrate complex. At the same time, the substrate also changes its conformation, which ensures higher efficiency of the enzymatic reaction.

2. Sequence of events during enzymatic catalysis

A. the stage of approaching and orienting the substrate relative to the active center of the enzyme

b. formation of an enzyme-substrate complex

V. substrate deformation and formation of an unstable enzyme-product complex

d. decomposition of the enzyme-product complex with the release of reaction products from the active center of the enzyme and release of the enzyme

3. The role of the active site in enzymatic catalysis

Only a small part of the enzyme comes into contact with the substrate, from 5 to 10 amino acid residues, forming the active center of the enzyme. The remaining amino acid residues ensure the correct conformation of the enzyme molecule for optimal chemical reactions. In the active site of the enzyme, the substrates are arranged so that the functional groups of the substrates involved in the reaction are in close proximity to each other. This arrangement of substrates reduces the activation energy, which determines the catalytic efficiency of enzymes.

There are 2 main mechanisms of enzymatic catalysis:

1. acid-base catalysis

2. covalent catalysis

The concept of acid-base catalysis explains enzymatic activity by the participation of acidic groups (proton donors) and/or basic groups (proton acceptors) in a chemical reaction. The amino acid residues that make up the active center have functional groups that exhibit the properties of both acids and bases. These are cysteine, tyrosine, serine, lysine, glutamic acid, aspartic acid and histidine.

An example of acid-base catalysis is the oxidation of alcohol using the enzyme alcohol dehydrogenase.

Covalent catalysis is based on the attack of the “-” and “+” groups of the active center of the enzyme by substrate molecules with the formation of a covalent bond between the substrate and the coenzyme. An example is the effect of serine proteases (pripsin, chemotrypsin) on the hydrolysis of peptide bonds during protein digestion. A covalent bond is formed between the substrate and the serine amino acid residue of the active site of the enzyme.

Catalysis is the process of accelerating a chemical reaction under the influence of catalysts that actively participate in it, but at the end of the reaction remain chemically unchanged. The catalyst accelerates the establishment of chemical equilibrium between the starting materials and reaction products. The energy required to start a chemical reaction is called activation energy. It is necessary so that the molecules participating in the reaction can enter a reactive (active) state. The mechanism of action of the enzyme is aimed at reducing the activation energy. This is achieved by dividing the reaction into separate steps or stages through the participation of the enzyme itself. Each new stage has a lower activation energy. The division of the reaction into stages becomes possible due to the formation of a complex of the enzyme with the starting substances, the so-called substrates ( S). Such a complex is called an enzyme-substrate complex ( ES). This complex is then cleaved to form the reaction product (P) and the unchanged enzyme ( E).

E + SESE + P

Thus, an enzyme is a biocatalyst that, by forming an enzyme-substrate complex, breaks the reaction into separate stages with a lower activation energy and thereby sharply increases the reaction rate.

4. Properties of enzymes.

All enzymes are of protein nature.

Enzymes have high molecular weight.

They are highly soluble in water and, when dissolved, form colloidal solutions.

All enzymes are thermolabile, i.e. optimum action 35 – 45 o C

According to their chemical properties, they are amphoteric electrolytes.

Enzymes are highly specific with respect to substrates.

Enzymes require a strictly defined pH value for their action (pepsin 1.5 - 2.5).

Enzymes have high catalytic activity (accelerate the reaction rate by 10 6 – 10 11 times).

All enzymes are capable of denaturation when exposed to strong acids, alkalis, alcohols, and heavy metal salts.

Specificity of enzyme action:

Based on the specificity of their action, enzymes are divided into two groups: those with absolute specificity and those with relative specificity.

Relative specificity observed when an enzyme catalyzes one type of reaction with more than one structure-like substrate. For example, pepsin breaks down all proteins of animal origin. Such enzymes act on a specific type of chemical bond, in this case a peptide bond. The action of these enzymes extends to a large number of substrates, which allows the body to get by with a small number of digestive enzymes.

Absolute specificity manifests itself when the enzyme acts on only one single substance and catalyzes only a certain transformation of this substance. For example, sucrase only breaks down sucrose.

Reversibility of action:

Some enzymes can catalyze both forward and reverse reactions. For example, lactate dehydrogenase, an enzyme that catalyzes the oxidation of lactate to pyruvate and the reduction of pyruvate to lactate.

The sequence of events in enzymatic catalysis can be described by the following diagram. First, a substrate-enzyme complex is formed. In this case, a change in the conformations of the enzyme molecule and the substrate molecule occurs, the latter is fixed in the active center in a tense configuration. This is how the activated complex is formed, or transition state, is a high-energy intermediate structure that is energetically less stable than the parent compounds and products. The most important contribution to the overall catalytic effect is made by the process of stabilization of the transition state - the interaction between amino acid residues of the protein and the substrate, which is in a tense configuration. The difference between the free energy values ​​for the initial reactants and the transition state corresponds to the free energy of activation (ΔG #). The reaction rate depends on the value (ΔG #): the smaller it is, the greater the reaction rate, and vice versa. Essentially, the DG represents an “energy barrier” that must be overcome for a reaction to occur. Stabilizing the transition state lowers this “barrier” or activation energy. At the next stage, the chemical reaction itself occurs, after which the resulting products are released from the enzyme-product complex.

There are several reasons for the high catalytic activity of enzymes, which reduce the energy barrier to the reaction.

1. An enzyme can bind molecules of reacting substrates in such a way that their reactive groups will be located close to each other and from the catalytic groups of the enzyme (effect rapprochement).

2. With the formation of a substrate-enzyme complex, fixation of the substrate and its optimal orientation for breaking and formation of chemical bonds are achieved (effect orientation).

3. Binding of the substrate leads to the removal of its hydration shell (exists on substances dissolved in water).

4. Effect of induced correspondence between substrate and enzyme.

5. Stabilization of the transition state.

6. Certain groups in the enzyme molecule can provide acid-base catalysis(transfer of protons in the substrate) and nucleophilic catalysis(formation of covalent bonds with the substrate, which leads to the formation of structures that are more reactive than the substrate).

One example of acid-base catalysis is the hydrolysis of glycosidic bonds in the murein molecule by lysozyme. Lysozyme is an enzyme present in the cells of various animals and plants: in tear fluid, saliva, chicken protein, milk. Lysozyme from chicken eggs has a molecular weight of 14,600 Da, consists of one polypeptide chain (129 amino acid residues) and has 4 disulfide bridges, which ensures high stability of the enzyme. X-ray structural analysis of the lysozyme molecule showed that it consists of two domains forming a “gap” in which the active center is located. Along this “gap” the hexosaccharide binds, and the enzyme has its own site for binding each of the six sugar rings of murein (A, B, C, D, E and F) (Fig. 6.4).

The murein molecule is held in the active site of lysozyme mainly due to hydrogen bonds and hydrophobic interactions. In close proximity to the site of hydrolysis of the glycosidic bond, there are 2 amino acid residues of the active center: glutamic acid, occupying the 35th position in the polypeptide, and aspartic acid, the 52nd position in the polypeptide (Fig. 6.5).

The side chains of these residues are located on opposite surfaces of the “cleft” in close proximity to the attacked glycosidic bond—at a distance of approximately 0.3 nm. The glutamate residue is in a non-polar environment and is not ionized, and the aspartate residue is in a polar environment; its carboxyl group is deprotonated and participates as a hydrogen acceptor in a complex network of hydrogen bonds.

The hydrolysis process is carried out as follows. The protonated carboxyl group of the Glu-35 residue provides its proton to the glycosidic oxygen atom, which leads to the rupture of the bond between this oxygen atom and the C 1 atom of the sugar ring located in site D (stage of general acid catalysis). As a result, a product is formed that includes the sugar rings located in regions E and F, which can be released from the complex with the enzyme. The conformation of the sugar ring located in region D is distorted, taking on the conformation half-chairs, in which five of the six atoms forming the sugar ring lie practically in the same plane. This structure corresponds to the transition state conformation. In this case, the C 1 atom turns out to be positively charged and the intermediate product is called a carbonium ion (carbocation). The free energy of the transition state decreases due to the stabilization of the carbonium ion by the deprotonated carboxyl group of the Asp-52 residue (Fig. 6.5).

At the next stage, a water molecule enters the reaction and replaces the disaccharide residue diffusing from the region of the active center. The proton of the water molecule goes to Glu-35, and the hydroxyl ion (OH -) to the C 1 atom of the carbonium ion (stage of general basic catalysis). As a result, the second fragment of the cleaved polysaccharide becomes a reaction product (chair conformation) and leaves the active center region, and the enzyme returns to its original state and is ready to carry out the next disaccharide cleavage reaction (Fig. 6.5).