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. In this case, 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.

Enzymes play a key role in metabolism. They speed up reactions by increasing their rate constants.

Let's consider the energy profile of an ordinary reaction (Fig. 12.I), taking place in a solution according to the collision mechanism A + IN -> R.

Product education R occurs if the energy of colliding molecules of the starting substances A And IN exceeds the energy barrier. Obviously, you can speed up this reaction if you somehow reduce the activation energy &.E ZKG

The general scheme of an enzymatic reaction, as is known, includes the formation of a single enzyme-substrate complex, in the active center of which the old bonds are broken and new bonds are formed with the appearance of the product.

Various theoretical models of the mechanism of action of enzymes propose different ways to lower the reaction barrier in the enzyme-substrate complex. As a result of the fixation of the substrate on the enzyme, there is a slight decrease in the entropy of the reagents compared to their free state. This in itself facilitates further chemical interactions between the active groups in the enzyme-substrate complex, which must be strictly oriented to each other. It is also assumed that the excess sorption energy, which is released when the substrate binds,

Rice. 12.1.

does not completely transform into heat. The sorption energy can be partially stored in the protein part of the enzyme, and then concentrated on the attacked bond in the area of ​​the formed enzyme-substrate contacts.

Thus, it is postulated that the sorption energy is used to create a low-entropy energetically intense conformation in the enzyme-substrate complex and thereby contributes to the acceleration of the reaction. However, experimental attempts to detect elastic deformations that could be stored in the protein globule of the enzyme without dissipating into heat for a sufficiently long time between catalytic acts (10 10 -3 s) were not successful. Moreover, necessary for

In catalysis, mutual orientation and convergence of the cleavable bond of the substrate and active groups in the center of the enzyme occur spontaneously, due to the intramolecular mobility of different, including active, groups of the enzyme and substrate. Such a rapprochement does not require the formation of any energetically unfavorable contacts. This conclusion follows from the analysis of nonvalent interactions in the active centers of a number of enzymes (a-chymotrypsin, lysozyme, ribonuclease, carboxyneptidase). Thus, the conformational tension in the enzyme-substrate complex itself is not a necessary source of energy and the driving force of catalysis.

Other models suggest that in the protein globule there is a non-dissipative transfer of thermal vibration energy from the outer layers of the protein to the attacked bond in the active center. However, there is no serious evidence for this, except for the statement that the enzyme must be “designed” so that its structure ensures the coherent nature of the propagation of fluctuational changes in conformation without thermal losses along certain degrees of freedom.

In addition to the lack of experimental evidence, a common drawback of these models is that they do not explicitly take into account an important factor - the spontaneous intramolecular mobility of the protein.

A step forward in this regard has been made in the conformational-relaxation concept of enzymatic catalysis. In it, the appearance of the product is considered as a result of successive conformational changes in the enzyme-substrate complex induced by initial changes in the electronic state in the active center of the enzyme. Initially, for a short time (10 |2 - 10 13 s), electronic-vibrational interactions occur, affecting only the selected chemical bonds of the substrate and functional groups of the enzyme, but not the rest of the protein globule.

As a result, a conformational nonequilibrium state is created, which relaxes to a new equilibrium with the formation of a product. The relaxation process occurs slowly and is directed, including the stages of product elimination and relaxation of the free enzyme molecule to the initial equilibrium state. The coordinate of the enzymatic reaction coincides with the coordinate of conformational relaxation. Temperature affects conformational mobility, and not the number of active collisions of free reactant molecules, which simply does not take place in an already formed enzyme-substrate complex.

Due to the large differences in speeds, fast electronic interactions in the active center, which occur over short distances, and slower conformational-dynamic changes in the protein part can be considered separately.

At the first stage of catalysis, the stochastic nature of the dynamics of the enzyme protein globule and the diffusion of the substrate to the active center lead to the formation of a strictly defined configuration, including the functional groups of the enzyme and the chemical bonds of the substrate. For example, in the case of hydrolysis of a peptide bond, the reaction requires simultaneous attack of the substrate by two groups of the active center - nucleophilic and electrophilic.

Example 12.1. In Fig. 12.2 shows the relative position of the cleavable peptide bond of the substrate and the side chains ser- 195, gis-51. The atom of the ser-195 residue is located at a distance of 2.8 A against the carbonyl carbon C1, and the proton of the hydroxyl group, without breaking the hydrogen bond with the N atom gis-51, is located at a distance of 2.0 A above the nitrogen atom of the fissile group. When this and only this configuration occurs, a chemical act of catalysis occurs. Formally, this corresponds to the simultaneous collision of several molecules, which is extremely unlikely in a solution.

The question arises: what is the probability of spontaneous formation of this kind of reactive configuration in a densely structured environment due to conformational fluctuations of several groups occurring according to the laws of limited diffusion?

Calculations show that there is a definite probability of several groups simultaneously falling into the “reactionary”

Rice. 12.2.

an area of ​​a certain radius where they are brought closer together at short distances. This probability depends mainly on the diffusion coefficient and the number of degrees of freedom of functional groups “searching” for each other in a limited space. For example, when hydrolyzing a peptide bond, it is necessary to create a favorable orientation for the two groups of the active site relative to certain areas of the substrate. Each of the groups has three degrees of freedom, and taking into account the vibrations of the substrate molecule, the total number of degrees of freedom N- 6 - 7. This is typical for enzymatic processes.

It turns out that under normal conditions the average time for the formation of such an active configuration is t ~

10 2 - 1SIc, which coincides with the enzyme turnover times under conditions of substrate saturation. In a solution for a similar reaction, this time is much longer, even with significant diffusion coefficients. The reason is that, once in a limited area in a densely structured environment, functional groups “find” each other and approach each other over short distances before they “scatter” in different directions, as happens in a solution. At the same time, the value m - 10~ 2 - 1CHc is much greater than the relaxation times of individual groups, which is a consequence of the rather stringent steric conditions for the reaction to occur. An increase in the number of functional groups and the necessary simultaneous contacts between them leads to an increase in the time to achieve a multicenter active configuration. The overall rate of enzymatic catalysis is determined precisely by the time of formation of the desired conformation during the spontaneous approach of the corresponding groups in the active center. The subsequent electronic interactions occur much faster and do not limit the overall rate of catalysis.

There are a number of features of enzymes that facilitate the conversion of the substrate at the active site. As a rule, the microenvironment of the active center with its amino acid residues is more hydrophobic than the surrounding aqueous environment. This reduces the value of the dielectric constant of the active center (e

The high local concentration of peptide bond dipoles creates electric fields in the active center with a strength of the order of thousands and hundreds of thousands of volts per centimeter. Thus, oriented polar groups create an intraglobular electric field that affects the Coulomb interactions in the active center.

The mechanisms of electronic transitions themselves in the active configuration require the use of quantum chemistry methods to be deciphered. Overlapping of electron orbitals can lead to a redistribution of electron density, the appearance of an additional charge on the antibonding orbital of the attacked bond in the substrate and its weakening.

This is exactly what happens during the hydrolysis of a peptide bond in a tetrahedral complex (see Fig. 12.2). The flow of electron density from Ofoj-cep-195 to the antibonding orbital in the peptide bond occurs due to the interaction of the lone pair of electrons 0[ 95 5 with the p-electrons of the C 1 atom of the peptide bond. In this case, the lone nitrogen pair of the amine group is pushed out of the peptide

Rice. 12.3.

N=C" bond, which loses its double character and is weakened as a result.

At the same time, the decrease in electron density from 0.95 weakens the H-O^ bond. But then the interaction between the enzyme H and the amine group N and its protonation with the transition of the proton from 0"[ h5 to gis-57. In turn, this again increases the interaction of Oj9 5 with the peptide group, etc.

Thus, a unique situation is created in a tetrahedic complex when several monomolecular reactions occur simultaneously, mutually accelerating each other. Synchronous movement of charge and proton between ser- 195, gis-57, peptide bond ensures high efficiency of the process. The catalytic act brings together three separate bimolecular reactions into a single cooperative system, leading to the cleavage of the peptide bond - an event that is unlikely in solution. Natural conformational rearrangements are indicated in the system and, as a result, deacylation of the enzyme and protonation of the atom occurs 0} 95 .

The principle of the formation of a polyfunctional closed system of atomic groups in the active configuration is also fulfilled in other enzyme-substrate complexes (Fig. 12.3).

In enzymatic catalysis, the multistage nature of substrate transformations, which is unlikely in solution, is ensured due to their synchronous cooperative occurrence in a single multifunctional system.

Replacing ineffective sequential activation stages with a coordinated process formally leads to a decrease in the activation energy of the entire reaction. Let us note once again that, strictly speaking, the physical meaning of the concept of “activation energy” in enzymatic processes does not correspond to that for reactions in solutions that proceed through the mechanism of active collisions of free molecules.

The sequence of events in enzymatic catalysis can be described by the following scheme. 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).

Catalysis is the process of accelerating a chemical reaction under the influence of catalysts that actively participate in it, but remain chemically unchanged by the end of the reaction. 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.

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 enzymes, 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.