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 performance chemical reaction. 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 hydrolysis peptide bonds when digesting proteins. A covalent bond is formed between the substrate and the serine amino acid residue of the active site of the enzyme.
Catalysts- substances that change the rate of a chemical reaction, but themselves remain unchanged. Biological catalysts are called enzymes.
Enzymes (enzymes)- biological catalysts of a protein nature, synthesized in cells and accelerating chemical reactions under normal body conditions by hundreds and thousands of times.
Substrate- a substance on which an enzyme acts.
Apoenzyme- the protein part of the protein enzyme molecule.
Coenzymes (cofactors)- the non-protein part of the enzyme, plays an important role in the catalytic function of enzymes. They may contain vitamins, nucleotides, etc.
Enzyme active site- a section of an enzyme molecule with a specific structure that binds and converts the substrate. In the molecules of simple enzyme proteins (proteins), they are built from amino acid residues and can include various functional groups (-COOH, -NH 2, -SH, -OH, etc.). In the molecules of complex enzymes (proteins), in addition to amino acids, non-protein substances (vitamins, metal ions, etc.) participate in the formation of the active center.
Allosteric center of the enzyme- a section of an enzyme molecule to which specific substances can bind, changing the structure of the enzyme and its activity.
Enzyme activators- molecules or ions that increase enzyme activity. For example, hydrochloric acid is an activator of the enzyme pepsin; Calcium ions Ca++ are activators of muscle ATPase.
Enzyme inhibitors- molecules or ions that reduce enzyme activity. For example, Hg ++ and Pb ++ ions inhibit the activity of almost all enzymes.
Activation energy- an additional amount of energy that molecules must have in order for their collision to lead to interaction and the formation of a new substance.
Mechanism of action of enzymes- is due to the ability of enzymes to lower the energy barrier of a reaction due to interaction with the substrate and the formation of an intermediate enzyme-substrate complex. To carry out a reaction with the participation of an enzyme, less energy is required than without it.
Thermal lability of enzymes– dependence of enzyme activity on temperature.
Temperature optimum of enzymes- temperature range from 37° to 40°C, at which the greatest activity of enzymes in the human body is observed.
Enzyme specificity - the ability of an enzyme to catalyze a specific chemical reaction.
Relative enzyme specificity- the ability to catalyze the transformation of a group of substrates of similar structure that have a certain type of connection. For example, the enzyme pepsin catalyzes the hydrolysis of various food proteins by breaking the peptide bond.
Absolute (strict) specificity of the enzyme- the ability to catalyze the transformation of only one substrate of a certain structure. For example, the enzyme maltase catalyzes the hydrolysis of only maltose.
Proenzyme- inactive form of the enzyme. For example, the proenzyme of pepsin is pepsinogen.
Coenzyme A, or coenzyme acetylation (CoA)- a coenzyme of many enzymes that catalyze reactions of addition of acetyl groups to other molecules. It contains vitamin IN 3 .
NAD (nicotinamide adenine dinucleotide)- coenzyme of biological oxidation enzymes, carrier of hydrogen atoms. It contains vitamin PP (nicotinamide).
Flavin adenine dinucleotide (FAD)- the non-protein part of flavin-dependent dehydrogenases, which is associated with the protein part of the enzyme. Participates in redox reactions, contains vitamin IN 2 .
Enzyme classes:
Oxidoreductases- enzymes that catalyze redox reactions. These include dehydrogenases and oxidases.
Transferases- enzymes that catalyze reactions that transfer atoms or groups of atoms from one substance to another.
Hydrolases- enzymes that catalyze hydrolysis reactions of substances.
Lyases- enzymes that catalyze reactions of non-hydrolytic elimination of groups of atoms from the substrate or breaking of the carbon chain of a compound.
Isomerases- enzymes that catalyze the formation of isomers of substances.
Ligases (synthetases)- enzymes that catalyze the biosynthesis reactions of various substances in the body.
The mechanism of action of enzymes can be considered from two positions: from the point of view of changes in the energy of chemical reactions and from the point of view of events in the active center.
A. Energy changes during chemical reactions
Any chemical reactions proceed in accordance with two basic laws of thermodynamics: the law of conservation of energy and the law of entropy. According to these laws, the total energy chemical system and its environment remains constant, while the chemical system tends to decrease order (increase entropy). To understand the energy of a chemical reaction, it is not enough to know the energy balance of the reagents entering and exiting the reaction; it is necessary to take into account energy changes during the process of a given chemical reaction and the role of enzymes in the dynamics of this process. Consider the decomposition reaction of carbonic acid:
H2CO3 → H20 + CO2.
Carbonic acid is weak; the reaction of its decomposition will proceed under normal conditions if the molecules of carbonic acid have an energy exceeding a certain level, called the activation energy Ea (Fig. 2-10).
Activation energy is the additional amount of kinetic energy required for the molecules of a substance to react.
When this energy barrier is reached, changes occur in the molecule causing redistribution chemical bonds and the formation of new compounds. Molecules possessing Ea are said to be in a transition state. The energy difference between the initial reagent H2CO3 and the final compounds H2O and CO2 is called the change free energy DG reactions. H2O and CO2 molecules are more stable substances than H2CO3, i.e. have less energy and practically do not react under normal conditions. The energy released as a result of this reaction is dissipated in the form of heat into the environment.
How more molecules has energy exceeding the level Ea, the higher the rate of the chemical reaction. You can increase the rate of a chemical reaction by heating. This increases the energy of the reacting molecules. However, high temperatures are destructive for living organisms, so enzymes are used in cells to speed up chemical reactions. Enzymes provide a high rate of reactions under optimal conditions existing in the cell by lowering the level of Ea. Thus, enzymes reduce the height of the energy barrier, as a result the number of reactive molecules increases, and therefore the reaction rate increases.
In the mechanism of enzymatic catalysis, the formation of unstable intermediate compounds is of decisive importance - the enzyme-substrate complex ES, which undergoes transformation into an unstable transition complex EP, which almost instantly disintegrates into a free enzyme and the reaction product.
Thus, biological catalysts (enzymes) do not change free energy.
An enzyme, performing the function of a catalyst for a chemical reaction, obeys the general laws of catalysis and has all the properties characteristic of non-biological catalysts, but also has distinctive properties associated with the structural features of enzymes.
The similarity between enzymes and non-biological catalysts is that:
Enzymes catalyze energetically possible reactions;
· the energy of the chemical system remains constant;
·during catalysis, the direction of the reaction does not change;
Enzymes are not consumed during the reaction.
The differences between enzymes and non-biological catalysts are that:
· the rate of enzymatic reactions is higher than reactions catalyzed by non-protein catalysts;
Enzymes are highly specific;
· the enzymatic reaction takes place in the cell, i.e. at a temperature of 37 °C, constant atmospheric pressure and physiological pH;
The speed of the enzymatic reaction can be adjusted.
Mechanisms of enzyme action
In general terms, it all comes down to the complementary interaction of enzyme and substrate. In this case, the functional groups of the substrate interact with their corresponding functional groups of the enzyme. The presence of substrate specificity is explained by two hypotheses:
1. Fisher's theory(model of “rigid matrix”, “key-lock”) - the active center of the enzyme strictly corresponds to the configuration of the substrate and does not change when it is attached. This model explains absolute specificity well, but not group specificity.
2. In 1958, Daniel Koshland proposed a modification of the “key-lock” model. Enzymes are generally not rigid, but flexible molecules. The active site of an enzyme can change conformation after binding a substrate. The amino acid side groups of the active site assume a position that allows the enzyme to perform its catalytic function. In some cases, the substrate molecule also changes conformation after binding at the active site. Unlike the key-lock model, the induced-fit model explains not only the specificity of enzymes, but also the stabilization of the transition state. This model is called the “glove hand”.
Kinetics of enzymatic reactions. Dependence of the rate of enzymatic reactions on temperature, pH of the environment, enzyme concentrations, and substrate. The concept of pH and temperature optimums, physiological and clinical diagnostic significance. Determination of the Michaelis constant and its clinical and diagnostic significance.
The kinetics of an enzymatic reaction (i.e., the dependence of the reaction rate on its conditions) is determined primarily catalyst properties.
Enzyme kinetics studies the patterns of influence of the chemical nature of reacting substances (enzymes, substrates) and the conditions of their interaction (concentration, pH, temperature, presence of activators or inhibitors) on the rate of enzymatic reactions. The main goal of studying the kinetics of enzymatic reactions is to obtain information that can help elucidate the molecular mechanism of enzyme action.
Dependence of the rate of enzymatic reaction on the amount of enzymes:
When an enzymatic reaction is carried out under conditions of excess substrate, the reaction rate will depend on the concentration of the enzyme. The graphical dependence of such a reaction has the form of a straight line. However, the amount of enzyme is often impossible to determine in absolute terms, so in practice they use conditional values characterizing the activity of the enzyme: one international unit of activity (IU) corresponds to the amount of enzyme that catalyzes the conversion of 1 µmol of substrate in 1 min under optimal conditions for the enzymatic reaction. Optimal conditions are individual for each enzyme and depend on the temperature of the environment, the pH of the solution, in the absence of activators and inhibitors.
Rice. Dependence of product accumulation (A) and substrate loss (B) on the time (duration) of the reaction. The rate of an enzymatic reaction is determined by the change in the concentration of the product or substrate per unit time.
The period of an enzymatic reaction is characterized by a nonlinear accumulation of product (or loss of substrate) depending on the reaction time.
unit of enzyme activity: 1 katal (kat), corresponding to the amount of catalyst that converts 1 mole of substrate in 1 s. The number of catalyses is determined by the formula:
The international unit of enzymatic activity ME is related to the cathal by the following equalities:
1 cat = 1 mol S/c = 60 mol S/min = 60x10 6 µmol/min = 6x10 7 ME,
1 ME = 1 µmol/min = 1/60 µmol/s = 1/60 µkat = 16.67 nkat.
In medical practice, enzyme activity is often assessed. international units activity - ME. To estimate the number of enzyme molecules among other proteins of a given tissue, the specific activity (sp. ac.) of the enzyme is determined, numerically equal to the number of enzyme activity units (pME) in the tissue sample divided by the mass (mg) of the protein in this tissue:
The specific activity is used to judge the purification of the enzyme: the fewer foreign proteins, the higher the specific activity.
Dependence of the rate of enzymatic reaction on the temperature of the medium
Temperature rises to certain limits influences the rate of an enzymatic reaction, similar to the effect of temperature on any chemical reaction. As the temperature increases, the movement of molecules accelerates, which leads to an increase in the likelihood of interaction between reactants. In addition, temperature can increase the energy of reacting molecules, which also speeds up the reaction. However, the rate of a chemical reaction catalyzed by enzymes has its own temperature optimum, the excess of which is accompanied by a decrease in enzymatic activity resulting from thermal denaturation of the protein molecule.
For most human enzymes, the optimal temperature is 37-38 °C. However, thermostable enzymes also exist in nature. For example, Taq polymerase isolated from microorganisms living in hot springs is not inactivated when the temperature rises to 95 °C. This enzyme is used in scientific and practical medicine for the molecular diagnosis of diseases using the polymerase chain reaction (PCR) method.
Rice. Dependence of the enzymatic reaction rate (V) on temperature.
Dependence of the rate of enzymatic reaction on the pH of the medium
For each enzyme there is a pH value at which its maximum activity is observed. Deviation from the optimal pH value leads to a decrease in enzymatic activity.
The effect of pH on enzyme activity is associated with ionization functional groups amino acid residues of a given protein, ensuring the optimal conformation of the active center of the enzyme. When pH changes from optimal values, the ionization of the functional groups of the protein molecule changes. For example, when the environment is acidified, free amino groups (NH 3 +) are protonated, and when alkalization occurs, a proton is removed from carboxyl groups (COO -). This leads to a change in the conformation of the enzyme molecule and the conformation of the active center; consequently, the attachment of the substrate, cofactors and coenzymes to the active center is disrupted. In addition, the pH of the environment can affect the degree of ionization or spatial organization of the substrate, which also affects the affinity of the substrate for the active site. With a significant deviation from the optimal pH value, denaturation of the protein molecule can occur with a complete loss of enzymatic activity.
The optimum pH value is different for different enzymes. Enzymes that work under acidic environmental conditions (for example, pepsin in the stomach or lysosomal enzymes) acquire a conformation that ensures the enzyme operates at acidic pH values. However, most enzymes in the human body have an optimum pH close to neutral, coinciding with physiological significance pH. 
Dependence of the rate of enzymatic reaction on the amount of substrate
If the concentration of enzymes is left constant, changing only the amount of substrate, then the graph of the rate of the enzymatic reaction is described by a hyperbola.
As the amount of substrate increases, the initial speed increases. When the enzyme becomes completely saturated with substrate, i.e. the maximum possible formation of an enzyme-substrate complex occurs at a given enzyme concentration, and the highest rate of product formation is observed. A further increase in the substrate concentration does not lead to an increase in product formation, i.e. the reaction rate does not increase. This state corresponds to the maximum reaction speed Vmax.
Thus, the enzyme concentration is the limiting factor in the formation of the product. This observation formed the basis of enzyme kinetics developed by scientists L. Michaelis and M. Menten in 1913.
The enzymatic process can be expressed by the following equation:
where k 1 is the rate constant for the formation of the enzyme-substrate complex; k -1 is the rate constant of the reverse reaction, the decomposition of the enzyme-substrate complex; k 2 is the rate constant for the formation of the reaction product.
The following ratio of rate constants (k -1 + k 2)/k 1 is called the Michaelis constant and denoted K m.
The reaction rate is proportional to the concentration of the enzyme-substrate ES complex, and the rate of ES formation depends on the substrate concentration and the concentration of free enzyme. The concentration of ES is affected by the rate of formation and decay of ES.
Highest speed reactions are observed when all enzyme molecules are in complex with the substrate, i.e. in the enzyme-substrate complex ES, i.e. [E] = .
The dependence of the rate of an enzymatic reaction on the substrate concentration is expressed by the following equation
| V max [S] |
| K m + [S] |
This equation is called the Michaelis-Menten equation.
In the case where the reaction rate is half the maximum, K m = [S] Thus, the Michaelis constant is numerically equal to the substrate concentration at which half the maximum rate is achieved.
The Michaelis-Menten equation is the basic equation of enzyme kinetics, describing the dependence of the rate of an enzymatic reaction on the concentration of the substrate.
If the substrate concentration is significantly greater than K m (S >> K m), then an increase in the substrate concentration by K m has practically no effect on the sum (K m + S) and it can be considered equal to the substrate concentration. Consequently, the reaction rate becomes equal to the maximum speed: V = V max. Under these conditions, the reaction has zero order, i.e. does not depend on the substrate concentration. We can conclude that V max is a constant value for a given enzyme concentration, independent of the substrate concentration.
If the substrate concentration is significantly less than K m (S<< K m), то сумма (K m + S) примерно равна К m , следовательно, V = V max [S]/K m , т.е. в данном случае скорость реакции прямо пропорциональна концентрации субстрата (реакция имеет первый порядок).
Rice. Dependence of reaction rate (V) on substrate concentration S.
V max and K m are the kinetic characteristics of the enzyme efficiency.
· V max characterizes the catalytic activity of the enzyme and has the dimension of the rate of the enzymatic reaction mol/l, i.e. determines the maximum possibility of product formation at a given enzyme concentration and under conditions of excess substrate. K m characterizes the affinity of a given enzyme for a given substrate and is a constant value that does not depend on the concentration of the enzyme. The less
· Km, the greater the affinity of the enzyme for a given substrate, the higher the initial reaction rate and vice versa, the greater Km, the lower the initial reaction rate, the lower the affinity of the enzyme for the substrate.
The following steps can be distinguished in an enzymatic reaction:
1. Attachment of a substrate (S) to an enzyme (E) to form an enzyme-substrate complex (E-S).
2. Conversion of the enzyme-substrate complex into one or more transition complexes (E-X) in one or more steps.
3. Conversion of the transition complex into an enzyme-product (E-P) complex.
4. Separation of final products from the enzyme.
Mechanisms of catalysis
| Donors | Acceptors |
|
UNS |
-SOO - -NH 2 -S- -O- |
1. Acid-base catalysis– in the active center of the enzyme there are groups of specific amino acid residues that are good donors or acceptors of protons. Such groups are powerful catalysts for many organic reactions.
2. Covalent catalysis– enzymes react with their substrates, forming, using covalent bonds, very unstable enzyme-substrate complexes, from which reaction products are formed during intramolecular rearrangements.
Types of Enzyme Reactions
1. Ping-pong type– the enzyme first interacts with substrate A, removing any chemical groups from it and converting it into the corresponding product. Substrate B is then attached to the enzyme, receiving these chemical groups. An example is the reaction of transfer of amino groups from amino acids to keto acids - transamination.
Ping-pong enzymatic reaction
2. Type of sequential reactions– substrates A and B are sequentially added to the enzyme, forming a “ternary complex”, after which catalysis occurs. The reaction products are also sequentially cleaved from the enzyme.
Enzymatic reaction according to the "sequential reactions" type
3. Type of random interactions– substrates A and B are added to the enzyme in any order, randomly, and after catalysis they are also cleaved off.
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 that form 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 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).
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