A). 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.
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. In reactions catalyzed by enzymes 1 and 2, the initial rate of the reaction catalyzed by enzyme 1 is lower than the rate of the reaction catalyzed by enzyme 2, since the tangent of the tangent to the reaction profile curve drawn from the "O" point of the second enzyme is higher, as in the case of accumulation of product (A) and loss of substrate (B). The speed at any time t is determined by the tangent of the tangent to the reaction profile at time t. The time period of an enzymatic reaction is characterized by a linear accumulation of product (or loss of substrate) depending on the duration of the reaction. The period of an enzymatic reaction is characterized by a nonlinear accumulation of product (or loss of substrate) depending on the reaction time.
The number of nME activity units is determined by the formula:
B). Dependence of the rate of enzymatic reaction on the temperature of the medium
Increasing the temperature to certain limits affects the rate of enzymatic
reaction is 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 speed chemical reaction, catalyzed by enzymes, has its own temperature optimum, the excess of which is accompanied by a decrease in enzymatic activity arising due to 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.
IN). Dependence of the rate of enzymatic reaction on the amount of substrate
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 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.
The highest reaction rate is 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 concentration of the substrate is expressed by the following equation (the mathematical derivation of this formula can be found in textbooks on enzymatic kinetics):
V = Vmax[S] / Km + [S]
This equation is called the Michaelis-Menten equation.
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 Km (S >> Km), then an increase in the substrate concentration by the value of Km has virtually no effect on the sum (Km + S) and it can be considered equal to the substrate concentration. Consequently, the reaction rate becomes equal to the maximum speed: V = Vmax. Under these conditions, the reaction has zero order, i.e. does not depend on the substrate concentration. We can conclude that Vmax is a constant value for a given enzyme concentration, independent of the substrate concentration.
If the substrate concentration is significantly less than Km(S<< Km), то сумма (Km + S) примерно равна Кm, следовательно, V = Vmax[S]/Km, т.е. в данном случае скорость реакции прямо пропорциональна концентрации субстрата (реакция имеет первый порядок).
Vmax and Km are kinetic characteristics of enzyme efficiency.
Vmax 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. Km 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 smaller Km, the greater the affinity of the enzyme for a given substrate, the higher the initial reaction rate, and vice versa, the larger Km, the lower the initial reaction rate, the lower the affinity of the enzyme for the substrate.
KINETICS OF ENZYMATIVE REACTIONS
Vfr is determined by the amount of substance that is converted per unit of time. V of these reactions depends on the influence of external factors (temperature, pH, exposure to natural and foreign compounds, etc.).
Vfr is a measure of catalytic activity and is simply referred to as enzyme activity.
Enzyme activity can only be measured indirectly:
1) by the amount of converted S;
2) increase in concentration P per unit time.
To express enzyme concentration use:
a) the unit of measurement of enzymes is the amount of enzyme that catalyzes the conversion of 1 µmol S per minute. [µmol/min];
b) 1 catal (cat) - the amount of enzymes capable of causing the conversion of 1 mole of S into P in 1 second. [mol/s].
1 cat = 6×107E; 1E = 16.67 (n cat)
To express enzyme activity, use:
a) specific activity of enzymes is the number of enzymes per 1 mg or the number of cat. per 1 kg of protein;
b) molecular activity or turnover number is the number of molecules S undergoing conversion by one molecule E per 1 minute.
One molecule of erythrocyte catalase breaks down 5 × 106 molecules of H2O2 in 1 minute.
Specificity of enzyme action
The concept of the E S complex and ACP are closely related to the special property of enzymes - their specificity. According to the degree of specificity (in descending order) there are:
I. Stereochemical substrate specificity - in this case, enzymes catalyze only 1 form of S (1 isomer). For example, fumarate hydratase catalyzes only the conversion of fumaric acid, but does not catalyze the conversion of its isomer, maleic acid.
II. Absolute substrate specificity - E is converted only by 1S. For example, urease converts only urea.
III. Absolute group S specificity. Enzymes act on a group of similar S-b. For example, alcohol DG converts not only ethanol, but also other aliphatic alcohols.
IV. Relative group S specificity. The enzyme does not act on a group of S molecules, but on certain bonds of certain S groups. For example, pepsin and trypsin are specific for peptide bonds in various proteins.
V. Relative S specificity. The enzyme catalyzes, turning into S-b, belonging to various groups of chemical compounds. For example, the enzyme cytochrome-450 catalyzes hydroxylation reactions of up to 7000 different S-b. This is the least specific enzyme system.
There are two theories to explain enzyme specificity.
E. Fisher's hypothesis is the “key and lock” hypothesis or the “template” hypothesis. According to Fischer, an enzyme is a rigid structure, the ACP of which is an exact “cast” of S. If S fits E like a key to a lock, then the reaction will occur. If S is slightly changed (the "key"), then it does not correspond to the ACF (the "lock"), and the reaction becomes impossible. Although this explanation is logical, Fisher's hypothesis does not explain what absolute and relative group specificity are then based on. For example, cytochrome-450 combines with such a large number of S-b, different in structure.
These external contradictions are explained by the Koshland hypothesis, or the forced correspondence hypothesis. According to Koshland, the enzyme molecule is not “rigid”, but flexible, the structure and configuration of the enzyme and its ACP begin to change the moment the enzyme attaches to S or other ligands. During the formation of an E-S complex, in addition to geometric complementarity, electrostatic complementarity also occurs, which occurs due to the pairing of oppositely charged molecules E and S. In reality, apparently, both variants of addition take place.
Koshland's hypothesis allows us to explain why the transformation of close analogues of S-in occurs. If the “false” substrate (quasi-S) differs from the natural one and ACP takes on a conformation close to the true substrate, then the arrangement of catalytic groups in such an E-S complex will allow the reaction to occur. The enzyme does not seem to notice this “deception,” although the reaction does not proceed as quickly as with the true substrate. If the configuration of the quasi-substrate does not allow the correct positioning of the catalytic group, then in this case the reaction will not proceed. Those. if the range of conformational rearrangements is limited to one only possible one, then the enzyme is highly specific, and if the possibilities of ACP rearrangement are great, then the enzyme also works on quasi-substrates.
Dependence of Vfr on pH environment
Each enzyme has its own optimum pH, at which Vfr is maximum. A pH deviation in one direction or another leads to a decrease in enzyme activity. Most enzymes have a pH of ~7.0, that is, it coincides with physiological pH values.
At the optimal pH value, the functional groups of ACP and S itself are in the most preferred form for bonding. Some enzymes have an optimal pH that differs sharply from physiological values; pepsin is 100% active at pH = 1.5-2.5; arginase – at pH = 10.
Dependence of Vfr on temperature
With increasing environmental temperature, Vfr increases, reaching optimal values of ~ 20-40ºС for most enzymes.
The thermolability of enzymes is associated with their protein structure: when the temperature rises to 40-50ºC and above, they denature.
For some enzymes, denaturation occurs at 0ºC.
For any chemical reactions, with an increase in temperature for every 10ºC, the V of the reaction increases by 2-3 times; for enzymatic reactions this coefficient is lower - 2 or even less. Exception: the thermostable enzyme adenymate cyclase can withstand temperatures of 100ºC, and the enzyme catalase is active at 0ºC.
Dependence of Vfr on concentration. S.
The mechanism of action of enzymes is described by the Michaelis-Menten equation. The dependence of Vfr on [S] can be established graphically.
a) according to the Michaelis curve: the smaller Km, the greater Vm and the higher the affinity of E for S.
Vmax corresponds to the state of complete saturation of the enzyme S-vol.
in solution there is an excess of E (3 mol S, 5 mol E) this is the site of saturation of the enzyme S-vol.
b) the Lainciver-Burk reciprocal method, where the dependence of Vfr on [S] is calculated in reciprocal quantities.
Regulation of enzyme activity.
Enzymes are catalysts with controlled activity, so Vfr can be controlled through enzymes. Regulation of activity can be carried out through the interaction of enzymes with various biological components or foreign compounds (drugs, poisons), which are called modifiers. If in the presence of a modifier Vfr increases, then such modifiers are called activators, and if it decreases, they are called inhibitors.
Activation of enzymes.
There are several types of enzyme activation.
1. Activation by influencing the subunits of enzyme molecules. Some enzymes have a SN in the form of 2 subunits: catalytic and regulatory. When saving an emergency situation, the ACF is hidden.
For example, many enzymes in the body are produced as proenzymes or zymogens, that is, in an inactive state. As needed, a certain number of them are activated. For example, inactive trypsinogen is converted into active trypsin by the enzyme enterokinase.
2. Ions influence the activation of enzymes:
a) cations - their effect is more specific than anions. Cations themselves can act as prosthetic groups in enzymes (Fe in cytochrome) or by their presence influence the enzyme, activating it. For example, carbonic anhydrase is activated in the presence of Zn+2.
b) anions - act less specifically and usually affect the 2nd stage of the d.f. – disintegration of the ES complex. However, sometimes anions are direct activators of enzymes. For example, Cl– activates inactive pepsinogen and converts it into active pepsin.
3. Activation by protecting enzymes from the inactivating influence of various influences. Provided with specific substances that prevent negative effects on enzymes.
Enzyme inhibition.
Substances that cause partial or complete inhibition of enzymes are called inhibitors (I). Inhibitors have the property of binding tightly to the enzyme. On this basis, inhibition is distinguished: reversible and irreversible.
With reversible inhibition, I and E interact. If the inhibitor is somehow neutralized (for example, by dialysis), then the activity of E is restored. If this cannot be achieved, then we are talking about irreversible inhibition.
Reversible inhibition
competitive non-competitive
Competitive inhibition can be caused by substances with a structure similar to that of true S.
I and S compete for ACP, and the complex with the enzyme forms the compound that has more molecules. Either I or S binds to the enzyme; for such inhibition, the equation is valid: .
During competitive inhibition, a ternary E S I complex is NEVER formed, which is how this type of inhibition differs from others.
For example, DG succinate is included in farms. CTK systems. Its natural S is succinate. Inhibitors can be oxaloacetate, malonate (quasi-substrates).
When in excess, the inhibitor binds in polarized groups to ACP succinate DG.
With competitive inhibition, Vmax never changes, but Km does. The slope of the curves in the presence of I increases, as a result Km increases
Based on the results of the experiment using the Michaelis-Menten curve, it is possible to establish the competitive nature of I (by increasing Km and stability of Vmax). The nature of this curve also indicates that the process is reversible, that is, by increasing [S], the time to reach Vmax can be reduced.
The competitive inhibition method has found wide application in medical practice.
Para-aminobenzoic acid and sulfonamide have a similar structure. The bacterial cell uses p-ABA to synthesize folic acid, which is a component of bacterial enzymes. S/a blocks the action of enzymes that synthesize folic acid, as a result, bacterial growth stops.
Non-competitive inhibition is reversible inhibition when I interacts not with ACP, but with other functional groups of enzymes, that is, in this case, I has no structural similarity to S. The addition of such an inhibitor reduces the activity of the enzyme, and not its affinity for S, that is the inhibitor does not change Km, but reduces max. Vfr.
With this type of inhibition, inactive low-dissociation complexes E I or E I S are formed. For example, the action of HCN, other chemical compounds that bind Me ions or other functional groups in the enzyme molecule.
Mixed inhibition (or partially non-competitive type) - a decrease in Vmax is combined with an increase in Km.
In this case, an E I S complex is formed, and the S in it undergoes a slow catalytic transformation.
Substrate inhibition is a decrease in Vfr with a significant increase in [S]. Initially, with an increase in [S], Vfr increases, reaching its maximum, but with a further increase in [S], Vfr begins to fall.
The mechanism of the inhibitory effect of excess S is varied. Most often, this is the interaction of intermediate compounds E S with one or more molecules of S, resulting in the formation of an inactive compound, then
there is a complex that does not produce reaction products.
Methods for regulating enzyme activity
In a living organism, reactions of synthesis, decomposition and interconversion of thousands of different substances simultaneously occur. All these many reactions are regulated in the body through various mechanisms, the most important of which are:
a) feedback-type regulation; usually characteristic of synthesis reactions. The accumulation of reaction products above the permissible level has a strong inhibitory effect on the first stage of the process:
b) allosteric regulation of enzyme activity - characteristic only of a special group of enzymes with SN, which have regulatory centers for binding allosteric effectors. Negative effectors inhibit the conversion of S and act as allosteric inhibitors. Positive effectors, on the contrary, accelerate Vfr, therefore they are classified as allosteric activators.
The mechanism of action of allosteric inhibitors on an enzyme is to change the ACP of this enzyme. A decrease in Vfr is either a consequence of an increase in Km, or a result of a decrease in Vmax, at the same saturating concentrations of S. Allosteric activators, on the contrary, facilitate the conversion of S into ACP, which is accompanied by either a decrease in Km or an increase in Vmax.
Compartmentalization is a phenomenon in which membranes are used to spatially separate
a) an enzyme from its S (for example, lysomal enzymes from the substances on which they act in the cytoplasm);
b) processes that are mutually incompatible at the same time. The synthesis of fatty acids occurs in the soluble part of the cytoplasm, and the breakdown of fatty acids occurs in the mitochondria.
Enzyme kinetics studies the rate of reactions catalyzed by enzymes depending on various conditions (concentration, temperature, pH, etc.) of their interaction with the substrate.
However, enzymes are proteins that are sensitive to the influence of various external influences. Therefore, when studying the rate of enzymatic reactions, they mainly take into account the concentrations of reacting substances, and try to minimize the influence of temperature, pH of the environment, activators, inhibitors and other factors and create standard conditions. Firstly, this is the pH value of the environment that is optimal for a given enzyme. Secondly, it is recommended to maintain a temperature of 25°C, where possible. Thirdly, complete saturation of the enzyme with the substrate is achieved. This point is especially important because at low substrate concentrations, not all enzyme molecules participate in the reaction (Fig. 6.5, A), which means the result will be far from the maximum possible. The greatest power of the catalyzed reaction, other things being equal, is achieved if each enzyme molecule participates in the transformation, i.e. at a high concentration of the enzyme-substrate complex (Fig. 6.5, V). If the substrate concentration does not ensure complete saturation of the enzyme (Fig. 6.5, b), then the rate of the reaction does not reach its maximum value.
Rice. 65.
A - at low substrate concentration; 6 - with insufficient substrate concentration; V - when the enzyme is completely saturated with substrate
The rate of an enzymatic reaction measured under the above conditions and complete saturation of the enzyme with the substrate is called maximum rate of enzymatic reaction (V).
The rate of the enzymatic reaction, determined when the enzyme is not completely saturated with the substrate, is denoted v.
Enzyme catalysis can be simplified by the following diagram:
where F is an enzyme; S - substrate; FS - enzyme-substrate complex.
Each stage of this process is characterized by a certain speed. The unit of measurement for the rate of an enzymatic reaction is the number of moles of substrate converted per unit of time(same as the speed of a normal reaction).
The interaction of the enzyme with the substrate leads to the formation of an enzyme-substrate complex, but this process is reversible. The rates of forward and reverse reactions depend on the concentrations of the reactants and are described by the corresponding equations:
In a state of equilibrium, equation (6.3) is valid, since the rates of the forward and reverse reactions are equal.
Substituting the speed values of the forward (6.1) and reverse (6.2) reactions into equation (6.3), we obtain the equality:
The state of equilibrium is characterized by an appropriate equilibrium constant K p, equal to the ratio of the constants of the forward and reverse reactions (6.5). The reciprocal of the equilibrium constant is called substrate constant Ks, or the dissociation constant of the enzyme-substrate complex:
From equation (6.6) it is clear that the substrate constant decreases at high concentrations of the enzyme-substrate complex, i.e. with great stability. Consequently, the substrate constant characterizes the affinity of the enzyme and substrate and the ratio of the rate constants for the formation and dissociation of the enzyme-substrate complex.
The phenomenon of enzyme saturation with substrate was studied by Leonor Michaelis and Maud Mepten. Based on mathematical processing of the results, they derived equation (6.7), which received their names, from which it is clear that at a high substrate concentration and a low value of the substrate constant, the rate of the enzymatic reaction tends to the maximum. However, this equation is limited because it does not take into account all parameters:
The enzyme-substrate complex during the reaction can undergo transformations in different directions:
- dissociate into parent substances;
- transform into a product from which the enzyme is separated unchanged.
Therefore, to describe the overall action of the enzymatic process, the concept Michaelis constants Kt, which expresses the relationship between the rate constants of all three reactions of enzymatic catalysis (6.8). If both terms are divided by the reaction rate constant for the formation of the enzyme-substrate complex, we obtain expression (6.9):
An important corollary follows from equation (6.9): the Michaelis constant is always greater than the substrate constant by the amount k 2 /k v
Numerically K t equal to the concentration of the substrate at which the reaction rate is half the maximum possible speed and corresponds to the saturation of the enzyme with the substrate, as in Fig. 6.5, b. Since in practice it is not always possible to achieve complete saturation of the enzyme with the substrate, it is precisely K t used for comparative characterization of the kinetic characteristics of enzymes.
The rate of the enzymatic reaction when the enzyme is not completely saturated with the substrate (6.10) depends on the concentration of the enzyme-substrate complex. The proportionality coefficient is the reaction constant for the release of the enzyme and product, since this changes the concentration of the enzyme-substrate complex:
After transformations, taking into account the above dependencies, the rate of the enzymatic reaction when the enzyme is not completely saturated with the substrate is described by equation (6.11), i.e. depends on the concentrations of the enzyme, substrate and their affinity K s:
The graphical dependence of the rate of an enzymatic reaction on the concentration of the substrate is not linear. As is obvious from Fig. 6.6, with increasing substrate concentration, an increase in enzyme activity is observed. However, when maximum saturation of the enzyme with the substrate is achieved, the rate of the enzymatic reaction becomes maximum. Therefore, the rate-limiting factor for the reaction is the formation of an enzyme-substrate complex.
Practice has shown that substrate concentrations, as a rule, are expressed in values much less than unity (10 6 -10 3 mol). It is quite difficult to operate with such quantities in calculations. Therefore, G. Lineweaver and D. Burke proposed to express the graphical dependence of the rate of an enzymatic reaction not in direct coordinates, but in inverse ones. They proceeded from the assumption that for equal quantities their inverses are also equal:
Rice. 6.6.
After transforming expression (6.13), we obtain an expression called Lineweaver-Burk equation (6.14):
The graphical dependence of the Lineweaver-Burk equation is linear (Fig. 6.7). The kinetic characteristics of the enzyme are determined as follows:
- the segment cut off on the ordinate axis is equal to 1/V;
- the segment cut off on the abscissa axis is equal to -1 /To t.
Rice. 6.7.
It is believed that the Lineweaver-Burk method makes it possible to determine the maximum reaction rate more accurately than in direct coordinates. Valuable information regarding enzyme inhibition can also be gleaned from this graph.
There are other ways to transform the Michaelis-Menten equation. Graphic dependencies are used to study the influence of various external influences on the enzymatic process.
Kinetics of enzymatic reactions. This branch of enzymology studies the influence of chemical and physical factors on the rate of enzymatic reactions. In 1913, Michaelis and Menten created the theory of enzymatic kinetics, based on the fact that the enzyme (E) interacts with the substrate (S) to form an intermediate enzyme-substrate complex (ES), which further decomposes into the enzyme and the reaction product according to the equation:
Each stage of interaction between the substrate and the enzyme is characterized by its own rate constants. The ratio of the sum of the rate constants for the decomposition of the enzyme-substrate complex to the rate constant for the formation of the enzyme-substrate complex is called the Michaelis constant (Km). They determine the affinity of the enzyme for the substrate. The lower the Michaelis constant, the higher the affinity of the enzyme for the substrate, the higher the rate of the reaction it catalyzes. Based on the Km value, catalytic reactions can be divided into fast (Km 106 mol/l or less) and slow (Km 102 to 106).
The rate of an enzymatic reaction depends on temperature, reaction medium, concentration of reactants, amount of enzyme and other factors.
1. Let us consider the dependence of the reaction rate on the amount of enzyme. Provided there is an excess of substrate, the reaction rate is proportional to the amount of enzyme, but with an excess amount of enzyme, the increase in the reaction rate will decrease, since there will no longer be enough substrate.
2. The rate of chemical reactions is proportional to the concentration of reacting substances (law of mass action). This law also applies to enzymatic reactions, but with certain restrictions. At constant
In large quantities of the enzyme, the reaction rate is indeed proportional to the concentration of the substrate, but only in the region of low concentrations. At high substrate concentrations, the enzyme becomes saturated with the substrate, that is, a moment comes when all enzyme molecules are already involved in the catalytic process and there will be no increase in the reaction rate. The reaction rate reaches the maximum level (Vmax) and then no longer depends on the substrate concentration. The dependence of the reaction rate on the substrate concentration should be determined in that part of the curve that is below Vmax. Technically, it is easier to determine not the maximum speed, but ½ Vmax. This parameter is the main characteristic of the enzymatic reaction and makes it possible to determine the Michaelis constant (Km).
Km (Michaelis constant) is the concentration of the substrate at which the rate of the enzymatic reaction is half the maximum. From this we derive the Michaelis–Menten equation for the rate of an enzymatic reaction.
The rate of enzymatic reactions depends on the concentration of the enzyme, substrate, temperature, pH, and the presence of activators and inhibitors.
Under conditions of excess substrate, the reaction rate directly proportional enzyme concentration (Fig. 3.2).
Rice. 3.2. Dependence of reaction rate on enzyme concentration.
Dependence of reaction speed on substrate concentration presented in Figure 3.3.
Rice. 3.3. Dependence of reaction rate on substrate concentration.
There are 3 sections on the graph. At low substrate concentration (section A) the reaction rate is directly proportional to the substrate concentration and obeys first order kinetics. Location on b(mixed order reaction) this dependence is violated. Location on c the reaction rate is maximum and does not depend on the substrate concentration.
An enzymatic reaction is characterized by the formation of an enzyme-substrate complex, which breaks down to form the free enzyme and reaction product.
In this equation, k 1 is the rate constant for the formation of the enzyme-substrate complex, k 2 is the dissociation constant of the enzyme-substrate complex to form a free enzyme and substrate, and k 3 is the rate constant for the dissociation of the enzyme-substrate complex to the free enzyme and reaction product.
Michaelis and Menten proposed an equation that describes the dependence of the reaction rate on the substrate concentration.
v is the reaction rate at a given substrate concentration; Ks – dissociation constant of the enzyme-substrate complex; Vmax – maximum reaction speed.
Ks=k -2 /k 1 i.e. the ratio of the reverse reaction constant to the forward reaction constant.
However, this equation describes only the section A on the graph and does not take into account the influence of reaction products on the rate of the enzymatic process.
Haldane and Briggs replaced the dissociation constant in the equation with the Michaelis constant (Km).
Michaelis constant numerically equal to substrate concentration, at which the reaction rate is half the maximum. The Michaelis constant characterizes the affinity of the enzyme and substrate. A high affinity of an enzyme for a substrate is characterized by a low Km value and vice versa.
Using the graph proposed by Michaelis and Menten is inconvenient. For a more convenient graphical representation, G. Lineweaver and D. Burke transformed the Haldane and Briggs equation using the method of double reciprocals, based on the principle that if there is equality between two quantities, then the reciprocals will also be equal.
Graphic representation of the dependence of the reaction rate on pH has a bell shape. The pH value at which the enzyme exhibits maximum activity is called optimum pH(Fig. 5.4 A) . For most enzymes, the optimum pH is 6-8. The exception is pepsin, whose optimum is 2.0. When the pH changes in one direction or another from the optimum, the reaction rate decreases due to the ionization of the functional groups of the enzyme and substrate, which disrupts the formation of the enzyme-substrate complex.
Rice. 3.4. Dependence of the reaction rate on pH (A) and temperature (B).
The rate of a chemical reaction increases by 2 times with increasing temperature by 10°C. However, due to the protein nature of the enzyme, with a further increase in temperature, denaturation of the enzyme occurs. The temperature at which the reaction rate is maximum is called temperature optimum(Fig. 3.4. B) . For most enzymes, the optimum temperature is 37-40°C. The exception is muscle myokinase, which can withstand heating up to 100°C.
Enzyme activators– these are substances 1) forming the active center of the enzyme (Co 2+, Mg 2+, Zn 2+, Fe 2+, Ca 2+); 2) facilitating the formation of the enzyme-substrate complex (Mg 2+); 3) reducing SH groups (glutathione, cysteine, mercaptoethanol); 4) stabilizing the native structure of the protein-enzyme. Enzymatic reactions are usually activated by cations (in the periodic table from 19 to 30). Anions are less active, although chlorine ions and the anions of some other halogens can activate pepsin, amylase, and adenylate cyclase. Proteins can be activators: apoprotein A-I (LCAT), apoprotein C-II (LPL).
Mechanism of action of activators:
1) participate in the formation of the active center of enzymes;
2) facilitate the binding of the substrate and the enzyme;
3) participate in the formation of the native structure of the enzyme.
Inhibitors– substances that cause partial or complete inhibition of reactions catalyzed by enzymes.
Inhibitors are classified into nonspecific And specific. The action of nonspecific inhibitors is not related to the mechanism of action of enzymes. These inhibitors cause denaturation of the enzyme protein (heat, acids, alkalis, salts of heavy metals, etc.).
Specific inhibitors affect the mechanism of action of enzymes. Specific inhibitors are divided into 2 groups: reversible and irreversible. Irreversible inhibitors cause a permanent, irreversible change or modification of the functional groups of the enzyme through tight or covalent binding. This group includes: 1) metal inhibitors enzymes (HCN, RCN, HF, CO, etc.). These compounds bind to metals with variable valence (Cu or Fe), as a result of which the process of electron transfer along the respiratory chain of enzymes is disrupted. Therefore, these inhibitors are called respiratory poisons. 2) inhibitors of enzymes containing SH groups(monoidoacetate, diiodoacetate, iodoacetamide, arsenic and mercury compounds). 3) inhibitors of enzymes containing an OH group in the active center (organophosphorus compounds, insecticides). These inhibitors inhibit, first of all, the activity of cholinesterase, an enzyme that plays a primary role in the activity of the nervous system.
Reversible inhibition can be quantified using the Michaelis-Menten equation. Reversible inhibitors are divided into competitive and non-competitive.
Competitive inhibitors- These are substances similar in structure to the substrate. The inhibitor binds to the active site of the enzyme and prevents the formation of the enzyme-substrate complex.
A classic example of competitive inhibition is the inhibition of succinate dehydrogenase by malonic acid. Succinate dehydrogenase catalyzes the oxidation of succinic acid (succinate) by dehydrogenation to fumaric acid.
If malonic acid (an inhibitor) is added to the medium, then, as a result of its structural similarity to the true substrate succinate, it will react with the active site to form an enzyme-inhibitor complex, but the reaction will not occur.
The effect of the inhibitor is eliminated by increasing substrate concentration. With competitive inhibition, the kinetics of enzymatic reactions changes: Km increases, V max remains constant(Fig. 3.5).
Rice. 3.5. Effect of competitive inhibitors on the rate of enzymatic reaction
The competitive inhibition method has found application in medical practice as antimetabolites.
For example, sulfonamide drugs are used to treat some infectious diseases caused by bacteria. These drugs are structurally similar to para-aminobenzoic acid, which the bacterial cell uses to synthesize folic acid, which is necessary for the life of bacteria. Due to this structural similarity, sulfonamide blocks the action of the enzyme by displacing para-aminobenzoic acid from the complex with the enzyme that synthesizes folic acid.
Non-competitive inhibitors – substances that are not structurally similar to substrates. Noncompetitive inhibitors bind not to the active site, but to another location in the enzyme molecule, for example, in the allosteric center. This changes the conformation of the active center in such a way that the interaction of the substrate with it is disrupted.
For non-competitive inhibition: V max decreases, but K m does not change(Fig. 3.6).