Almost all biochemical reactions are enzymatic. Enzymes(biocatalysts) are protein substances activated by metal cations. About 2000 different enzymes are known, and about 150 of them have been isolated, some of which are used as medicines. Trypsin and chymotrypsin are used to treat bronchitis and pneumonia; pepsin - for the treatment of gastritis; plasmin - for the treatment of heart attack; Pancreatin – for the treatment of the pancreas. Enzymes differ from conventional catalysts: (a) by higher catalytic activity; (b) high specificity, i.e. selectivity of action.
The mechanism of a single-substrate enzymatic reaction can be represented by the following diagram:
where E is an enzyme,
S - substrate,
ES - enzyme-substrate complex,
P is the reaction product.
The characteristic of the first stage of the enzymatic reaction is Michaelis constant (K M). K M is the reciprocal of the equilibrium constant:
The Michaelis constant (K M) characterizes the stability of the enzyme-substrate complex (ES). The lower the Michaelis constant (K M), the more stable the complex.
The rate of an enzymatic reaction is equal to the rate of its rate-limiting stage:
where k 2 is the rate constant, called number of revolutions or molecular activity of the enzyme.
molecular enzyme activity(k 2) is equal to the number of substrate molecules undergoing transformations under the influence of one enzyme molecule in 1 minute at 25 0 C. This constant takes values in the range: 1·10 4< k 2 < 6·10 6 мин‾ 1 .
For urease, which accelerates the hydrolysis of urea, k 2 = 1.85∙10 6 min‾ 1 ; for adenosine triphosphatase, which accelerates ATP hydrolysis, k 2 = 6.24∙10 6 min‾ 1 ; for catalase, which accelerates the decomposition of H 2 O 2, k 2 = 5∙10 6 min‾ 1.
However, the kinetic equation of the enzymatic reaction in the form in which it is given above is practically impossible to use due to the impossibility of experimentally determining the concentration of the enzyme-substrate complex (). Expressed in terms of other quantities that are easily determined experimentally, we obtain the kinetic equation of enzymatic reactions, called by the Michaelis-Menten equation (1913):
,
where the product k 2 [E] total is a constant value, which is denoted (maximum speed).
Respectively: 
Let us consider special cases of the Michaelis-Menten equation.
1) At low substrate concentration K M >> [S], therefore
which corresponds to the kinetic equation of the first order reaction.
2) At high substrate concentration K m<< [S], поэтому
which corresponds to the kinetic equation of the zero-order reaction.
Thus, at a low substrate concentration, the rate of the enzymatic reaction increases with increasing substrate content in the system, and at a high substrate concentration, the kinetic curve reaches a plateau (the reaction rate does not depend on the substrate concentration) (Fig. 30).
Figure 30. - Kinetic curve of an enzymatic reaction
If [S] = K M, then
which allows you to graphically determine the Michaelis constant K m (Fig. 31).
Figure 31. - Graphical definition of the Michaelis constant
The activity of enzymes is influenced by: (a) temperature, (b) acidity of the medium, (c) the presence of inhibitors. The effect of temperature on the rate of an enzymatic reaction is discussed in Chapter 9.3.
The influence of the acidity of the medium on the rate of the enzymatic reaction is presented in Figure 32. The maximum enzyme activity corresponds to the optimal pH value (pH opt).
Figure 32. - Effect of solution acidity on enzyme activity
For most enzymes, optimal pH values coincide with physiological values (7.3 - 7.4). However, there are enzymes whose normal functioning requires a strongly acidic (pepsin - 1.5 - 2.5) or sufficiently alkaline environment (arginase - 9.5 - 9.9).
Enzyme inhibitors- these are substances that occupy part of the active centers of enzyme molecules, as a result of which the rate of the enzymatic reaction decreases. Heavy metal cations, organic acids and other compounds act as inhibitors.
Lecture 11
The structure of the atom
There are two definitions of the term "atom". Atom is the smallest particle of a chemical element that retains its chemical properties.
Atom is an electrically neutral microsystem consisting of a positively charged core and a negatively charged electron shell.
The doctrine of the atom has come a long way of development. The main stages in the development of atomistics include:
1) natural philosophical stage - the period of formation of the concept of the atomic structure of matter, not confirmed by experiment (5th century BC - 16th century AD);
2) the stage of formation of the hypothesis about the atom as the smallest particle of a chemical element (XVIII-XIX centuries);
3) the stage of creating physical models that reflect the complexity of the structure of the atom and make it possible to describe its properties (beginning of the 20th century)
4) the modern stage of atomistics is called quantum mechanical. Quantum mechanics is a branch of physics that studies the movement of elementary particles.
PLAN
11.1. Structure of the nucleus. Isotopes.
11.2. Quantum mechanical model of the electron shell of an atom.
11.3. Physicochemical characteristics of atoms.
Structure of the nucleus. Isotopes
Atomic nucleus is a positively charged particle consisting of protons, neutrons and some other elementary particles.
It is generally accepted that the main elementary particles of the nucleus are protons and neutrons. Proton (p) – is an elementary particle whose relative atomic mass is 1 amu and its relative charge is + 1. Neutron (n) – This is an elementary particle that has no electric charge and whose mass is equal to the mass of a proton.
99.95% of the mass of an atom is concentrated in the nucleus. Between elementary particles there are special nuclear forces of extension, which significantly exceed the forces of electrostatic repulsion.
The fundamental characteristic of an atom is charge his kernels, equal to the number of protons and coinciding with the atomic number of the element in the periodic table of chemical elements. A set (type) of atoms with the same nuclear charge is called chemical element. Elements with numbers from 1 to 92 are found in nature.
Isotopes- these are atoms of the same chemical element containing the same number of protons and different numbers of neutrons in the nucleus.
where mass number (A) is the mass of the nucleus, z is the charge of the nucleus.
Each chemical element is a mixture of isotopes. As a rule, the name of isotopes coincides with the name of a chemical element. However, special names have been introduced for hydrogen isotopes. The chemical element hydrogen is represented by three isotopes:
Number p Number n
Protium N 1 0
Deuterium D 1 1
Tritium T 1 2
Isotopes of a chemical element can be both stable and radioactive. Radioactive isotopes contain nuclei that spontaneously break down, releasing particles and energy. The stability of a nucleus is determined by its neutron-proton ratio.
Once in the body, radionuclides disrupt the most important biochemical processes, reduce immunity, and doom the body to illness. The body protects itself from the effects of radiation by selectively absorbing elements from the environment. Stable isotopes take precedence over radioactive isotopes. In other words, stable isotopes block the accumulation of radioactive isotopes in living organisms (Table 8).
S. Shannon’s book “Nutrition in the Atomic Age” provides the following data. If a blocking dose of ~100 mg of stable isotope iodine is taken no later than 2 hours after I-131 enters the body, radioiodine uptake in the thyroid gland will be reduced by 90%.
Radioisotopes are used in medicine
for the diagnosis of certain diseases,
· for the treatment of all forms of cancer,
· for pathophysiological studies.
Table 8 - Blocking effect of stable isotopes
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, since at a low substrate concentration, not all enzyme molecules participate in the reaction (Fig. 6.5, A), which means the result will be far from the maximum possible. The highest power of the catalyzed reaction, other things being equal, is achieved if each enzyme molecule is involved in the transformation, i.e. at a high concentration of the enzyme-substrate complex (Fig. 6.5, V). If the concentration of the substrate 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 the 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 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 coefficient of proportionality is the reaction constant for the release of the enzyme and the 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 the maximum saturation of the enzyme with the substrate is reached, the rate of the enzymatic reaction becomes maximum. Therefore, the factor limiting the reaction rate is the formation of an enzyme-substrate complex.
Practice has shown that the concentrations of substrates, 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 the enzymatic reaction not in direct coordinates, but in reverse. They proceeded from the assumption that for equal quantities, their reciprocals 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.
This branch of enzymology studies the influence of various factors on the rate of an enzymatic reaction. Considering the general equation for enzymatic catalysis of the reversible reaction of converting one substrate into one product (1),
The main factors influencing the rate of an enzymatic reaction should be named: substrate concentration [S], enzyme concentration [E], and reaction product concentration [P].
The interaction of some enzymes with their substrate can be described by a hyperbolic curve of the dependence of the rate of enzymatic reaction V on the concentration of the substrate [S] (Fig. 19):
Fig. 19. Dependence of the rate of enzymatic reaction on the concentration of the substrate.
On this curve, three sections can be distinguished, which can be explained by the provisions of the mechanism of interaction of the enzyme with the substrate: OA - a section of directly proportional dependence of V on [S], the active centers of the enzyme are gradually filled with substrate molecules with the formation of an unstable complex ES; section AB - curvilinear dependence of V on [S], complete saturation of the active centers of the enzyme with substrate molecules has not yet been achieved. The ES complex is unstable before reaching the transition state; the probability of reverse dissociation to E and S is still high; section BC - the dependence is described by a zero-order equation, the section is parallel to the [S] axis, complete saturation of active enzymes with substrate molecules has been achieved, V=V max.
The characteristic shape of the curve is described mathematically by the Briggs-Haldane equation:
V=V max ● [S]/ Km + [S] (2),
where Km is the Michaelis-Menten constant, numerically equal to the substrate concentration at which the rate of the enzymatic reaction is equal to half V max .
The lower the K m of the enzyme, the higher the affinity of the enzyme for the substrate, the faster the transition state for the substrate is reached, and it turns into a reaction product. The search for Km values for each of the enzyme's substrates with group specificity is important in determining the biological role of this enzyme in the cell.
For most enzymes, it is impossible to construct a hyperbolic curve (Fig. 19). In this case, the double reciprocal method (Lineweaver-Burk) is used, i.e. a graphical dependence of 1/[V] on 1/[S] is plotted (Fig. 20). The method of constructing such curves in an experiment is very convenient when studying the effect of various types of inhibitors on the activity of enzymes (see the text below).
Fig.20. Graph of 1/[V] versus 1/[S] (Lineweaver-Burk method),
where y is the cut-off section - , and x is the cut-off section - 
, tangent of angle α - .
Dependence of the rate of enzymatic reaction V on the enzyme concentration [E].
This graphical dependence (Fig. 21) is considered at optimal temperature and pH of the environment, at substrate concentrations significantly higher than the saturation concentration of the active centers of the enzyme.
Rice. 21. The influence of enzyme concentration on the rate of enzymatic reaction.
Dependence of the rate of an enzymatic reaction on the concentration of a cofactor or coenzyme. For complex enzymes, it should be taken into account that a deficiency of coenzyme forms of vitamins in case of hypovitaminosis and a violation of the intake of metal ions into the body necessarily lead to a decrease in the concentration of the corresponding enzymes necessary for the course of metabolic processes. Therefore, it should be concluded that the activity of the enzyme is directly dependent on the concentration of the cofactor or coenzyme.
The influence of product concentration on the rate of enzymatic reaction. For reversible reactions occurring in the human body, it must be taken into account that the products of the direct reaction can be used by the enzyme as substrates for the reverse reaction. Therefore, the direction of flow and the moment of reaching Vmax depend on the ratio of the concentrations of the initial substrates and reaction products. For example, the activity of alanine aminotransferase, which catalyzes the transformation:
Alanine + Alpha-ketoglutarate ↔ Pyruvate + Glutamate
depends in the cell on the concentration ratio:
[alanine + alpha-ketoglutarate] / [pyruvate + glutamate].
MECHANISM OF ENZYME ACTION. THEORIES OF ENZYME CATALYSIS
Enzymes, like non-protein catalysts, increase the rate of a chemical reaction due to their ability to reduce the activation energy of this reaction. The activation energy of an enzymatic reaction is calculated as the difference between the energy value in the system of the ongoing reaction that has reached the transition state and the energy determined at the beginning of the reaction (see graphical dependence in Fig. 22).
Rice. 22. Graphical dependence of the energy state of a chemical reaction without an enzyme (1) and in the presence of an enzyme (2) on the reaction time.
The work of V. Henry and, in particular, L. Michaelis, M. Menten on studying the mechanism of monosubstrate reversible enzymatic reactions made it possible to postulate that enzyme E first reversibly and relatively quickly combines with its substrate S to form an enzyme-substrate complex (ES):
E+S<=>ES (1)
The formation of ES occurs due to hydrogen bonds, electrostatic, hydrophobic interactions, in some cases covalent, coordination bonds between the side radicals of amino acid residues of the active center and the functional groups of the substrate. In complex enzymes, the function of contact with the substrate can also be performed by the non-protein part of the structure.
The enzyme-substrate complex then disintegrates in a second, slower, reversible reaction to produce reaction product P and free enzyme E:
ES<=>EP<=>E+P (2)
Currently, thanks to the work of the above-mentioned scientists, as well as Keilin D., Chance B., Koshland D. (the theory of “induced correspondence”), there are theoretical provisions about four main points in the mechanism of action of an enzyme on a substrate, which determine the ability of enzymes to accelerate chemical reactions:
1. Orientation and approach . The enzyme is able to bind a substrate molecule in such a way that the bond attacked by the enzyme is not only located in close proximity to the catalytic group, but also correctly oriented with respect to it. The likelihood that the ES complex will reach the transition state through orientation and proximity is greatly increased.
2. Stress and Strain : induced correspondence. The attachment of a substrate can cause conformational changes in the enzyme molecule, which lead to tension in the structure of the active center, and also somewhat deform the bound substrate, thereby facilitating the achievement of a transition state by the ES complex. A so-called induced correspondence arises between molecules E and S.
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 to P in 1 sec. [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 only catalyzes 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, DG alcohol 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 different proteins.
V. Relative S specificity. The enzyme catalyzes, turning into S-in, belonging to various groups of chemical compounds. For example, the cytochrome-450 enzyme catalyzes hydroxylation reactions of up to 7000 different S-v. 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 (“key”), then it does not correspond to ACF (“lock”), and the reaction becomes impossible. Despite the logic of such an explanation, Fisher's hypothesis does not explain what then absolute and relative group specificity are based on. For example, cytochrome-450 combines with so many S-in, 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 diverse. 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.
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.
