Properties of enzymes
1. Dependence of reaction rate on temperature
The dependence of enzyme activity (reaction rate) on temperature is described bell curve with maximum speed at values optimal temperature for a given enzyme. An increase in the reaction rate as the optimal temperature is approached is explained by an increase in the kinetic energy of the reacting molecules.
Dependence of reaction rate on temperature
The law about increasing the reaction rate by 2-4 times with an increase in temperature by 10°C is also valid for enzymatic reactions, but only within the range of 55-60°C, i.e. up to temperatures denaturation proteins. As the temperature drops, enzyme activity decreases, but does not disappear completely.
As an exception, there are enzymes of some microorganisms that exist in the water of hot springs and geysers; their optimum temperature approaches the boiling point of water. An example of weak activity at low temperatures is the hibernation of some animals (gophers, hedgehogs), whose body temperature drops to 3-5°C. This property of enzymes is also used in surgical practice during operations on the chest cavity, when the patient is cooled to 22°C.
Enzymes can be very sensitive to temperature changes:
- Siamese cats have a black muzzle, tips of ears, tail, and paws. In these areas the temperature is only 0.5°C lower than in the central regions of the body. But this allows the enzyme that forms the pigment in the hair follicles to work; at the slightest increase in temperature, the enzyme is inactivated,
- the opposite case - when the ambient temperature drops in the white hare, the pigment-forming enzyme is inactivated and the hare gets a white coat,
- antiviral protein interferon begins to be synthesized in cells only when body temperature reaches 38°C,
There are also unique situations:
- For most people, an increase in body temperature of 5°C (up to 42°C) is incompatible with life due to an imbalance in the rate of enzymatic reactions. At the same time, some athletes were found to have a body temperature of about 40°C during marathon running, with the maximum body temperature recorded being 44°C.
2. Dependence of reaction rate on pH
The dependency is also described bell curve with maximum speed at optimal for a given enzyme pH value.
This feature of enzymes is essential for the body in its adaptation to changing external and internal conditions. Shifts in pH outside and inside the cell play a role in the pathogenesis of diseases, changing the activity of enzymes in various metabolic pathways.
For each enzyme, there is a certain narrow pH range of the environment, which is optimal for the manifestation of its highest activity. For example, the optimal pH values for pepsin are 1.5-2.5, trypsin 8.0-8.5, salivary amylase 7.2, arginase 9.7, acid phosphatase 4.5-5.0, succinate dehydrogenase 9.0.
Dependence of reaction rate on pH value
The dependence of activity on the acidity of the medium is explained by the presence of amino acids in the structure of the enzyme, the charge of which changes with a shift in pH (glutamate, aspartate, lysine, arginine, histidine). A change in the charge of the radicals of these amino acids leads to a change in their ionic interaction during the formation of the tertiary structure of the protein, a change in its charge and the appearance of a different configuration of the active center and, therefore, the substrate binds or does not bind to the active center.
Changes in enzyme activity with a pH shift may also cause adaptive functions. For example, in the liver, gluconeogenesis enzymes require a lower pH than glycolytic enzymes, which is successfully combined with the acidification of body fluids during fasting or physical activity.
For most people, shifts in blood pH beyond 6.8-7.8 (with the norm being 7.35-7.45) are incompatible with life due to an imbalance in the rate of enzymatic reactions. At the same time, some marathon runners showed a decrease in blood pH at the end of the distance to 6.8-7.0. And yet they remained functional!
3. Dependence on the amount of enzyme
As the number of enzyme molecules increases, the reaction rate increases continuously and is directly proportional to the amount of enzyme, because more enzyme molecules produce more product molecules.
ENZYMATIVE REACTION KINETICS
studies the patterns of the passage of enzymatic reactions over time, as well as their mechanism; chapter chemical kinetics.
Catalytic the cycle of conversion of substance S (substrate) into product P under the action of enzyme E proceeds with the formation of intermediates. conn. X i:
Where ki- rate constants of individual elementary stages,
formation of the enzyme-substrate complex X 1 (ES, Michaelis complex).
At a given temperature, the speed of the reaction depends on the concentrations of the enzyme, substrate and composition of the medium. There are stationary, pre-stationary and relaxation kinetics of enzymatic reactions.
Stationary kinetics. In a stationary state via intermediate connections. (dX i/dt= 0, i = 1, ..., n) and with an excess of substrate, where [S] 0 and [E] 0 are the initial concentrations, respectively. substrate and enzyme, the kinetics of the process is characterized by a constant, time-invariant level of concentrations. conn., and the expression for the process speed v 0, called initial stationary speed, has the form (Michaelis-Menten equation):
(1)
where the values of k cat and K m -> functions of rate constants of elementary stages and are given by the equations:
The value of k cat
called K m -> effective catalytic process rate constant, parameter
Michaelis constant. k cat value determined by quantities
max. slow stages of catalytic districts and sometimes called number of revolutions of the enzyme (enzyme system); k cat
characterizes the number of catalytic cycles performed by the enzyme system per unit time. Naib. common, having the value k cat. for specific substrates in the range of 10 2 -10 3 s -1. Typical values of the Michaelis constant lie in the range 10 -3 - 10 -4 M.
(2)
At high concentrations of the substrate, when, i.e., the rate of circulation does not depend on the concentration of the substrate and reaches a constant value, called. Max. speed. Graphically, the Michaelis-Menten equation is a hyperbole. It can be linearized using the method of double reciprocals (Linewere-Burk method), i.e., constructing the dependence 1/v on 1/[S] 0, or other methods. The linear form of equation (1) has the form: It allows you to determine graphically the values K m
and v max (Fig. 1).
Rice. 1. Graph of linear transformation of the Michaelis - Menten equation in double reciprocals (according to Lineweaver - Burke). Magnitude K m > It allows you to determine graphically the values is numerically equal to the concentration of the substrate, at which the rate of circulation is equal, therefore
often serves as a measure of the affinity of the substrate and the enzyme, but this is only valid if Magnitude Quantities vary depending on pH values. This is due to the ability of the enzyme molecule groups involved in catalysis to change their ionization state and, thereby, their catalytic activity. efficiency. In the simplest case, a change in pH results in the protonation or deprotonation of at least two ionizable groups of the enzyme involved in catalysis. If, in this case, only one form of the enzyme-substrate complex (for example, ESH) out of three possible forms (ES, ESH and ESH 2) is capable of being converted into a product of the solution, then the dependence of the rate on pH is described by the formula:
Where f = 1 + / And f" = 1 +
+K" b />-T. called pH-functions of Michaelis, and K a, K b Quantities K" a, K" b -> ionization constants of groups a and bresp. free enzyme and enzyme-substrate complex. In lg coordinates - pH this dependence is presented in Fig. 2, and the tangents of the angles of inclination of the tangents to the ascending, independent of pH, and descending branches of the curve should be equal to +1, 0 and -1, respectively. From such a graph you can determine the values pK a groups involved in catalysis.
Rice. 2. Dependence of catalytic constants from pH to logarithmic. coordinates
The speed of the enzymatic reaction does not always obey equation (1). One of the most common cases is the participation of allosteric in the reaction. enzymes (see Enzyme regulators), for which the dependence of the degree of saturation of the enzyme on [S] 0 is non-hyperbolic. character (Fig. 3). This phenomenon is due to the cooperativity of substrate binding, i.e., when the binding of a substrate on one of the sites of the enzyme macromolecule increases (positive cooperativity) or decreases (negative cooperativity) the affinity for the substrate of another site.
Rice. H Dependence of the degree of saturation of the enzyme with the substrate on the concentration of the substrate with positive (I) and negative (II) cooperativity, as well as in its absence (III).
Pre-steady-state kinetics. With rapid mixing of enzyme and substrate solutions in the time interval of 10 -6 -10 -1 s, one can observe transient processes preceding the formation of a stable stationary state. In this pre-stationary mode, when using a large excess of substrate, the differential system. The equation describing the kinetics of the processes is linear. The solution of this type of linear differential system. The equation is given by the sum of the exponential terms. So, for kinetic scheme presented above, the kinetics of product accumulation has the form:
where A i ->, b, and n -> functions of elementary rate constants; -roots of the corresponding characteristic. level.
The reciprocal quantity is called characteristic process time:
For a river flowing with the participation of nintervals. connection, you can get ncharacteristics. times
The study of the kinetics of the enzymatic reaction in a pre-stationary mode allows us to get an idea of the detailed mechanism of catalytic reactions. cycle and determine the rate constants of the elementary stages of the process.
Experimentally, the kinetics of the enzymatic reaction in a prestationary mode is studied using the stopped jet method (see. Jet kinetic methods), allowing mixing of the components of the solution within 1 ms.
Relaxation kinetics. With a rapid disturbing effect on the system (change in temperature, pressure, electric field), the time required for the system to achieve a new equilibrium or stationary state depends on the speed of the processes that determine the catalytic reaction. enzymatic cycle.
The system of equations describing the kinetics of the process is linear if the displacement from the equilibrium position is small. The solution of the system leads to the dependences of the concentrations of the components, dec. stages of the process in the form of a sum of exponential terms, the exponents of which have the character of relaxation times. The result of the study is a spectrum of relaxation times corresponding to the number of intervals. connections participating in the process. The relaxation times depend on the rate constants of the elementary stages of the processes.
Relaxation techniques kinetics make it possible to determine the rate constants of individual elementary stages of transformation of intermediates. Methods for studying relaxation kinetics vary. resolution: ultrasound absorption - 10 -6 -10 -10
s, temperature jump - 1O -4 -10 -6 s, electrical method. impulse - 10 -4 -10 -6 s, pressure jump - 10 -2 s. When studying the kinetics of enzymatic reactions, the method of temperature jump found application.
Macrokinetics of enzymatic processes. Development of methods for producing heterogeneous catalysts by immobilizing enzymes on decomp. media (see Immobilized enzymes) necessitated the analysis of the kinetics of processes taking into account the mass transfer of the substrate. The kinetics of reactions have been studied theoretically and experimentally, taking into account the effects of the diffusion layer and for systems with intradiffusion difficulties during the distribution of the enzyme within the carrier.
Under conditions where the kinetics of the process is affected by the diffusion transfer of the substrate, catalytic. system efficiency decreases. The efficiency factor is equal to the ratio of the product flow density under conditions of enzymatic flow with a diffusively reduced substrate concentration to the flow that could be realized in the absence of diffusion restrictions. In the purely diffusion region, when the rate of the process is determined by the mass transfer of the substrate, the efficiency factor for systems with external diffusion inhibition is inversely proportional to the diffusion modulus:
Where
thickness of the diffusion layer, D - coefficient. substrate diffusion.
For systems with intradiffusion inhibition in first-order regions
where Ф T- dimensionless modulus (Thiele modulus).
When analyzing the kinetic patterns in enzymatic reactors are widely theoretical. and experiment. “ideal” reactor models have been developed: flow reactor (flow reactor with ideal mixing), flow reactor with ideal displacement, and membrane reactor.
Kinetics of multienzyme processes. In the body (cell), enzymes do not act in isolation, but catalyze chains of transformation of molecules. R-tions in multienzyme systems with kinetic. points of view can be considered as consistent. processes, specific a feature of which is the enzymes of each of the stages:
Where ,
resp. max, process speed and Michaelis constant i th stage of the district, respectively.
An important feature of the process is the possibility of forming a stable stationary state. The condition for its occurrence can be inequality > v 0 , where v 0 is the speed of the limiting stage, characterized by the smallest rate constant and thereby determining the speed of everything that follows. process. In the steady state, the concentrations of metabolites after the limiting stage are less than the Michaelis constant of the corresponding enzyme.
Specific the group of multienzyme systems consists of systems that carry out oxidation-reduction. r-tions with the participation of protein electron carriers. Carriers form specific structures, complexes with a deterministic sequence of electron transfer. Kinetic. the description of this kind of systems considers the state of circuits with decomposition as an independent variable. degree of electron population.
Application. F.r. K. is widely used in research practice to study the mechanisms of action of enzymes and enzyme systems. A practically significant area of enzyme science is engineering enzymology, operates with the concepts of F. r. for optimization of biotechnol. processes.
Lit.: Poltorak O. M., Chukhrai E. S., Physico-chemical foundations of enzymatic catalysis, M., 1971; Berezin I.V., Martinek K., Fundamentals of physical chemistry of enzymatic catalysis, M., 1977; Varfolomeev S. D., Zaitsev S. V., Kinetic methods in biochemical research, M.. 1982. S. D. Varfolomeev.
Chemical encyclopedia. - M.: Soviet Encyclopedia. Ed. I. L. Knunyants. 1988 .
See what "ENZYMATIVE REACTION KINETICS" is in other dictionaries:
Catalytic radio cyclic a process consisting of a number of elementary movements, the speeds of which are described by the law of mass action. This law has a simple form for ideal gas mixtures, ideal liquids and ideal surface layers.... ... Chemical encyclopedia
Kinetics of chemical reactions, the study of chemical processes, the laws of their occurrence in time, speeds and mechanisms. The most important areas of modern chemistry and chemical science are associated with studies of the kinetics of chemical reactions... ... Great Soviet Encyclopedia
CHEMICAL KINETICS- (from the Greek kinesis movement), a department of theoretical chemistry devoted to the study of the laws of chemistry. reactions. Several types of chemicals can be identified. interactions and, first of all, to distinguish reactions occurring in a homogeneous (homogeneous) medium from reactions... ... Great Medical Encyclopedia
- (biocatalysis), acceleration of biochemical. rations with the participation of protein macromolecules called enzymes. F. k. is a type of catalysis, although the term fermentation (fermentation) has been known since ancient times, when there was no concept of chemistry. catalysis. First… … Chemical encyclopedia
- (from the Latin re prefix meaning reverse action, and actio action), the transformation of some into (initial compounds) into others (products of the diet) with the immutability of atomic nuclei (as opposed to nuclear reactions). Initial compounds in R. x. sometimes called... ... Chemical encyclopedia
- (from Latin fermentum starter) (enzymes), proteins that act as catalysts in living organisms. Basic functions of F. to accelerate the conversion of substances entering the body and formed during metabolism (to renew cellular structures, to ensure its ... Chemical encyclopedia
- (from the Greek pharmakon medicine and kinetikos setting in motion), studies kinetic. patterns of processes occurring with lek. Wed vom in the body. Basic pharmacokinetic processes: absorption, distribution, metabolism and excretion (removal).... ... Chemical encyclopedia
Lecture No. 6
Basics of enzymatic catalysis.
A brief history of the study of the kinetics of enzymatic reactions. Dependence of the rate of enzymatic reaction on temperature, pH, enzyme concentration and substrate concentration. Derivation of the Michaelis-Menten equation. Enzyme activity. The catalytic constant is the number of revolutions of the enzyme. Maximum speed of enzymatic reaction (V max). Dissociation constant of the enzyme-substrate complex (K s). Michaelis-Menten constant (K m).
A brief history of the study of the kinetics of enzymatic reactions.
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.
The general principles of chemical reaction kinetics also apply to enzymatic reactions.
The earliest attempt to mathematically describe enzymatic reactions was made by Duclos in 1898. Brown (1902) and, independently of him, Henri (1903) first hypothesized the formation of an enzyme-substrate complex during the reaction. This assumption was based on three experimental facts:
1. papain forms an insoluble compound with fibrin (Wurtz, 1880);
2. sucrose protects the invertase enzyme from thermal denaturation (O'Sullivan and Thompson, 1890);
3. Fischer in 1898-1899 showed that enzymes are stereochemically specific catalysts.
MICHAELIS, Leonor
Leonor Michaelis is a German biochemist and organic chemist, the founder of the kinetics of enzymatic processes. The main works are devoted to the study of enzymatic reactions. In 1913, he introduced a constant (Michaelis constant) into the equation of the dependence of the rate of an enzymatic reaction on the concentration of the substrate in a steady state (Michaelis–Menten equation).
Dependence of the rate of enzymatic reaction on temperature, pH, enzyme concentration and substrate concentration.
Preliminary experiments to study the kinetics of enzymatic reactions showed that the rate of the reaction E + S E + P, contrary to theoretical expectations, does not depend on the concentration of the enzyme and substrate as in the case of a conventional second-order reaction.
At the same time, three main criteria that are also characteristic of inorganic catalysts are applicable to enzymes, namely:
1. They remain unchanged after the reaction, i.e. when released, they can react again with new substrate molecules (although side effects of environmental conditions on the activity of the enzyme cannot be ruled out).
2. Enzymes can act in negligibly small concentrations (for example, one molecule of the enzyme renin, contained in the mucous membrane of the calf’s stomach, curdles about 10 6 molecules of milk caseinogen in 10 minutes at a temperature of 37 ° C). The presence or absence of an enzyme or any other catalyst does not affect the value of the equilibrium constant and free energy (ΔG).
3. Catalysts only increase the rate at which the system approaches thermodynamic equilibrium, without shifting the equilibrium point.
The activity of the enzyme is influenced by all those factors that can cause a change in its structure, namely, such factors include:
3. Forces acting in fluid media (hydrodynamic forces, hydrostatic pressure and surface tension)
4. Chemical agents (alcohol, urea or hydrogen peroxide, etc.)
5. Irradiation (light, sound, ionizing radiation)
6. Various chemical compounds that bind to enzymes can change the rate of reactions catalyzed by enzymes.
Sometimes a decrease in catalytic activity, caused, for example, by a change in pH, is reversible. In such cases, a return to initial conditions is accompanied by restoration of enzyme activity. An irreversible change in enzyme activity is also possible.
Let's consider the influence of various factors on the rate of an enzymatic reaction.
Effect of temperature
One of the basic equations of chemical kinetics is the Arrhenius equation, which expresses the dependence of the reaction rate constant on temperature:
However, the temperature range of enzymatic reactions for which the Arrhenius equation is applicable is very narrow for most enzymes. What happens if we try to force the enzyme to catalyze the process even faster by raising the temperature above the physiologically acceptable one? At high temperatures, when the process of thermal inactivation of the enzyme begins to dominate, the dependence of the reaction rate on temperature, described by the Arrhenius equation, is violated - namely, after a certain temperature maximum, the reaction rate quickly drops to zero. On the other hand, when the temperature drops below 0°C, the reaction rate also drops to zero, i.e. the reaction stops completely. Thus, enzymatic reactions have a bell-shaped dependence of the reaction rate on temperature, which is explained by the superposition of two effects - an increase in the reaction rate with increasing temperature and acceleration of thermal denaturation of the protein molecule, leading to inactivation of the enzyme at high temperatures. Denaturation of most proteins begins in the temperature range from 45 to 50°C and is completed very quickly at 55°C. The cessation of enzymatic reactions at low temperatures is due to the fact that aqueous solutions freeze.
Thus, thermolability, or sensitivity to increased temperature, is one of the characteristic properties of enzymes that sharply distinguishes them from inorganic catalysts. At a temperature of 90°C, almost all enzymes lose their activity (the only exceptions are two enzymes - myokinase, which can withstand heating up to 100°C, and DNA polymerase from thermophilic bacteria at a maximum of 90°C). The optimal temperature for the action of most enzymes in warm-blooded animals is 40-45°C; under these conditions, the reaction rate is maximum due to an increase in the kinetic energy of the reacting molecules. At low temperatures (4°C and below), enzymes, as a rule, are not destroyed, although their activity drops almost to zero. This phenomenon is caused by a change in the structure of the active center of the enzyme due to a decrease in the density of water. In all cases, the time of exposure to the appropriate temperature is important. Currently, for pepsin, trypsin and a number of other enzymes, the existence of a direct relationship between the rate of enzyme inactivation and the degree of protein denaturation has been proven. Thus, the effect of temperature on the rate of an enzymatic reaction obeys the Arrhenius equation only in a relatively narrow temperature range of 4-45°C. Outside this range, the rates of enzymatic reactions sharply decrease, which is taken into account and used in food technologies and during the storage of food products and medicines containing enzymes. Give examples.
Influence of pH of the environment.
Enzymes, like all proteins, are made up of amino acids. Depending on the pH, the radicals of some amino acids, and therefore the protein as a whole, can acquire a charge. Charged groups are often included in the active sites of enzymes, since a number of mechanisms of enzymatic catalysis are based on acid or base catalysis. A necessary condition for the implementation of acid or base catalysis may be the presence of a certain charge on the ionizable groups of the active center. It follows that the catalytically active form of the enzyme exists only in one strictly defined state of ionization, and depending on the pH, a larger or smaller part of the total enzyme present in the mixture can be converted into it.
The dependence of enzyme activity on pH is bell-shaped with a rather narrow maximum. For different enzymes, the pH value at which the enzyme has maximum activity is different. In experiments, the study of pH dependences of enzymatic reactions is often used to study the number and properties of ionogenic groups.
When determining the dependence of enzyme activity on the concentration of hydrogen ions, the reaction is carried out at different pH values of the medium, usually at an optimal temperature and the presence of sufficiently high (saturating) concentrations of the substrate. The figure and table show the optimal pH values for a number of enzymes.
Rice. Dependence of enzyme activity on pH.
 |
From the data in table. 4.3 it can be seen that the pH optimum of enzyme action lies within physiological values. The exception is pepsin, whose pH optimum is 2.0 (at pH 6.0 it is not active and stable). This is explained, firstly, by the structural organization of the enzyme molecule and, secondly, by the fact that pepsin is a component of gastric juice containing free hydrochloric acid, which creates an optimal acidic environment for the action of this enzyme. On the other hand, the pH optimum of arginase lies in the highly alkaline zone (about 10.0); There is no such environment in liver cells; therefore, in vivo, arginase apparently does not function in its optimal pH zone.
According to modern concepts, the effect of changes in the pH of the environment on the enzyme molecule is to influence the state and degree of ionization of acidic and basic groups (in particular, the COOH group of dicarboxylic amino acids, the SH group of cysteine, the imidazole nitrogen of histidine, the NH 2 group of lysine, etc. ). With sharp shifts from the optimum pH of the environment, enzymes can undergo conformational changes, leading to loss of activity due to denaturation or a change in the charge of the enzyme molecule. At different pH values of the medium, the active center can be in a partially ionized or non-ionized form, which affects the tertiary structure of the protein and, accordingly, the formation of the active enzyme-substrate complex. In addition, the ionization state of substrates and cofactors is important.
Related information.
Enzyme reaction rate
The rate of an enzymatic reaction is measured by the amount of substrate converted per unit time or the amount of product formed. The speed is determined by the angle of inclination of the tangent to the curve at the initial stage of the reaction.
Rice. 2 Rate of enzymatic reaction.
The steeper the slope, the greater the speed. Over time, the reaction rate usually decreases, in large part as a result of decreasing substrate concentration.
Factors affecting enzymatic activity
F.'s action depends on a number of factors: temperature, environmental reaction (pH), enzyme concentration, substrate concentration, and the presence of specific activators and nonspecific or specific inhibitors.
Enzyme concentration
At high substrate concentrations and other factors remaining constant, the rate of the enzymatic reaction is proportional to the enzyme concentration.
Rice. 3 Dependence of the rate of enzymatic reaction on the concentration of the enzyme.
Catalysis always occurs under conditions where the enzyme concentration is much lower than the substrate concentration. Therefore, as the concentration of the enzyme increases, the rate of the enzymatic reaction also increases.
Temperature
The effect of temperature on the rate of an enzymatic reaction can be expressed through the temperature coefficient Q 10: Q 10 = (reaction rate at (x + 10) °C) / (reaction rate at x °C)
Between 0-40°C, the Q10 of an enzymatic reaction is 2. In other words, for every 10°C increase in temperature, the rate of the enzymatic reaction doubles.
Rice. 4 The influence of temperature on the activity of an enzyme such as salivary amylase.
As the temperature increases, the movement of molecules speeds up, and the molecules of the reacting substances are more likely to collide with each other. Consequently, the likelihood that a reaction between them will occur increases. The temperature that provides the greatest activity is called optimal. Beyond this level, the rate of the enzymatic reaction decreases, despite the increase in collision frequency. This occurs due to the destruction of the secondary and tertiary structures of the enzyme, in other words, due to the fact that the enzyme undergoes denaturation.
Rice. 5 The course of the enzymatic reaction at different temperatures.
When the temperature approaches or falls below freezing, the enzymes are inactivated, but denaturation does not occur. With increasing temperature, their catalytic activity is restored again.
Since proteins in the dry state are denatured much more slowly than hydrated proteins (in the form of a protein gel or solution), inactivation of phosphorus in the dry state occurs much more slowly than in the presence of moisture. Therefore, dry bacterial spores or dry seeds can withstand heating to much higher temperatures than the same spores or seeds in a moist state.
Substrate concentration
For a given enzyme concentration, the rate of the enzymatic reaction increases with increasing substrate concentration.
Rice. 6 Dependence of the rate of enzymatic reaction on the concentration of the substrate.
The theoretical maximum reaction rate Vmax is never reached, but there comes a point when a further increase in the substrate concentration no longer entails any noticeable change in the reaction rate. This should be explained by the fact that at high substrate concentrations, the active centers of phosphorus molecules are practically saturated at any given moment. Thus, no matter how much excess substrate is available, it can combine with enzyme only after the previously formed enzyme-substrate complex dissociates into product and free enzyme. Therefore, at high substrate concentrations, the rate of the enzymatic reaction is limited by both the concentration of the substrate and the time required for dissociation of the enzyme-substrate complex.
At a constant temperature, any phosphorus works most effectively within a narrow pH range. The optimal pH value is the one at which the reaction proceeds at maximum speed.
Rice. 7 Dependence of enzyme activity on pH.
At higher and lower pH, F.'s activity decreases. The pH shift changes the charge of ionized acidic and basic groups, on which the specific shape of the phosphorus molecules depends. As a result, the shape of the phosphorus molecules changes, and primarily the shape of its active center. If the pH changes too sharply, F. denatures. The pH optimum characteristic of a given phosphorus does not always coincide with the pH of its immediate intracellular environment. This suggests that the environment in which F. is located regulates his activity to some extent.
Kinetics of enzymatic reactions. Kinetics studies the rates, mechanisms of reactions and the influence on them of factors such as concentrations of enzymes and substrates, temperature, pH of the environment, the presence of inhibitors or activators.
At a constant substrate concentration, the reaction rate is directly proportional to the enzyme concentration. The graph of the dependence of the rate of an enzymatic reaction on the concentration of the substrate has the form of an equilateral hyperbola.
Dependence of the rate of enzymatic reaction on the concentration of enzyme (a) and substrate (b)
The dependence of the rate of an enzymatic reaction on the concentration of the substrate is described Michaelis-Menten equation:
where V is the steady-state rate of the biochemical reaction; Vmax - maximum speed; Km - Michaelis constant; [S] - substrate concentration.
If the substrate concentration is low, i.e. [S]<< Кm, то [S] в знаменателе можно пренебречь.
Then 
Thus, at low substrate concentrations, the reaction rate is directly proportional to the substrate concentration and is described by a first-order equation. This corresponds to the initial straight section of the curve V = f[S] (Figure b).
At high substrate concentrations [S] >> Km, when Km can be neglected, the Michaelis-Menten equation takes the form, i.e. V=Vmax.
Thus, at high substrate concentrations, the reaction rate becomes maximum and is described by a zero-order equation. This corresponds to the section of the curve V =f [S] parallel to the abscissa axis.
At substrate concentrations numerically comparable to the Michaelis constant, the reaction rate increases gradually. This is quite consistent with the ideas about the mechanism of the enzymatic reaction:
where S is the substrate; E - enzyme; ES - enzyme-substrate complex; P - product; k1 is the rate constant for the formation of the enzyme-substrate complex; k2 is the rate constant for the decay of the enzyme-substrate complex with the formation of initial reagents; k3 is the rate constant for the decay of the enzyme-substrate complex with the formation of a product.
Substrate conversion rate with the formation of product (P) is proportional to the concentration of the enzyme-substrate complex. At low substrate concentrations in solution there is a certain number of free enzyme molecules (E) not bound into a complex (ES). Therefore, as the substrate concentration increases, the concentration of the complexes increases, and therefore the rate of product formation also increases. At high substrate concentrations, all enzyme molecules are bound into an ES complex (the phenomenon of enzyme saturation), therefore, a further increase in the substrate concentration practically does not increase the concentration of the complexes and the rate of product formation remains constant.
Thus, the physical meaning of the maximum rate of an enzymatic reaction becomes clear. Vmax is the rate at which an enzyme reacts when it exists entirely as an enzyme-substrate complex..
The Michaelis constant numerically corresponds to the substrate concentration at which the steady-state speed is equal to half the maximum. This constant characterizes the dissociation constant of the enzyme-substrate complex:
Physical meaning of the Michaelis constant in that it characterizes the affinity of the enzyme for the substrate. Km has small values when k1 > (k2 + k3), i.e. the process of formation of the ES complex prevails over the processes of ES dissociation. Consequently, the lower the Km value, the greater the affinity of the enzyme for the substrate. And, conversely, if Km is of great importance, then (k2 + k3) > k1 and ES dissociation processes predominate. In this case, the affinity of the enzyme for the substrate is low.
Enzyme inhibitors and activators . Enzyme inhibitors are called substances that reduce enzyme activity. Any denaturing agents (for example, heavy metal salts, acids) are nonspecific enzyme inhibitors.
Reversible inhibitors- these are compounds that interact non-covalently with the enzyme. Irreversible inhibitors- these are compounds that specifically bind the functional groups of the active center and form covalent bonds with the enzyme.
Reversible inhibition is divided into competitive and non-competitive. Competitive inhibition suggests structural similarity between the inhibitor and the substrate. The inhibitor takes up space in the active site of the enzyme, and a significant number of enzyme molecules are blocked. Competitive inhibition can be removed by increasing the substrate concentration. In this case, the substrate displaces the competitive inhibitor from the active site.
Reversible inhibition may be non-competitive in relation to the substrate. In this case, the inhibitor does not compete for the site of attachment to the enzyme. The substrate and inhibitor bind to different centers, so it becomes possible to form the IE complex, as well as the ternary IES complex, which can decompose to release the product, but at a lower rate than the ES complex.
By the nature of his action inhibitors are divided into:
- specific,
- nonspecific.
Specific inhibitors exert their effect on the enzyme by joining with a covalent bond in the active center of the enzyme and turning it off from the scope of action.
Nonspecific inhibition involves the effect of denaturing agents on the enzyme (salts of heavy metals, urea, etc.). In this case, as a result of the destruction of the quaternary and tertiary structure of the protein, the biological activity of the enzyme is lost.
Enzyme activators- These are substances that increase the rate of enzymatic reactions. Most often, metal ions (Fe2+, Fe3+, Cu2+, Co2+, Mn2+, Mg2+, etc.) act as activators. There are metals found in metalloenzymes, which are cofactors, and acting as enzyme activators. Cofactors can bind tightly to the protein part of the enzyme; as for activators, they are easily separated from the apoenzyme. Such metals are obligatory participants in the catalytic act, determining the activity of the enzyme. Activators enhance the catalytic effect, but their absence does not prevent the enzymatic reaction from proceeding. As a rule, the metal cofactor interacts with negatively charged groups of the substrate. A metal with variable valence takes part in the exchange of electrons between the substrate and the enzyme. In addition, they take part in the formation of a stable transition conformation of the enzyme, which contributes to faster formation of the ES complex.
Regulation of enzyme activity . One of the main mechanisms for regulating metabolism is the regulation of enzyme activity. One example is allosteric regulation, regulation through activators and inhibitors. It often happens that the end product of a metabolic pathway is an inhibitor of a regulatory enzyme. This type of inhibition is called retroinhibition, or inhibition based on the principle of negative feedback.
Many enzymes are produced as inactive proenzyme precursors and then activated at the right time through partial proteolysis. Partial proteolysis- cleavage of part of the molecule, which leads to a change in the tertiary structure of the protein and the formation of the active center of the enzyme.
Some oligomeric enzymes can change their activity due to associations - dissociations of subunits, included in their composition.
Many enzymes can be found in two forms: as a simple protein and as a phosphoprotein. The transition from one form to another is accompanied by a change in catalytic activity.
The rate of the enzymatic reaction depends on amount of enzyme, which in a cell is determined by the ratio of the rates of its synthesis and decay. This method of regulating the rate of an enzymatic reaction is a slower process than regulating enzyme activity.