The maximum rate of the enzymatic reaction. Enzymatic reaction rate
Kinetics of enzymatic reactions. This branch of enzymology studies the influence of chemical and physical factors on the rate of an enzymatic reaction. In 1913, Michaelis and Menten created a theory of enzymatic kinetics based on the fact that an enzyme (E) interacts with a substrate (S) to form an intermediate enzyme-substrate complex (ES), which then decomposes into an enzyme and a reaction product according to the equation:
Each stage of the interaction of the substrate with the enzyme is characterized by its own rate constants. The ratio of the sum of the rate constants for the degradation of an enzyme-substrate complex to the rate constant for the formation of an enzyme-substrate complex is called the Michaelis constant (Km). It determines 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 catalyzed by it. According to the Km value, catalytic reactions can be divided into fast (Km 106 mol/l and less) and slow (Km 102 to 106).
The rate of an enzymatic reaction depends on temperature, the reaction of the medium, the concentration of reactants, the amount of enzyme, and other factors.
1. Consider the dependence of the reaction rate on the amount of enzyme. Under the condition of an excess of the substrate, the reaction rate is proportional to the amount of the enzyme, but with an excess of the enzyme, the increase in the reaction rate will decrease, since the substrate will no longer be enough.
2. The rate of chemical reactions is proportional to the concentration of reactants (the law of mass action). This law also applies to enzymatic reactions, but with certain limitations. At constant
In the presence of enzymes, the reaction rate is indeed proportional to the concentration of the substrate, but only in the range of low concentrations. At high substrate concentrations, the enzyme becomes saturated with the substrate, i.e., there comes a moment 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 its 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 substrate concentration at which the rate of the enzymatic reaction is half the maximum. From this, the Michaelis–Menten equation for the rate of the enzymatic reaction is derived.
Enzymatic reaction rate
A measure of the rate of an enzymatic reaction is the amount of substrate that has undergone transformation per unit of time, or the amount of product formed. The rate is determined by the slope of the tangent to the curve at the initial stage of the reaction.
Rice. 2 Enzymatic reaction rate.
The steeper the slope, the greater the speed. Over time, the rate of the reaction usually decreases, mostly as a result of a decrease in the concentration of the substrate.
Factors affecting enzymatic activity
F.'s action depends on a number of factors: temperature, reaction of the medium (pH), enzyme concentration, substrate concentration, the presence of specific activators and nonspecific or specific inhibitors.
Enzyme concentration
At a high substrate concentration and other factors being constant, the rate of the enzymatic reaction is proportional to the enzyme concentration.
Rice. 3 Dependence of the rate of the 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, with an increase in the concentration of the enzyme, the rate of the enzymatic reaction also increases.
Temperature
The effect of temperature on the rate of an enzymatic reaction can be expressed in terms of the temperature coefficient Q 10: Q 10 = (reaction rate at (x + 10) ° C) / (reaction rate at x ° C)
Between 0-40°C, the Q 10 of the enzymatic reaction is 2. In other words, for every 10°C increase in temperature, the rate of the enzymatic reaction doubles.
Rice. 4 Effect of temperature on the activity of an enzyme such as salivary amylase.
As the temperature rises, the movement of molecules accelerates, and the molecules of the reactants are more likely to collide with each other. Consequently, the probability that a reaction between them will occur also increases. The temperature that provides the greatest activity is called optimal. Beyond this level, the rate of the enzymatic reaction decreases despite an increase in the frequency of collisions. This happens 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 the freezing point, the enzymes are inactivated, but denaturation does not occur. With an increase in temperature, their catalytic activity is restored again.
Since proteins in the dry state denature much more slowly than hydrated proteins (in the form of a protein gel or solution), F. inactivation 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
At a given enzyme concentration, the rate of the enzymatic reaction increases with increasing substrate concentration.
Rice. 6 Dependence of the rate of the 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 concentrations of the substrate, the active centers of F. molecules at any given moment turn out to be practically saturated. Thus, no matter how much excess substrate is present, it can combine with F. only after the previously formed enzyme-substrate complex dissociates into the product and free F. Therefore, at high concentrations of the substrate, the rate of the enzymatic reaction is limited both by the concentration of the substrate and the time required for the dissociation of the enzyme-substrate complex.
At a constant temperature, any F. works most efficiently within narrow pH limits. The optimum pH value is the one at which the reaction proceeds at the fastest rate.
Rice. 7 Dependence of enzyme activity on pH.
At higher and lower pH, F.'s activity decreases. A shift in pH changes the charge of ionized acidic and basic groups, on which the specific form of F molecules depends. As a result, the shape of F molecules, and primarily the shape of its active center, changes. At too sharp shifts pH Ph. denatures. The optimum pH characteristic of a given F. does not always coincide with the pH of its immediate intracellular environment. This suggests that the environment in which F. is located regulates its activity to some extent.
ENZYMATIC REACTION KINETICS
studies the patterns of flow in time of enzymatic p-tions, as well as their mechanism; chapter chemical kinetics.
catalytic the cycle of conversion in-va S (substrate) into the product P under the action of the enzyme E proceeds with the formation of an intermediate. conn. X i:
where ki- rate constants of individual elementary stages,
formation of an enzyme-substrate complex X 1 (ES, Michaelis complex).
At a given t-re, the rate of the p-tion depends on the concentrations of the enzyme, substrate, and the composition of the medium. There are stationary, pre-stationary and relaxation kinetics of enzymatic p-tions.
Stationary kinetics. In a stationary state on intermediate Comm. (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 in between. comp., and the expression for the rate of the process v 0 , called initial stationary speed, has the form (Michaelis-Menten equation):
(1)
where the values k cat and K m -> functions of the rate constants of elementary stages and are given by the equations:
The value of k cat
called efficient catalytic. process rate constant, parameter K m -> Michaelis constant. The value of k cat
determined by the quantities max. slow stages catalytic. districts and sometimes called. the number of revolutions of the enzyme (enzyme system); k cat
characterizes the number of catalytic. cycles performed by the enzyme system per unit of time. Naib. common, having a value of 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.
At high concentrations of the substrate, when that is, the rate of p-tion 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 (the Lineweaver-Burk method), i.e., building the dependence of 1/v on 1/[S] 0 , or other methods. The linear form of equation (1) has the form:
(2)
It allows you to determine graphically the values K m and v max (Fig. 1).
Rice. 1. Graph of the linear transformation of the Michaelis - Menten equation in double reciprocals (according to Lineweaver - Burke).
Value K m > numerically equal to the concentration of the substrate, at which the rate of p-tion is equal, therefore K m often serves as a measure of the affinity of the substrate and the enzyme, but this is only true if
Quantities K m > and change depending on the pH values. This is due to the ability of the groups of the enzyme molecule involved in catalysis to change their state of ionization and, thereby, their catalytic. efficiency. In the simplest case, a change in pH leads to 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 (ES, ESH and ESH 2) is able to turn into a solution product, then the dependence of the rate on pH is described by the f-loy:
where f= 1 + / and f" = 1 +
+K" b/>-t. called pH-functions of Michaelis, and K a, K b and K" a, K" b -> group ionization constants a and b, respectively. free enzyme and enzyme-substrate complex. In lg coordinates - pH this dependence is shown in fig. 2, and the tangents of the slopes of the tangents to the ascending, pH-independent, and descending branches of the curve should be +1, 0, and -1, respectively. From such a graph, one can determine the values RK a groups involved in catalysis.
Rice. 2. Dependence of the catalytic constants from pH to logarithmic. coordinates.
The rate of enzymatic p-tion is not always subject to equation (1). One of the most common cases - participation in the district of allosteric. enzymes (see enzyme regulators) for to-rykh 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 to one of the sites of the enzyme macromolecule increases (positive cooperativity) or decreases (negative cooperativity) 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).
Prestationary kinetics. With rapid mixing of enzyme and substrate solutions in the time interval of 10 -6 -10 -1 s, transient processes can be observed that precede the formation of a stable stationary state. In this pre-stationary mode, when using a large excess of the substrate, the differential system. ur-tion, describing the kinetics of processes, is linear. The solution of this type of system of linear differentials. ur-tion is given by the sum of the exponential terms. So, for the kinetic scheme presented above, the kinetics of accumulation of the product has the form:
where A i ->, b and n -> functions of elementary rate constants; -roots of the corresponding characteristic. ur-tion.
The reciprocal of , called. characteristic process time:
For p-tion, flowing with the participation of nintermediate. Comm., you can get ncharacteristic. times.
The study of the kinetics of the enzymatic district in the pre-stationary mode allows you to get an idea of the detailed mechanism of catalytic. cycle and determine the rate constants of the elementary stages of the process.
Experimentally, the kinetics of the enzymatic solution in the pre-stationary mode is studied using the stopped jet method (see Fig. jet kinetic methods), allowing to mix the components of the district within 1 ms.
Relaxation kinetics. With a rapid perturbing effect on the system (changes in t-ry, pressure, electric fields), the time it takes for the system to achieve a new equilibrium or stationary state depends on the speed of the processes that determine the catalytic. 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 decomp. 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 the spectrum of relaxation times corresponding to the number of intervals. Comm. involved in the process. The relaxation times depend on the rate constants of the elementary stages of the processes.
Relaxation methods kinetics make it possible to determine the rate constants of individual elementary stages of the transformation of intermediates. Methods for studying relaxation kinetics are different. resolution: ultrasound absorption - 10 -6 -10 -10
s, temperature jump - 1O -4 -10 -6 s, electric method. impulse - 10 -4 -10 -6 s, pressure jump - 10 -2 s. In the study of the kinetics of enzymatic p-tions, the application was found by the method of temperature jump.
Macrokinetics of enzymatic processes. Development of methods for obtaining heterogeneous catalysts by immobilization of 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 regularities of the kinetics of p-tions were studied theoretically and experimentally, taking into account the effects of the diffusion layer and for systems with intradiffusion difficulties in the distribution of the enzyme inside 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 the conditions of the flow of the enzymatic district with a diffusion-reduced substrate concentration to the flow, which could be realized in the absence of diffusion restrictions. In the purely diffusion region, when the process rate 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
diffusion layer thickness, D - coefficient. substrate diffusion.
For systems with intradiffusion deceleration in first-order p-tions
where f t- dimensionless module (Thiele module).
When analyzing the kinetic regularities in fermentation reactors wide theoretical. and experiment. "ideal" models of reactors, a flow reactor (a flow reactor of ideal mixing), a flow reactor with ideal displacement, and a membrane reactor, have been developed.
Kinetics of polyenzymatic processes. In the body (cell), enzymes do not act in isolation, but catalyze the chains of transformation of molecules. R-tion in polyenzymatic systems with kinetic. points of view can be seen as consistent. processes, specific a feature of to-rykh 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 the formation of a stable stationary state. The condition for its occurrence can be the inequality > v 0 , where v 0 is the rate of the limiting stage, characterized by the smallest rate constant and thus determining the rate of the entire sequence. process. In the stationary state, the concentration of metabolites after the limiting stage is less than the Michaelis constant of the corresponding enzyme.
Specific a group of polyenzymatic systems is made up of systems that carry out oxidizing.-restore. p-tion with the participation of protein electron carriers. Carriers form specific. structures, complexes with a deterministic electron transfer sequence. Kinetic the description of such systems is considered as an independent state variable of circuits with decomp. degree of population of electrons.
Application. F. r. widely used in research practice to study the mechanisms of action of enzymes and enzyme systems. Practically significant area of enzyme science is engineering enzymology, operates with the concepts of F. r. to. for optimization of biotechnol. processes.
Lit.: Poltorak O. M., Chukhrai E. S., Physical and chemical bases of enzymatic catalysis, M., 1971; Berezin IV, 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 rtion cyclic. a process consisting of a number of elementary rations, the velocities of which are described by the acting mass law. This law has a simple form for ideal gas mixtures, ideal p moats, and ideal surface layers. ... ... Chemical Encyclopedia
Kinetics of chemical reactions, the doctrine of chemical processes, the laws of their flow in time, speeds and mechanisms. The most important areas of modern chemistry and chemical ... ... are associated with the study of the kinetics of chemical reactions. Great Soviet Encyclopedia
KINETICS CHEMICAL- (from the Greek. kinesis movement), a department of theoretical chemistry devoted to the study of the laws of chemistry. reactions. There are several types of chem. interactions and, above all, to distinguish reactions occurring in a homogeneous (homogeneous) medium from reactions that ... ... Big Medical Encyclopedia
- (biocatalysis), acceleration of biochemical. rations with the participation of protein macromolecules called enzymes (enzymes). F. to. a kind of catalysis, although the term fermentation (fermentation) has been known since ancient times, when there was no concept of chemical. catalysis. First… … Chemical Encyclopedia
- (from Latin re prefix, meaning reverse action, and actio action), the transformation of some in in (initial compounds) into others (products of the reaction) with the invariability of the nuclei of atoms (unlike nuclear reactions). Initial connections in R. x. sometimes called ... ... Chemical Encyclopedia
- (from lat. fermentum leaven) (enzymes), proteins that act as catalysts in living organisms. Main functions of F. to accelerate the transformation into into, entering the body and formed during metabolism (to renew cellular structures, to ensure it ... Chemical Encyclopedia
- (from the Greek pharmakon medicine and kinetikos setting in motion), studies kinetic. patterns of processes occurring with lek. cfd in the body. Main pharmacokinetic. processes: absorption, distribution, metabolism and excretion (excretion). ... ... Chemical Encyclopedia
Introduction
One of the characteristic manifestations of life is the ability of living organisms to kinetically regulate chemical reactions, suppressing the desire to achieve thermodynamic equilibrium. Enzymatic kinetics studies the patterns of influence of the chemical nature of reacting substances (enzymes, substrates) and the conditions of their interaction (concentration, pH of the medium, temperature, presence of activators or inhibitors) on the rate of an enzymatic reaction. The main goal of studying the kinetics of enzymatic reactions is to obtain information that can help elucidate the molecular mechanism of enzyme action.
Dependence of the Enzymatic Reaction Rate on Substrate Concentration
enzyme substrate biochemical inhibitor
The general principles of chemical reaction kinetics apply to enzymatic reactions as well. It is known that any chemical reaction is characterized by a constant of thermodynamic equilibrium. It expresses the state of chemical equilibrium achieved by the system, and is denoted by Kr. So for the reaction:
the equilibrium constant is equal to the product of the concentrations of the formed substances divided by the product of the concentration of the starting substances. The value of the equilibrium constant is usually found from the ratio of the rate constants of the direct (k+1) and reverse (k-1) reactions, i.e.
At equilibrium, the rate of the forward reaction is:
v+1 = k+1[A]*[B]
equal to the rate of the reverse reaction:
v-1 = k-1[C]*[D],
those. v+1 = v-1
respectively k+1[A]*[B] = k-1[C]*[D],
Rice. one.
reactions from substrate concentration at constant concentration
enzyme
a - first-order reaction (at [S]<Кm скорость реакции пропорциональна концентрации субстрата); б - реакция смешанного порядка; в - реакция нулевого порядка, когда v = Vmaxi скорость реакции не зависит от концентрации субстрата.
Thus, the equilibrium constant is equal to the ratio of the rate constants of the forward and reverse reactions. The reciprocal of the equilibrium constant is called the substrate constant, or, in the case of an enzymatic reaction, the dissociation constant of the enzyme-substrate complex, and denoted by the symbol KS. So, in reaction
those. KS is equal to the ratio of the product of the concentration of the enzyme and the substrate to the concentration of the enzyme-substrate complex or the ratio of the rate constants of the reverse and forward reactions. It should be noted that the KS constant depends on the chemical nature of the substrate and the enzyme and determines the degree of their affinity. The lower the KS value, the higher the affinity of the enzyme for the substrate.
When studying the kinetics of enzymatic reactions, one important feature of these reactions (not characteristic of ordinary chemical reactions), which is associated with the phenomenon of saturation of the enzyme with a substrate, should be taken into account. At a low substrate concentration, the dependence of the reaction rate on the substrate concentration (Fig. 1) is almost linear and obeys first-order kinetics. This means that the reaction rate S -> P is directly proportional to the concentration of the substrate S and at any time t is determined by the following kinetic equation:
where [S] is the molar concentration of the substrate S; -d[S]/dt - the rate of loss of the substrate; k" is the rate constant of the reaction, which in this case has the dimension reciprocal to the unit of time (min-1 or s-1).
At a high substrate concentration, the reaction rate is maximum, becomes constant and does not depend on the substrate concentration [S]. In this case, the reaction obeys the zero-order kinetics v=k" (when the enzyme is completely saturated with the substrate) and is entirely determined by the concentration of the enzyme. In addition, there are second-order reactions, the rate of which is proportional to the product of the concentrations of the two reactants. Under certain conditions, when proportionality is violated, they say sometimes about reactions of a mixed order (see Fig. 1).
Studying the phenomenon of saturation, L. Michaelis and M. Menten developed a general theory of enzymatic kinetics. They proceeded from the assumption that the enzymatic process proceeds in the form of the following chemical reaction:
those. enzyme E interacts with substrate S with the formation of an intermediate complex ES, which then decomposes into a free enzyme and reaction product P. Mathematical processing based on the law of mass action made it possible to derive an equation named after the authors of the Michaelis-Menten equation, expressing the quantitative relationship between substrate concentration and enzymatic reaction rate:
where v is the observed reaction rate at a given substrate concentration [S]; KS - dissociation constant of the enzyme-substrate complex, mol/l; Vmax is the maximum reaction rate at full saturation of the enzyme with the substrate.
It follows from the Michaelis-Menten equation that at a high substrate concentration and a low KS value, the reaction rate is maximum, i.e. v=Vmax (zero order reaction, see Fig. 1). At a low substrate concentration, on the contrary, the reaction rate is proportional to the substrate concentration at any given moment (first-order reaction). It should be pointed out that the Michaelis-Menten equation in its classical form does not take into account the effect of the reaction products on the rate of the enzymatic process, for example, in the reaction
and is somewhat limited. Therefore, attempts have been made to improve it. So, the Briggs-Haldane equation was proposed:
where Km is the Michaelis constant, which is an experimentally determined quantity. It can be represented by the following equation:
Rice. 2. - Michaelis-Menten equation curve: hyperbolic
dependence of the initial rates of the reaction catalyzed by the enzyme
from substrate concentration
The numerator represents the rate constants for the decomposition of the ES complex in two directions (towards the initial E and S and towards the final reaction products E and P). The ratio k-1/ k+1 is the dissociation constant of the enzyme-substrate complex KS, then:
An important consequence follows from this: the Michaelis constant is always greater than the dissociation constant of the enzyme-substrate complex KS by the value k+2/k+1.
To determine the numerical value of Km, one usually finds the substrate concentration at which the enzymatic reaction rate V is half of the maximum Vmax, i.e. if V = 1/2 Vmax. Substituting the value of V into the Briggs-Haldane equation, we get:
dividing both sides of the equation by Vmax, we get
Thus, the Michaelis constant is numerically equal to the substrate concentration (mol/l) at which the rate of this enzymatic reaction is half of the maximum.
Determining the value of Km is important in elucidating the mechanism of action of effectors on the activity of enzymes, etc. The Michaelis constant can be calculated from the graph (Fig. 2). The segment on the abscissa corresponding to the speed equal to half the maximum will be Km.
It is inconvenient to use a graph constructed in direct coordinates of the dependence of the initial reaction rate v0 on the initial substrate concentration , since the maximum rate Vmax in this case is an asymptotic value and is not determined accurately enough.
Rice. 3.
For a more convenient graphical representation of experimental data, G. Lineweaver and D. Burke transformed the Briggs-Haldane equation using the double reciprocal method based on the principle that if there is an equality between any two quantities, then the reciprocals will also be equal. In particular, if
then after the transformation we get the equation:
which is called the Lineweaver-Burk equation. This is the straight line equation:
If now, in accordance with this equation, to build a graph in the coordinates 1/v(y) from l/[S](x), then we get a straight line (Fig. 3), the tangent of the slope angle which will be equal to the value Km/Vmax; the segment cut off by a straight line from the y-axis is l/Vmax (the reciprocal of the maximum speed).
If we continue the straight line beyond the y-axis, then the segment corresponding to the reciprocal of the Michaelis constant - 1/Km is cut off on the abscissa (see Fig. 3). Thus, the value of Km can be calculated from the data of the slope of the straight line and the length of the segment cut off from the ordinate axis, or from the length of the segment cut off from the abscissa axis in the region of negative values.
It should be emphasized that the values of Vmax, as well as the value of Km, can be determined more accurately than from a graph plotted in direct coordinates from a graph plotted using the double reciprocal method. Therefore, this method has found wide application in modern enzymology. Also proposed are similar graphical methods for determining Km and Vmax in the coordinates of v versus v/[S] and [S]/v versus [S].
Some limitations of the application of the Michaelis-Menten equation should be noted, due to the multiple forms of enzymes and the allosteric nature of the enzyme. In this case, the graph of the dependence of the initial reaction rate on the concentration of the substrate (kinetic
Rice. 4.
curve) does not have a hyperbolic shape, but a sigmoid character (Fig. 4) like the oxygen saturation curve for hemoglobin. This means that the binding of one substrate molecule at one catalytic site increases the binding of the substrate to another site, i.e. there is a cooperative interaction, as in the case of oxygen addition to 4 hemoglobin subunits. To estimate the substrate concentration at which the reaction rate is half the maximum, under conditions of the sigmoid nature of the kinetic curve, the transformed Hill equation is usually used:
where K" is the association constant; n is the number of substrate-binding centers.
With an increase in the temperature of the medium, the rate of the enzymatic reaction increases, reaching a maximum at some optimal temperature, and then drops to zero. For chemical reactions, there is a rule that with an increase in temperature by 10 ° C, the reaction rate increases by two to three times. For enzymatic reactions, this temperature coefficient is lower: for every 10°C, the reaction rate increases by a factor of 2 or even less. The subsequent decrease in the reaction rate to zero indicates the denaturation of the enzyme block. The optimal temperature values for most enzymes are in the range of 20 - 40 0 C. The thermolability of enzymes is associated with their protein structure. Some enzymes are already denatured at a temperature of about 40 0 C, but most of them are inactivated at temperatures above 40 - 50 0 C. Some enzymes are inactivated by cold, i.e. at temperatures close to 0°C, denaturation occurs.
An increase in body temperature (fever) accelerates biochemical reactions catalyzed by enzymes. It is easy to calculate that an increase in body temperature for every degree increases the reaction rate by about 20%. At high temperatures of about 39-40°C, the wasteful use of endogenous substrates in the cells of a diseased organism is required to replenish their intake with food. In addition, at a temperature of about 40°C, some of the very thermolabile enzymes can be denatured, which disrupts the natural course of biochemical processes.
Low temperature causes a reversible inactivation of enzymes due to a slight change in its spatial structure, but sufficient to disrupt the corresponding configuration of the active center and substrate molecules.
The dependence of the reaction rate on the pH of the medium
For most enzymes, there is a certain pH value at which their activity is maximum; above and below this pH value, the activity of these enzymes decreases. However, not in all cases the curves describing the dependence of enzyme activity on pH are bell-shaped; sometimes this dependence can also be expressed directly. The dependence of the enzymatic reaction rate on pH mainly indicates the state of the functional groups of the active center of the enzyme. Changing the pH of the medium affects the ionization of acidic and basic groups of amino acid residues of the active center, which are involved either in the binding of the substrate (in the contact area) or in its transformation (in the catalytic area). Therefore, the specific effect of pH can be caused either by a change in the affinity of the substrate for the enzyme, or by a change in the catalytic activity of the enzyme, or both.
Most substrates have acidic or basic groups, so pH affects the degree of ionization of the substrate. The enzyme preferably binds to either the ionized or non-ionized form of the substrate. Obviously, at optimal pH, both functional groups of the active center are in the most reactive state, and the substrate is in a form that is preferable for binding by these groups of the enzyme.
When constructing curves describing the dependence of enzyme activity on pH, measurements at all pH values are usually carried out under conditions of saturation of the enzyme with the substrate, since the K m value for many enzymes changes with pH.
The curve characterizing the dependence of enzyme activity on pH can have a particularly simple shape in those cases where the enzyme acts on electrostatically neutral substrates or substrates in which charged groups do not play a significant role in the catalytic act. An example of such enzymes is papain, as well as invertase, which catalyzes the hydrolysis of neutral sucrose molecules and maintains a constant activity in the pH range of 3.0-7.5.
The pH value corresponding to the maximum activity of the enzyme does not necessarily coincide with the pH value characteristic of the normal intracellular environment of this enzyme; the latter can be both above and below the pH optimum. This suggests that the effect of pH on enzyme activity may be one of the factors responsible for the regulation of enzymatic activity within the cell. Since the cell contains hundreds of enzymes, and each of them reacts differently to changes in pH, the pH value inside the cell is perhaps one of the important elements in the complex system of regulation of cellular metabolism.
