Factors that determine the activity of enzymes. Enzymatic activity of bacteria

Enzyme activity. Under enzyme activity understand its amount, which catalyzes the transformation of a certain amount of substrate per unit of time. To express the activity of enzyme preparations, two alternative units are used: international (IU) and catal (cat). Behind international unit of activity the amount of enzyme taken is such that it catalyzes the conversion of 1 µmol of the substrate into the product in 1 min under standard conditions (usually optimal). One rolled denotes the amount of enzyme catalyzing the conversion of 1 mol of substrate in 1 s (1 cat = 6 ∙ 10 7 IU). In the bimolecular reaction A + B = C + D, the amount of enzyme activity that catalyzes the conversion of 1 µmol A or B, or 2 µmol A (if B = A) in 1 min is taken as a unit of enzyme activity.

Often enzyme preparations are characterized by specific activity, which reflects the degree of purification of the enzyme. Specific activity is the number of units of enzyme activity per 1 mg of protein.

Molecular activity (number of enzyme turnovers) is the number of substrate molecules converted by one enzyme molecule in 1 min when the enzyme is completely saturated with the substrate. It is equal to the number of units of enzyme activity divided by the amount of enzyme, expressed in micromoles. The concept of molecular activity is applicable only to pure enzymes.

When the number of active centers in an enzyme molecule is known, the concept is introduced catalytic site activity . It is characterized by the number of substrate molecules that undergoes transformation in 1 min per one active center.

The activity of enzymes strongly depends on external conditions, among which the temperature and pH of the medium are of paramount importance. An increase in temperature in the range of 0–50°С usually leads to a gradual increase in enzymatic activity, which is associated with an acceleration of the processes of formation of the enzyme–substrate complex and all subsequent events of catalysis. For every 10°C increase in temperature, the rate of the reaction approximately doubles (van't Hoff's rule). However, a further increase in temperature (>50°C) is accompanied by an increase in the amount of inactivated enzyme due to the denaturation of its protein part, which is expressed in a decrease in activity. Each enzyme is characterized temperature optimum– the temperature value at which its greatest activity is recorded.

The dependence of enzyme activity on the pH value of the medium is complex. Each enzyme has its own optimum pH environments at which it is most active. As you move away from this value in one direction or another, the enzymatic activity decreases. This is due to a change in the state of the active center of the enzyme (decrease or increase in the ionization of functional groups), as well as the tertiary structure of the entire protein molecule, which depends on the ratio of cationic and anionic centers in it. Most enzymes have an optimum pH in the neutral range. However, there are enzymes that show maximum activity at pH 1.5 (pepsin) or 9.5 (arginase). When working with enzymes, the pH must be maintained with an appropriate buffer solution.

Enzyme activity is subject to significant fluctuations depending on exposure inhibitors(substances that partially or completely reduce activity) and activators(substances that increase activity). Their role is played by metal cations, some anions, carriers of phosphate groups, reducing equivalents, specific proteins, intermediate and final products of metabolism, etc.

Principles of enzymatic kinetics. The essence of kinetic studies is to determine the maximum rate of the enzymatic reaction ( V max) and Michaelis constant K M. Enzymatic kinetics studies the rates of quantitative transformations of some substances into others under the action of enzymes. The rate of an enzymatic reaction is measured by the loss of the substrate or the increase in the resulting product per unit of time, or by the change in the concentration of one of the adjacent forms of the coenzyme.

Influence enzyme concentration on the reaction rate is expressed as follows: if the concentration of the substrate is constant (assuming an excess of the substrate), then the reaction rate is proportional to the concentration of the enzyme. For kinetic studies, an enzyme concentration of 10–8 M of active centers is used. The optimal value of the enzyme concentration is determined from the graph of the dependence of enzyme activity on its concentration. The optimal value is considered to lie on the plateau of the obtained graph in the range of enzyme activity values ​​that depend little on its concentration (Fig. 4.3).

Rice. 4.3. The dependence of the rate of the enzymatic reaction

on enzyme concentration

To study the influence substrate concentration on the rate of the enzymatic reaction, first build a kinetic curve that reflects the change in the concentration of the substrate (S 1) or product (P 1) over time (Fig. 4.4) and measure the initial rate ( V 1) reactions as the tangent of the slope of the tangent to the curve at the zero point.

Rice. 4.4. Kinetic curves of the enzymatic reaction

By constructing kinetic curves for other concentrations of a given substrate (S 2 , S 3 , S 4 , etc.) or product (P 2 , P 3 , P 4 , etc.) and determining the initial rates ( V 2, V 3 , V 4, etc.) of the reaction, build a graph of the dependence of the initial rate of the enzymatic reaction on the concentration of the substrate (at a constant concentration of the enzyme), which has the form of a hyperbola (Fig. 4.5).

Rice. 4.5. Dependence of the initial rate of the enzymatic reaction

from substrate concentration

The kinetics of many enzymatic reactions is described by the Michaelis-Menten equation. At a constant enzyme concentration and low substrate concentrations[S] the initial reaction rate is directly proportional to [S] (Fig. 4.5). In this case, we speak of half-saturation of the enzyme with the substrate, when half of the enzyme molecules are in the form of an enzyme-substrate complex and the reaction rate V = 1/2V max. In relation to the substrate, the reaction has the 1st order (the reaction rate is directly proportional to the concentration of one reactant) or the 2nd order (the reaction rate is proportional to the product of the concentrations of the two reactants).

At high values substrate concentration[S] the reaction rate is almost independent of [S]: with a further increase in [S], the reaction rate grows more slowly and eventually becomes constant (maximum) (Fig. 4.5). In this case, complete saturation of the enzyme with the substrate is achieved, when all enzyme molecules are in the form of an enzyme-substrate complex and V = V max. With regard to the substrate, the reaction has the 0th order (the reaction rate does not depend on the concentration of the reactants).

In 1913, L. Michaelis and M. Menten proposed a simple model to explain such kinetics. According to this model, the formation of a specific enzyme-substrate complex is a necessary intermediate step in catalysis.

k 1 k 3

E + S ⇄ ES → E + P

Enzyme E combines with substrate S to form an ES complex. The rate constant of this process k 1 . The fate of the ES complex is twofold: it can either dissociate into enzyme E and substrate S with a rate constant k 2 , or undergo further transformation, forming product P and free enzyme E, with a rate constant k 3 . It is postulated that the reaction product is not converted into the original substrate. This condition is observed at the initial stage of the reaction, while the concentration of the product is low.

The rate of catalysis is determined in stationary conditions, when the concentration of intermediate products remains constant, while the concentration of starting materials and final products changes. This occurs when the rate of formation of the ES complex is equal to the rate of its decay.

You can introduce a new constant K M - Michaelis constant(mol/l), which is equal to

Michaelis–Menten equation, which expresses the quantitative relationship between the rate of the enzymatic reaction and the concentration of the substrate, has the form

(4.2)

This equation corresponds to a plot of reaction rate versus substrate concentration. At low substrate concentrations when [S] is much lower than K M, V = V max [S] / K M, i.e. the reaction rate is directly proportional to the concentration of the substrate. At high substrate concentrations when [S] is much higher than K M, V = V max , i.e., the reaction rate is maximum and does not depend on the concentration of the substrate.

If [S] = K M, then V = V max /2.

Thus, K M equal to the substrate concentration at which the reaction rate is half the maximum.

Michaelis constant (KM) and maximum reaction rate ( V max) are important velocity characteristics at different substrate concentrations. V max is a constant value for each enzyme, which makes it possible to evaluate the effectiveness of its action.

The Michaelis constant shows the affinity of the substrate for the enzyme (in the case when k 2 >> k 3): the lower K M, the greater the affinity and the higher the reaction rate, and vice versa. Each substrate is characterized by its KM value for a given enzyme, and their values ​​can be used to judge the substrate specificity of the enzyme. The Michaelis constant depends on the nature of the substrate, temperature, pH, ionic strength of the solution, and the presence of inhibitors.

Due to the fact that the definition V max and K M directly from the graphic dependence of Michaelis - Menten (Fig. 4.5) is ambiguous, they resort to the linearization of this equation. To do this, it is converted into such a form that it is graphically expressed as a straight line. There are several linearization methods, among which the Lineweaver-Burk and Edie-Hofstee methods are most commonly used.

transformation Lineweaver-Burke has the form

(4.3)

Build a dependency graph 1/ V = f(1/[S]) and get a straight line, the intersection of which with the y-axis gives the value 1/ V max ; the segment cut off by a straight line on the abscissa axis gives the value −1 / K M, and the tangent of the angle of inclination of the straight line to the abscissa axis is K M / V max (Fig. 4.6). This graph allows you to more accurately determine V max. As we will see below, valuable information regarding the inhibition of enzyme activity can also be extracted from this graph.

Rice. 4.6. Method for linearizing the Michaelis–Menten equation

(according to Lineweaver-Burke)

Method Edie – Hofsty is based on transforming the Michaelis–Menten equation by multiplying both sides by V max:

(4.4)

Graph in coordinates V And V/[S] is a straight line, the intersection of which with the y-axis gives the value V max, and the segment cut off by a straight line on the abscissa axis is the value V max / K M (Fig. 4.7). It makes it very easy to determine K M and V max , as well as detect possible deviations from linearity that were not detected in the previous graph.

Rice. 4.7. Method for linearizing the Michaelis–Menten equation

(according to Edie-Hofsty)

Inhibition of enzyme activity. The action of enzymes can be completely or partially suppressed by certain chemicals - inhibitors . By the nature of their action, inhibitors are divided into reversible and irreversible. This division is based on the binding strength of the inhibitor to the enzyme.

Reversible inhibitors - These are compounds that interact non-covalently with the enzyme and when they are removed, the activity of the enzyme is restored. Reversible inhibition can be competitive, non-competitive and non-competitive.

An example competitive inhibition is the action of structural analogues of the substrate, which can bind to the active site of the enzyme in a similar way as the substrate, but without turning into a product and preventing the interaction of the enzyme with the true substrate, i.e. there is competition between the substrate and the inhibitor for binding to the active site of the enzyme . As a result of the formation of enzyme-inhibitor (EI) complexes, the concentration of ES-complexes decreases and, as a result, the reaction rate decreases. In other words, a competitive inhibitor reduces the rate of catalysis by reducing the proportion of enzyme molecules that bind the substrate.

Measurement of reaction rates at different substrate concentrations makes it possible to distinguish competitive inhibition from noncompetitive inhibition. With competitive inhibition on the graph of dependence 1/ V=f(1/[S]) lines intersect the y-axis at one point 1/ V max regardless of the presence of an inhibitor, but in the presence of an inhibitor, the tangent of the slope of the straight line to the abscissa axis increases, i.e. V max does not change, while KM increases, which indicates a decrease in the affinity of the substrate for the enzyme in the presence of an inhibitor (Fig. 4.8). Therefore, at a sufficiently high concentration of the substrate under conditions of competition for the active site of the enzyme, when the substrate displaces the inhibitor from the active site, inhibition can be eliminated and the rate of the catalyzed reaction is restored. In this case, the Michaelis − Menten equation has the form

(4.5)

where [I] is the inhibitor concentration; K i is the inhibition constant.

The inhibition constant characterizes the affinity of the enzyme for the inhibitor and is the dissociation constant of the EI complex:

(4.6)

In the presence of a competitive inhibitor, the slope of the straight line to the x-axis increases by (1 + [I]/ K i).

Rice. 4.8. Competitive inhibition:

a - scheme; b - graphical expression according to Lineweaver - Burke

At non-competitive inhibition the inhibitor differs in structure from the substrate and binds not to the active, but to the allosteric site of the enzyme. This leads to a change in the conformation of the active site of the enzyme, which is accompanied by a decrease in the catalytic activity of the enzyme. Moreover, the inhibitor can bind not only to the free enzyme (E + I → EI), but also to the enzyme-substrate complex (ES + I → ESI). Both forms EI and ESI are not active. The substrate and inhibitor can be simultaneously bound by the enzyme molecule, but their binding sites do not overlap. The action of a non-competitive inhibitor is to reduce the number of enzyme turnovers, and not to reduce the proportion of substrate-bound enzyme molecules. The inhibitor does not prevent the formation of ES complexes, but inhibits the conversion of the substrate into the product. Consequently V max decreases, i.e., in the presence of an inhibitor, the intersection of the straight line with the y-axis will occur at a higher point (Fig. 4.9). To the same extent, the tangent of the angle of inclination of the straight line to the abscissa axis, equal to K M / V max I . K M in contrast to V max does not change, so non-competitive inhibition cannot be eliminated by increasing the substrate concentration.

Rice. 4.9. Noncompetitive inhibition:

a - scheme; b - graphical expression according to Lineweaver - Burke

Maximum reaction rate V max I in the presence of a noncompetitive inhibitor is described by the equation

(4.7)

In a particular case uncompetitive inhibition, when the inhibitor binds only to the ES-complex and does not bind to the free enzyme, on the dependence graph 1/ V = f(1/[S]) lines are parallel to each other and intersect the ordinate and abscissa axes at different points (Fig. 4.10).

Rice. 4.10. Uncompetitive inhibition:

a - scheme; b - graphical expression according to Lineweaver - Burke

irreversible inhibitors - these are highly reactive compounds of various chemical nature, which can interact with functionally important groups of the active center, forming strong covalent bonds. This leads to an irreversible loss of enzyme activity. In this regard, the Michaelis-Menten theory, based on the assumption that the attachment of an inhibitor to an enzyme is reversible, is not applicable in this case.

An example of irreversible inhibition is the interaction of enzymes with heavy metal ions, which are attached to the sulfhydryl groups of cysteine ​​residues of the enzyme and form mercaptides, which are practically non-dissociating compounds, or covalent modification of the enzyme under the action of alkylating agents.

The concept of enzyme activity

In everyday biochemical practice, the amount of the enzyme is practically not estimated, but only its activity. Activity is a broader concept than quantity. It implies primarily the result of the reaction, namely the loss of the substrate or the accumulation of the product. Naturally, one cannot ignore the time that the enzyme has worked and the number of enzyme molecules. But since it is usually unrealistic to calculate the number of enzyme molecules, the amount of biological material containing the enzyme (volume or mass) is used.

Thus, when determining the activity of enzymes, three variables must be taken into account simultaneously:

• the mass of the obtained product or the disappeared substrate;
• time spent on reaction;
• the amount of enzyme, but actually the mass or volume of biological material containing the enzyme.

To understand the relationship between these factors, a clear and simple example can be the construction of two buildings. Buildings are equated to the product of the reaction, workers are enzymes, let the team correspond to the volume of biological material. So, tasks from the 3rd grade:

1. A team of 10 people worked on the construction of one building, a team of 5 people worked on another building of the same kind. The construction was completed simultaneously and in full. Where is the activity of workers higher?
2. A team of 10 people worked on the construction of one building of 3 floors, another building of 12 floors - a team of 10 people. The construction was completed simultaneously and in full. Where is the activity of workers higher?
3. On the construction of one building of 5 floors, a team of 10 people worked, another of the same building - a team of 10 people. The construction of the first building took 20 days, the second was completed in 10 days. Where is the activity of workers higher?

Fundamentals of Enzyme Activity Quantification

1. Enzyme activity is expressed as the rate of accumulation of the product or the rate of loss of the substrate in terms of the amount of material containing the enzyme.

In practice, they usually use:

• units of quantity of a substance - mol (and its derivatives mmol, µmol), gram (kg, mg),
• units of time - minute, hour, second,
• units of mass or volume - gram (kg, mg), liter (ml).

Other derivatives are also actively used - catal (mol / s), the international unit of activity (IU, Unit) corresponds to μmol / min.

Thus, enzyme activity can be expressed, for example, in mmol/s×l, g/h×l, IU/l, cat/ml, etc.

For example, it is known

2. Creation of standard conditions to be able to compare the results obtained in different laboratories - optimal pH and fixed temperature, for example, 25°C or 37°C, adherence to the incubation time of the substrate with the enzyme.

Enzymes are catalysts for chemical reactions of a protein nature, differing in the specificity of their action in relation to the catalysis of certain chemical reactions. They are products of the biosynthesis of all living soil organisms: woody and herbaceous plants, mosses, lichens, algae, microorganisms, protozoa, insects, invertebrates and vertebrates, represented in the natural environment by certain aggregates - biocenoses.

The biosynthesis of enzymes in living organisms is carried out due to genetic factors responsible for the hereditary transmission of the type of metabolism and its adaptive variability. Enzymes are the working apparatus by which the action of genes is realized. They catalyze thousands of chemical reactions in organisms, which ultimately form cellular metabolism. Thanks to them, chemical reactions in the body are carried out at a high speed.

Currently, more than 900 enzymes are known. They are divided into six main classes.

1. Oxireductases catalyzing redox reactions.

2. Transferases catalyzing the reactions of intermolecular transfer of various chemical groups and residues.

3. Hydrolases catalyzing reactions of hydrolytic cleavage of intramolecular bonds.

4. Lyases that catalyze the addition of groups to double bonds and the reverse reactions of abstraction of such groups.

5. Isomerases catalyzing isomerization reactions.

6. Ligases that catalyze chemical reactions with the formation of bonds due to ATP (adenosine triphosphoric acid).

When living organisms die and rot, some of their enzymes are destroyed, and some, getting into the soil, retain their activity and catalyze many soil chemical reactions, participating in the processes of soil formation and in the formation of a qualitative sign of soils - fertility. In different types of soils under certain biocenoses, their own enzymatic complexes were formed, differing in the activity of biocatalytic reactions.

VF Kuprevich and TA Shcherbakova (1966) note that an important feature of soil enzymatic complexes is the orderliness of the action of existing groups of enzymes, which manifests itself in the fact that a number of enzymes representing different groups are simultaneously active; the formation and accumulation of compounds present in the soil in excess are excluded; excess accumulated mobile simple compounds (for example, NH 3) are temporarily bound in one way or another and sent to cycles culminating in the formation of more or less complex compounds. Enzymatic complexes are balanced self-regulating systems. Microorganisms and plants play the main role in this, constantly replenishing soil enzymes, since many of them are short-lived. The number of enzymes is indirectly judged by their activity over time, which depends on the chemical nature of the reacting substances (substrate, enzyme) and on the interaction conditions (concentration of components, pH, temperature, composition of the medium, action of activators, inhibitors, etc.).

This chapter discusses the participation in some chemical soil processes of enzymes from the class of hydrolases - the activity of invertase, urease, phosphatase, protease and from the class of oxyreductases - the activity of catalase, peroxidase and polyphenol oxidase, which are of great importance in the conversion of nitrogen- and phosphorus-containing organic substances, substances of a carbohydrate nature and in the processes of humus formation. The activity of these enzymes is a significant indicator of soil fertility. In addition, the activity of these enzymes in forest and arable soils of various degrees of cultivation will be characterized using sod-podzolic, gray forest, and sod-calcareous soils as an example.

CHARACTERISTICS OF SOIL ENZYMES

Invertase - catalyzes the reactions of hydrolytic cleavage of sucrose into equimolar amounts of glucose and fructose, also acts on other carbohydrates with the formation of fructose molecules - an energy product for the vital activity of microorganisms, catalyzes fructose transferase reactions. Studies by many authors have shown that the activity of invertase better than other enzymes reflects the level of soil fertility and biological activity.

Urease - catalyzes the reactions of hydrolytic cleavage of urea into ammonia and carbon dioxide. In connection with the use of urea in agronomic practice, it must be borne in mind that urease activity is higher in more fertile soils. It rises in all soils during periods of their greatest biological activity - in July - August.

Phosphatase (alkaline and acid) - catalyzes the hydrolysis of a number of organophosphorus compounds with the formation of orthophosphate. Phosphatase activity is inversely related to the provision of plants with mobile phosphorus, so it can be used as an additional indicator when determining the need for phosphate fertilizers to be applied to soils. The highest phosphatase activity is in the rhizosphere of plants.

Proteases are a group of enzymes, with the participation of which proteins are broken down into polypeptides and amino acids, then they are hydrolyzed to ammonia, carbon dioxide and water. In this regard, proteases are of great importance in the life of the soil, since they are associated with changes in the composition of organic components and the dynamics of nitrogen forms assimilated by plants.

Catalase - as a result of its activating action, hydrogen peroxide, which is toxic to living organisms, is split into water and free oxygen. Vegetation has a great influence on the catalase activity of mineral soils. As a rule, soils under plants with a powerful deep-penetrating root system are characterized by high catalase activity. A feature of catalase activity is that it changes little down the profile, has an inverse relationship with soil moisture and a direct relationship with temperature.

Polyphenol oxidase and peroxidase - they play an important role in the processes of humus formation in soils. Polyphenol oxidase catalyses the oxidation of polyphenols to quinones in the presence of free atmospheric oxygen. Peroxidase catalyzes the oxidation of polyphenols in the presence of hydrogen peroxide or organic peroxides. At the same time, its role is to activate peroxides, since they have a weak oxidizing effect on phenols. Further condensation of quinones with amino acids and peptides can occur with the formation of a primary humic acid molecule, which can further become more complex due to repeated condensations (Kononova, 1963).

It was noted (Chunderova, 1970) that the ratio of the activity of polyphenol oxidase (S) to the activity of peroxidase (D), expressed as a percentage (), is related to the accumulation of humus in soils, therefore this value is called the conditional coefficient of humus accumulation (K). In arable poorly cultivated soils of Udmurtia for the period from May to September, it was: in soddy-podzolic soil - 24%, in gray forest podzolized soil - 26% and in soddy-calcareous soil - 29%.

ENZYMATIVE PROCESSES IN SOILS

The biocatalytic activity of soils is in significant agreement with the degree of their enrichment with microorganisms (Table 11), depends on the type of soils, and varies across genetic horizons, which is associated with changes in the humus content, reaction, Red-Ox potential, and other indicators along the profile.

In virgin forest soils, the intensity of enzymatic reactions is mainly determined by the horizons of the forest litter, and in arable soils, by arable layers. Both in some and in other soils, all biologically less active genetic horizons located under the A or Ap horizons have a low activity of enzymes, which slightly changes in a positive direction when soils are cultivated. After the development of forest soils for arable land, the enzymatic activity of the formed arable horizon in comparison with the forest litter turns out to be sharply reduced, but as it is cultivated, it increases and, in highly cultivated species, approaches or exceeds that of the forest litter.

11. Comparison of biogenicity and enzymatic activity of soils in the Middle Cis-Urals (Pukhidskaya and Kovrigo, 1974)

The activity of biocatalytic reactions in soils changes. It is lowest in spring and autumn, and the highest is usually in July-August, which corresponds to the dynamics of the general course of biological processes in soils. However, depending on the type of soils and their geographical location, the dynamics of enzymatic processes is very different.

Control questions and tasks

1. What compounds are called enzymes? What are their production and significance for living organisms? 2. Name the sources of soil enzymes. What role do individual enzymes play in soil chemistry? 3. Give the concept of the enzymatic complex of soils and its functioning. 4. Give a general description of the course of enzymatic processes in virgin and arable soils.

Before discussing the properties of enzymes and the dependence of enzymes on any factors, it is necessary to define the concept enzyme activity.

In everyday biochemical practice, practically not estimated quantity enzyme, but only its activity. Activity is a broader concept than quantity. It means first of all reaction result, namely substrate loss or accumulationproduct. Naturally, this cannot be ignored. time, which worked the enzyme and number of molecules enzyme. But since the number of enzyme molecules is usually unrealistic to calculate, they use quantity biological material containing the enzyme (volume or mass).

Thus, when determining the activity of enzymes, it is necessary to simultaneously take into account three variables:

• weight the resulting product or the disappeared substrate,
• time spent on the reaction
• amount of enzyme, but actually the mass or volume of biological material containing the enzyme.

To understand the ratios of these factors, a clear and simple example can serve as the construction of two buildings. Building equate to the reaction product, workers are the enzymes brigade let it correspond to the volume of biological material. So, tasks from the 3rd grade:

1. A team of 10 people worked on the construction of one building, a team of 5 people worked on another building of the same building. The construction was completed simultaneously and in full. Where is the activity of workers higher?

2. A team of 10 people worked on the construction of one building of 3 floors, another building of 12 floors - a team of 10 people. The construction was completed simultaneously and in full. Where is the activity of workers higher?

3. A team of 10 people worked on the construction of one building of 5 floors, another of the same building - a team of 10 people. The construction of the first building took 20 days, the second was completed in 10 days. Where is the activity of workers higher?

Fundamentals of Enzyme Activity Quantification

1. Enzyme activity expressed in speed accumulation of the product or the rate of loss of the substrate in terms of amount of material containing an enzyme.

In practice, they usually use:

• units of quantity of a substance - mol (and its derivatives mmol, µmol), gram (kg, mg),
• units of time - minute, hour, second,
• units of mass or volume - gram (kg, mg), liter (ml).

Other derivatives are also actively used - catal (mol / s), the international unit of activity (IU, Unit) corresponds to μmol / min.

Thus, enzyme activity can be expressed, for example, in mmol/s×l, g/h×l, IU/l, cat/ml, etc.

For example, it is known

• that 1 g pepsin breaks down 50 kg of egg white in one hour - thus, its activity will be 50 kg / hour per 1 g of enzyme,
• if 1.6 ml of saliva breaks down 175 kg of starch per hour - activity salivary amylase will be 109.4 kg of starch per hour per 1 ml of saliva or 1.82 kg / min × g or 30.3 g of starch / s × ml.

2. Creation standard conditions, so that you can compare the results obtained in different laboratories - optimal pH and fixed temperature, for example, 25 ° C or 37 ° C, compliance with the time of incubation of the substrate with the enzyme.

The concept of enzymes

enzymes (enzymes) called soluble or membrane-bound proteins endowed with catalytic activity. ( In addition to proteins, some RNA (ribozymes) and antibodies (abzymes) can exhibit catalytic activity in the body, but they are thousands of times less effective than enzymes.) These names come from the Latin “fermentatio” - fermentation and the Greek “en zym” - inside the starter . They are reminiscent of the first sources of enzymes. Biochemistry, which studies enzymes, is called enzymology. In the diagrams and in the reaction equations, enzyme molecules are denoted by - E. Substances that are catalyzed by enzymes are called substrates (S). Products enzymatic reaction denote - R. Since enzymes are proteins, they are obtained in a homogeneous form in the same ways as other proteins. Enzymes are characterized by the physicochemical properties inherent in proteins.

The difference between enzymes and inorganic catalysts:

a) accelerate reactions much more efficiently;

b) endowed with high specificity of action;

c) are regulated under physiological conditions;

d) operate in mild conditions.

The structure of enzymes

Enzymes can be both simple and complex (conjugated) proteins, which may include lipids, carbohydrates, metal ions, nitrogenous bases, vitamin derivatives. In the body, enzymes can function both in a soluble state and in the form of insoluble complexes or be part of biological membranes.

The distinguishing feature of enzymes is the presence active center. Active Center - it is a unique combination of space-contiguous amino acid residues that provides:

a) recognition of the substrate molecule,

b) binding of the substrate to the enzyme,

c) implementation of the catalytic transformation (in the case of a complex enzyme, the coenzyme, which is part of the active center, also takes part in the act of catalysis).

An active site occurs when a protein folds and assumes its native (active) conformation. The structure of the active center may change upon interaction with the substrate. According to the figurative expression of D. Koshland, the substrate approaches the active center like a hand to a glove.

One enzyme molecule, especially if it consists of several subunits, may contain more than one active site.

There are two sites in the active center. The first site is responsible for the recognition and binding of the substrate. It is called the substrate-binding site or anchoring area. The second section is called catalytic, it consists of amino acid residues that take part in the act of catalysis.

Enzymes are proteins that vary greatly in molecular weight and structural complexity. An example of a small molecule enzyme is ribonuclease, which consists of a single subunit with a molecular weight of 13,700 Da. (The amino acid sequence of ribonuclease has been determined. In 1969, ribonuclease was synthesized in the laboratory of B. Merrifield in New York.) Many enzymes consist of several subunits, for example, lactate dehydrogenase consists of four subunits of two types. To date, several multienzyme complexes are known, consisting of dozens of different subunits and several types of coenzymes. For example, the pyruvate dehydrogenase complex consists of 60 subunits of three types and five types of cofactors. The molecular weight of such a complex is 2.3 * 10 6 - 10 * 10 6 Da, depending on the source of the enzyme. The enzyme molecule may be smaller than the substrate molecule. For example: the molecules of the enzymes amylase and ribonuclease are smaller than the molecules of their substrates - starch and RNA.

The protein part of complex enzymes is catalytically inactive and is called apoenzyme. The binding of an apoenzyme to a non-protein component leads to the formation of a catalytically active enzyme (holoenzyme):

Many enzymes contain a metal ion in their composition, which can perform various functions:

a) participate in the binding of the substrate and the process of its catalytic conversion;

b) promote the attachment of the coenzyme to the enzyme molecule;

c) stabilize the tertiary structure of the enzyme (eg Ca 2+ in amylase);

d) by binding to the substrate, form a true substrate, on which the enzyme acts.

Many coenzymes are derivatives of vitamins, so metabolic disorders in vitamin deficiency are due to a decrease in the activity of certain enzymes.

Some enzymes, in addition to the active site, contain allosteric (regulatory) center - a region of a protein globule, outside the active center, where substances that regulate enzymatic activity can bind. These substances are called allosteric effectors (allosteric activators or inhibitors). As a result of the binding of the effector to the allosteric center, a change in the protein structure occurs, leading to a change in the spatial arrangement of amino acid residues in the active center and, as a result, to a change in the enzymatic activity.

Enzymes that occur in the same organism and catalyze the same chemical reaction, but with a different primary protein structure, are called isoenzymes. Isoenzymes differ from each other in such physicochemical properties as molecular weight, thermal stability, substrate specificity, electrophoretic mobility. The nature of the appearance of isoenzymes is diverse, but most often due to differences in the structure of the genes encoding these isoenzymes or their subunits. For example, the enzyme lactate dehydrogenase (LDH), which catalyzes the reversible reaction of lactate oxidation to pyruvate, has four subunits of two types M and H, the combination of these subunits underlies the formation of five LDH isoenzymes (Fig. 1). To diagnose diseases of the heart and liver, it is necessary to study the isoenzyme spectrum of LDH in the blood serum, since LDH 1 and LDH 2 are active in the heart muscle and kidneys, and LDH 4 and LDH 5 are active in skeletal muscles and liver.

Fig.1 The structure of various LDH isoenzymes.

Measurement of enzymatic activity

Determination of enzyme activity is carried out by measuring the rate of catalyzed reactions. The rate of enzymatic reactions is measured by the decrease in the concentration of the substrate or the increase in the concentration of the product per unit of time:

v = -ΔС S /Δτ , v = ΔC P /Δτ ,

Where ∆С S is the change in the molar concentration of the substrate (mol/l),

∆C P- change in the molar concentration of the reaction product (mol / l),

Δτ - time change (min, sec).

It is desirable to carry out kinetic studies at a saturating concentration of the substrate; otherwise, the enzyme will not be able to exhibit maximum activity.

Units of enzyme activity:

Enzyme International Unit (U)- this is the amount of enzyme that catalyzes the conversion of 1 μmol of the substrate in 1 minute at a temperature of 25 ° C and the optimum pH of the medium.

In the SI system, the unit of an enzyme is rolled (kat)- this is the amount of enzyme that catalyzes the transformation of one mole of the substrate in 1 second. It is easy to calculate that:

1 U \u003d (1 * 10 -6 M) / 60 s \u003d 1.67 * 10 -8 M s-1 \u003d 1.67 * 10 -8 cat \u003d 16.7 ncat.

Often defined specific activity enzyme preparations by dividing the activity of the portion of the enzyme preparation, expressed in (U), by the weight of the portion in milligrams:

And beats \u003d U / mass of the drug (mg)

When enzymes are purified, the specific activity increases. By increasing the specific activity, one can judge the efficiency of the purification steps and the purity of the enzyme preparation.

To assess the activity of highly purified, homogeneous enzyme preparations by dividing the number of international units (U) of the enzyme in the sample by the amount of enzyme substance (µmol) in this sample, calculate molar activity(speed). In physical terms, molar activity is the number of substrate molecules that undergo transformation on one enzyme molecule in 1 minute or 1 second. For example: for urease, which accelerates the hydrolysis of urea, the molar activity is 30,000, for trypsin - 102, for glucose oxidase - 17,000 cycles per second.

Enzyme Properties

4.1. Mechanism of action. Enzymes do not shift the equilibrium of the catalyzed reactions towards the formation of products, thus, the equilibrium constant of the reaction remains constant. Like all catalysts, enzymes only reduce the time it takes to reach this equilibrium. In most cases, enzymes speed up reactions by 10 7 - 10 14 times. The effectiveness of enzymatic catalysis is based on a strong decrease in the activation energy of the reaction due to the transformation of the substrate into the product through transition states.

4.2. Specificity of action. The specificity of binding to the substrate and the pathways of the enzymatic reaction are determined by the apoenzyme. The specificity of the action of enzymes determines the directed metabolism in the body.

Enzymes are said to have narrow substrate specificity if they act on a very small range of substrates. Sometimes you can talk about absolute substrate specificity, for example, catalase catalyzes only one reaction - the decomposition of hydrogen peroxide:

For most enzymes, relative (broad, group) substrate specificity when they catalyze a group of similar reactions. For example, alcohol dehydrogenase catalyzes the conversion of alcohols to aldehydes, and methanol, ethanol, propanol, and other alcohols can act as substrates. It is interesting that alcohol dehydrogenase can also oxidize non-linear alcohols, as well as an alcohol group that is part of complex molecules; in particular, this enzyme can catalyze the conversion of retinol to retinal. Naturally, enzymes endowed with broad substrate specificity catalyze substrate transformations with varying efficiencies.

Enzymes are also endowed stereochemical specificity: their active site recognizes substrate molecules by their spatial configuration. For example, L-amino acid oxidases are active only on L-amino acids and have absolutely no effect on their D-analogues. For oxidative deamination of D-amino acids in living organisms, there are D-amino acid oxidases that do not act on L-amino acids. It is the ability of the active site to bind to certain stereoisomers of the substrate that underlies the functioning of such enzymes as racemases, which convert one stereoisomer to another.

Specificity of transformation pathways is that one substrate under the action of different enzymes can be converted into products that differ in structure and role in metabolism.

Here's an example: L-amino acid oxidases act on L-amino acids, converting them into alpha-keto acids with the formation of ammonia and hydrogen peroxide.

L-amino acid decarboxylases bind to the same substrates, but catalyze a different reaction: decarboxylation with the formation of biogenic amines and the release of carbon dioxide.

Another example is the possibility of converting glucose-6 phosphate under the action of various enzymes, along one of the possible metabolic pathways:

4.3. Thermolability .

Like many proteins, enzymes undergo thermal denaturation with increasing temperature, which leads to a violation of the native conformation of the enzyme and a change in the structure of the active site. Mammalian enzymes begin to noticeably denature at temperatures above 40°C.

In connection with the foregoing, it is desirable to store enzyme preparations at low temperatures. One of the best ways to preserve enzymes is to lyophilize them (dry at temperatures below -70° C. under vacuum), partially denature them with ammonium salts, and place them in a refrigerator.

4.4. The dependence of the reaction rate on temperature. The rate of enzymatic reactions, like any chemical reactions, depends on temperature. When the temperature rises by 10 o C, the reaction rate increases by 2-4 times according to the van't Hoff rule. However, at temperatures above 40°C, the denaturation of enzymes becomes significant, which leads to a decrease in the total activity (Fig. 2):

Rice. 2. Dependence of the enzymatic reaction rate on temperature.

4.5. The dependence of the reaction rate on pH. The dependence of the enzymatic reaction rate on pH has a bell-shaped form (Fig. 3). The pH values ​​at which the highest rate of the enzymatic reaction is observed are called optimal (pH-optimum). The nature of the curves and the value of the pH optimum depend on the nature of the charged groups of the substrate and the charged groups of the enzyme (especially those included in the active center). The pH optimum for most enzymes lies in the range from 6.0 to 8.0 (Fig. 3).

Rice. 3. Dependence of the enzymatic reaction rate on pH.

However, there are exceptions, for example, pepsin is most active at pH 1.5 - 2.0, and alkaline phosphatase at pH 10.0 - 10.5 (Fig. 4)

Rice. 4. Dependences of the enzymatic reaction rate (v) on the pH of the medium.

At extreme (very low or very high) pH values, the tertiary structure of the enzyme molecule is disturbed, leading to a loss of enzymatic activity.

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