Measurement of enzymatic activity. Metabolism and Enzymes

Concept of enzymes

Enzymes (enzymes) are 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 leaven . They are reminiscent of the first sources of enzymes. Biochemistry, which studies enzymes, is called enzymology. In diagrams and reaction equations, enzyme molecules are designated - E. Substances whose transformations are catalyzed by enzymes are called substrates (S). Products enzymatic reaction means - R. Since enzymes are proteins, they are obtained in a homogeneous form using the same methods as other proteins. Enzymes are characterized by physicochemical properties inherent in proteins.

Difference between enzymes and inorganic catalysts:

a) speed up reactions much more efficiently;

b) endowed with high specificity of action;

c) are subject to regulation under physiological conditions;

d) operate under mild conditions.

The structure of enzymes

Enzymes can be both simple and complex (conjugated) proteins, which may include lipids, carbohydrates, metal ions, nitrogenous bases, and 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.

A distinctive feature of enzymes is the presence active center. Active center - This is a unique combination of amino acid residues close in space, which provides:

a) recognition of the substrate molecule,

b) binding of the substrate to the enzyme,

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

The active site occurs when the 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 regions in the active center. The first region is responsible for recognition and binding of the substrate. It is called the substrate-binding site or anchoring site. The second section is called the catalytic section; it contains 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 that can perform various functions:

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

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

c) stabilize the tertiary structure of the enzyme (for example, Ca 2+ in amylase);

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

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

Some enzymes contain, along with an active center, 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 structure of the protein occurs, leading to a change in the spatial arrangement of amino acid residues in the active center and, ultimately, to a change in enzymatic activity.

Enzymes found in the same organism and catalyzing the same chemical reaction, but with different primary protein structures, are called isoenzymes. Isoenzymes differ from each other in such physicochemical properties as molecular weight, thermal stability, substrate specificity, and electrophoretic mobility. The nature of the appearance of isoenzymes is varied, 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 the liver.

Fig. 1 Structure of various LDH isoenzymes.

Enzyme activity measurement

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

v = -ΔС S /Δτ , v = ΔC P /Δτ ,

Where ΔС S– change in the molar concentration of the substrate (mol/l),

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

Δτ - time change (min, sec).

It is advisable to carry out kinetic studies at saturating concentrations of the substrate, otherwise the enzyme will not be able to exhibit maximum activity.

Enzyme activity units:

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

The SI unit of enzyme is rolled (kat)– this is the amount of enzyme that catalyzes the conversion of one mole of substrate in 1 second. It is easy to calculate that:

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

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

A beat = U/weight of the drug (mg)

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

To assess the activity of highly purified, homogeneous enzyme preparations, the number of international units (U) of the enzyme in the sample is divided by the amount of enzyme substance (μmol) in this sample, calculated molar activity(speed). In physical terms, molar activity is the number of substrate molecules undergoing 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, trypsin - 102, glucose oxidase - 17,000 cycles per second.

Properties of enzymes

4.1. Mechanism of action. Enzymes do not shift the equilibrium of 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 reduction in the activation energy of the reaction due to the conversion of the substrate into the product through transition states.

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

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

Most enzymes are characterized by relative (broad, group) substrate specificity when they catalyze a group of similar reactions. For example, alcohol dehydrogenase catalyzes the transformation of alcohols into aldehydes, and methanol, ethanol, propanol and other alcohols can act as substrates. An interesting fact is that alcohol dehydrogenase can oxidize nonlinear alcohols, as well as the 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 the transformation of substrates with varying efficiencies.

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

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 oxidase act on L-amino acids, converting them into alpha-keto acids with the formation of ammonia and hydrogen peroxide.

L-amino acid decarboxylase 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. Thermal lability .

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

In connection with the above, it is advisable to store enzyme preparations at low temperatures. One of the best ways to preserve enzymes is to lyophilize them (dry at temperatures below -70 o C in a vacuum), convert them to a partially denatured state using ammonium salts and place them in the refrigerator.

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

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

4.5. Dependence of reaction rate on pH. The dependence of the enzymatic reaction rate on pH is bell-shaped (Fig. 3). The pH values ​​at which the highest rate of 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 optimum pH for most enzymes lies in the range from 6.0 to 8.0 (Fig. 3).

Rice. 3. Dependence of the rate of enzymatic reaction 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. Dependence 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 disrupted, leading to loss of enzymatic activity.

Related information.

Concept of enzyme activity

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

Thus, when determining enzyme activity, three variables must be taken into account simultaneously:

• the mass of the resulting 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 relationships between these factors, a clear and simple example can be the construction of two buildings. Let's equate buildings to the reaction product, workers are enzymes, and let the team correspond to the volume of biological material. So, problems from 3rd grade:

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

Basics of Enzyme Activity Quantification

1. Enzyme activity is expressed in 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 - mole (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 so that results obtained in different laboratories can be compared - optimal pH and fixed temperature, for example, 25 ° C or 37 ° C, observing the incubation time of the substrate with the enzyme.

Enzyme activity. Under enzyme activity understand the amount of it that 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 substrate into a product in 1 minute under standard conditions (usually optimal). One skated denotes the amount of enzyme that catalyzes the conversion of 1 mole of substrate in 1 s (1 cat = 6 ∙ 10 7 IU). In the bimolecular reaction A + B = C + D, a unit of enzyme activity is taken to be the amount that catalyzes the conversion of 1 µmol of A or B, or 2 µmol of A (if B = A), in 1 minute.

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 (enzyme turnover number) - the number of substrate molecules that are converted by one enzyme molecule in 1 minute when the enzyme is completely saturated with the substrate. It is equal to the number of enzyme activity units 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 activity of the catalytic center . It is characterized by the number of substrate molecules that undergo transformation in 1 minute per active center.

The activity of enzymes is highly dependent on external conditions, among which temperature and pH of the environment are of paramount importance. An increase in temperature in the range of 0−50°C usually leads to a smooth increase in enzymatic activity, which is associated with the acceleration of the formation of the enzyme-substrate complex and all subsequent catalytic events. For every 10°C increase in temperature, the reaction rate 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 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 is characterized optimum pH environment, at which it exhibits maximum activity. As you move away from this value in one direction or another, enzymatic activity decreases. This is explained by a change in the state of the active center of the enzyme (a 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 a pH optimum in the neutral range. However, there are enzymes that exhibit maximum activity at pH 1.5 (pepsin) or 9.5 (arginase). When working with enzymes, it is necessary to maintain the pH using 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 an enzymatic reaction ( V max) and Michaelis constants K M. Enzyme 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 substrate or the increase of the resulting product per unit of time, or by a change in the concentration of one of the adjacent forms of coenzyme.

Influence enzyme concentration on the reaction rate is expressed as follows: if the substrate concentration is constant (provided there is an excess of substrate), then the reaction rate is proportional to the enzyme concentration. For kinetic studies, an enzyme concentration of 10 - 8 M active sites is used. The optimal value of the enzyme concentration is determined from a graph of the dependence of enzyme activity on its concentration. The optimal value is considered to lie on the plateau of the resulting graph in the range of enzyme activity values ​​that are slightly dependent on its concentration (Fig. 4.3).

Rice. 4.3. Dependence of the rate of enzymatic reaction

on enzyme concentration

To study the influence substrate concentration to determine the rate of an enzymatic reaction, first construct a kinetic curve reflecting the change in the concentration of the substrate (S 1) or product (P 1) over time (Fig. 4.4) and measure the initial speed ( V 1) reactions as the tangent of the angle of inclination of the tangent to the curve at the zero point.

Rice. 4.4. Kinetic curves of 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.) reactions, 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 an enzymatic reaction

on substrate concentration

The kinetics of many enzymatic reactions is described by the Michaelis–Menten equation. At 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. With respect to the substrate, the reaction is 1st order (the reaction rate is directly proportional to the concentration of one reactant) or 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 and 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 respect to the substrate, the reaction has 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. 1 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. 3

k

E + S ⇄ ES → E + P 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. Enzyme E combines with substrate S, forming the ES complex. The rate constant for this process is 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. 1 . The fate of the ES complex is twofold: it can either dissociate into enzyme E and substrate S with a rate constant 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. 2, or undergo further transformation, forming product P and free enzyme E, with a rate constant

3. It is postulated that the reaction product does not transform into the original substrate. This condition is met at the initial stage of the reaction, while the concentration of the product is low. The rate of catalysis is determined in inpatient conditions

, when the concentration of intermediate products remains constant, while the concentration of starting substances and final products varies. 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

(4.2)

, expressing the quantitative relationship between the rate of the enzymatic reaction and the concentration of the substrate, has the form This equation corresponds to a graph of reaction rate versus substrate concentration. At low substrate concentrations V = V, when [S] is much lower than K M, max [S] / K M, i.e. the reaction rate is directly proportional to the concentration of the substrate. At high substrate concentrations 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 (K M) and maximum reaction rate ( V max) are important speed characteristics at different substrate concentrations. V max – a constant value for each enzyme allows you to evaluate the effectiveness of its action.

The Michaelis constant shows the affinity of the substrate for the enzyme (in the case when 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. 2 >> 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. 3): the smaller KM, the greater the affinity and the higher the reaction rate, and vice versa. Each substrate is characterized by its own 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 graphical Michaelis–Menten relationship (Fig. 4.5) is ambiguous, they resort to linearization of this equation. To do this, it is converted into such a form that it can be expressed graphically as a straight line. There are several linearization methods, among which the Lineweaver–Burk and Edy–Hofstee methods are most often used.

Conversion Lineweaver–Burk looks like

(4.3)

Build a graph of dependence 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 the 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 equal to 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 inhibition of enzyme activity can also be gleaned from this graph.

Rice. 4.6. Linearization method for the Michaelis–Menten equation

(according to Lineweaver - Burke)

Method Edie–Hofstee 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 the 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 , and also identify possible deviations from linearity that were not detected in the previous graph.

Rice. 4.7. Linearization method for the Michaelis–Menten equation

(according to Edi – Hofstee)

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

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

Example competitive inhibition is the effect of structural analogues of the substrate, which can bind to the active center of the enzyme in a similar way as the substrate, without however 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 center 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.

Measuring reaction rates at different substrate concentrations allows one to distinguish competitive from non-competitive inhibition. With competitive inhibition on the dependence graph 1/ V=f(1/[S]) straight lines intersect the ordinate 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 angle of inclination of the straight line to the abscissa axis increases, i.e. V max does not change, but KM increases, indicating a decrease in the affinity of the substrate for the enzyme in the presence of an inhibitor (Fig. 4.8). Consequently, 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 – 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 tangent of the angle of inclination of the straight line to the x-axis increases by the amount (1 + [I]/ K i).

Rice. 4.8. Competitive inhibition:

a – diagram; b – graphic 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 center of the enzyme. This leads to a change in the conformation of the active center 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 inactive. The substrate and inhibitor can be simultaneously bound by the enzyme molecule, but their binding sites do not overlap. The effect of a non-competitive inhibitor is to reduce the number of enzyme turnovers, and not to reduce the proportion of enzyme molecules that bind the substrate. 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 ordinate axis will occur at a higher point (Fig. 4.9). The tangent of the angle of inclination of the straight line to the abscissa axis increases to the same extent, equal to K M / V maxI. K M in contrast to V max does not change, so noncompetitive inhibition cannot be eliminated by increasing the substrate concentration.

Rice. 4.9. Non-competitive inhibition:

a – diagram; b – graphic expression according to Lineweaver – Burke

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

(4.7)

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

Rice. 4.10. Non-competitive inhibition:

a – diagram; b – graphic expression according to Lineweaver – Burke

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

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

(activators - increase, inhibitors - decrease) Protein enzymes are synthesized on ribosomes, and RNA - in the nucleus.

The terms “enzyme” and “enzyme” have long been used as synonyms (the former mainly in Russian and German scientific literature, the latter in English and French).
The science of enzymes is called enzymology, and not enzymology (so as not to mix the roots of words in Latin and Greek).

History of the study

Term enzyme proposed in the 17th century by the chemist van Helmont when discussing the mechanisms of digestion.

In the end XVIII - early XIX centuries it was already known that meat is digested by gastric juice, and starch is converted into sugar under the action of saliva. However, the mechanism of these phenomena was unknown

Enzymes are widely used in the national economy - food, textile industries, and in pharmacology.

Classification of enzymes

Based on the type of reactions they catalyze, enzymes are divided into 6 classes according to the hierarchical classification of enzymes (CF, Enzyme Commission code). The classification was proposed by the International Union of Biochemistry and Molecular Biology. Each class contains subclasses, so that the enzyme is described by a set of four numbers separated by dots. For example, pepsin has the EU name 3.4.23.1. The first number roughly describes the mechanism of the reaction catalyzed by the enzyme:

• CF 1: Oxidoreductases, catalyzing oxidation or reduction. Example: catalase, alcohol dehydrogenase
• CF 2: Transferases, catalyzing the transfer of chemical groups from one substrate molecule to another. Among transferases, kinases that transfer a phosphate group, usually from an ATP molecule, are especially distinguished.
• CF 3: Hydrolases, catalyzing the hydrolysis of chemical bonds. Example: esterase, pepsin, trypsin, amylase, lipoprotein lipase
• CF 4: Lyases, catalyzing the breaking of chemical bonds without hydrolysis with the formation of a double bond in one of the products.
• CF 5: Isomerases, catalyzing structural or geometric changes in the substrate molecule.
• CF 6: Ligases, catalyzing the formation of chemical bonds between substrates due to ATP hydrolysis. Example: DNA polymerase

Kinetic studies

Saturation curve of a chemical reaction illustrating the relationship between substrate concentration [S] and reaction rate v

The simplest description of the kinetics of single-substrate enzymatic reactions is the Michaelis-Menten equation (see figure). To date, several mechanisms of enzyme action have been described. For example, the action of many enzymes is described by the ping-pong mechanism.

Structure and mechanism of action of enzymes

The activity of enzymes is determined by their three-dimensional structure.

Like all proteins, enzymes are synthesized in the form of a linear chain of amino acids, which folds in a certain way. Each sequence of amino acids folds in a special way, and the resulting molecule (protein globule) has unique properties. Several protein chains can be combined to form a protein complex. The tertiary structure of proteins is destroyed by heat or exposure to certain chemicals.

To catalyze a reaction, an enzyme must bind to one or more substrates. The protein chain of the enzyme folds in such a way that a gap, or depression, is formed on the surface of the globule where substrates bind. This region is called the substrate binding site. It usually coincides with or is close to the active site of the enzyme. Some enzymes also contain binding sites for cofactors or metal ions.

Some enzymes have small molecule binding sites and may be substrates or products of the metabolic pathway in which the enzyme enters. They decrease or increase the activity of the enzyme, which creates the opportunity for feedback.

The active centers of some enzymes are characterized by the phenomenon of cooperativity.

Specificity

Enzymes generally exhibit high specificity for their substrates. This is achieved by partial complementarity between the shape, charge distribution and hydrophobic regions on the substrate molecule and the substrate binding site on the enzyme. The enzymes exhibit high levels of stereospecificity, regioselectivity and chemoselectivity.

Key-lock model

Koshland's induced correspondence conjecture

A more realistic situation is in the case of induced correspondence. Wrong substrates - too big or too small - do not fit the active site

In 1890, Emil Fischer proposed that the specificity of enzymes is determined by the exact match between the shape of the enzyme and the substrate. This assumption is called the key-lock model. The enzyme combines with the substrate to form a short-lived enzyme-substrate complex. However, although this model explains the high specificity of enzymes, it does not explain the phenomenon of transition state stabilization that is observed in practice.

Induced correspondence model

In 1958, Daniel Koshland proposed a modification of the key-lock model. Enzymes are generally not rigid, but flexible molecules. The active site of an enzyme can change conformation upon binding of a substrate. The amino acid side groups of the active site assume a position that allows the enzyme to perform its catalytic function. In some cases, the substrate molecule also changes conformation after binding at the active site. Unlike the key-lock model, the induced-fit model explains not only the specificity of enzymes, but also the stabilization of the transition state.

Modifications

Many enzymes undergo modifications after the synthesis of the protein chain, without which the enzyme does not fully exhibit its activity. Such modifications are called post-translational modifications (processing). One of the most common types of modification is the addition of chemical groups to side residues of the polypeptide chain. For example, the addition of a phosphoric acid residue is called phosphorylation and is catalyzed by the enzyme kinase. Many eukaryotic enzymes are glycosylated, that is, modified by oligomers of carbohydrate nature.

Another common type of post-translational modification is cleavage of the polypeptide chain. For example, chymotrypsin (a protease involved in digestion) is obtained by cleaving a polypeptide region from chymotrypsinogen. Chymotrypsinogen is an inactive precursor to chymotrypsin and is synthesized in the pancreas. The inactive form is transported to the stomach, where it is converted to chymotrypsin. This mechanism is necessary in order to avoid the splitting of the pancreas and other tissues before the enzyme enters the stomach. The inactive enzyme precursor is also called a "zymogen".

Enzyme cofactors

Some enzymes perform the catalytic function on their own, without any additional components. However, there are enzymes that require non-protein components to carry out catalysis. Cofactors can be either inorganic molecules (metal ions, iron-sulfur clusters, etc.) or organic (for example,

1. Features of enzymatic reactions

2. Effect of temperature on enzyme activity

3. Effect of pH on enzyme activity

4. Enzyme activators and inhibitors

I. All enzymatic reactions have 4 features

· High enzyme activity;

· Reversibility of enzyme action;

· Specificity of enzyme action;

· Lability (sensitivity).

High enzyme activity. Enzymes cause a high rate of enzymatic reaction, which is characterized by the number of enzyme turnovers - this is the number of substrate molecules that are converted into reaction products under the action of one enzyme molecule per unit time. For example, alcohol dehydrogenase has an activity of 4700 units, phosphorylase - 50,000 units, a-amylase - 16,000 units.

Reversibility of enzyme action established by Danilevsky. The reversibility of enzyme action refers to the formation of the ES complex and its disintegration, i.e. reactions involving an enzyme can go both in one direction (biosynthesis) and in the opposite direction (decomposition).

Specificity of enzyme action- each enzyme acts only on its specific substrate or group of related substrates. For example, invertase acts on sucrose; a-amylase – only for starch and dextrins; proteases - to proteins.

There are two points of view explaining the specificity of enzyme action. According to E. Fischer’s figurative idea, “an enzyme approaches the substrate like a key to a lock,” i.e. The topography of the active site of the enzyme is not only highly ordered, but also rigidly fixed. The active site of an enzyme corresponds to the topography of only one single substrate. The second point of view, proposed by D. Koshland, is the theory of induced correspondence between enzyme and substrate: the conformation of the enzyme, especially its active center, is capable of certain modifications. Depending on the conformational mobility of the active site, the enzyme is able to interact with either few or a wide variety of substrates. In other words, at the moment of formation of the ES complex, changes occur in the structure of both the enzyme and the substrate. As a result, they adapt to each other.

The specificity of enzymes plays an important role in the metabolic process in a living organism. (If the enzyme did not have unique properties, then there would be no metabolism in a living organism)

Based on specificity, enzymes are divided into 2 groups:

· Absolute specificity - the enzyme acts only on one single substance or catalyzes only a certain transformation of this substance;

· Relative or group specificity - enzymes act simultaneously on many substrates that have a number of common structural properties.

Lability (sensitivity) – all enzymes are sensitive to increased temperature and low pH values, at which loss of enzyme activity occurs.

II. The most important factor on which enzyme activity depends is temperature.

Graphically, the dependence of the rate of an enzymatic reaction on temperature is as follows:

At 0°C, and even more so at temperatures below 0°C, the action of most enzymes stops. An increase in temperature (curve 1) above 0°C promotes an increase in enzyme activity (the number of collisions of reacting substances increases). At a certain temperature the enzyme exhibits maximum activity. For most enzymes, the optimal operating temperature is 40-50°C. A further increase in temperature leads to inactivation of enzymes (decreased activity) due to thermal denaturation of the protein molecule (curve 2).

The change in reaction rate with every 10°C increase in temperature is expressed by the temperature coefficient Q 10 . The temperature coefficient is the ratio of the reaction rate at a given temperature v t +10 to the reaction rate at a temperature 10 ° C below this:

The value of Q 10 for chemical reactions lies in the ranges 2-4, for an enzymatic reaction - between 1 and 2; Q 10 of enzymatic reactions decreases markedly with increasing temperature.

III. Each enzyme exerts its action within a fairly narrow pH zone. The graphical dependence of enzyme activity on pH has the form:

In an acidic environment, at low pH values, it has the form EH 2 +, in this form it is inactive. At optimum pH, the enzyme has maximum activity and is in the EH form; When the medium is alkalized, the enzyme takes on the E - form.

Optimal activity corresponds to a certain pH region, and each enzyme has its own optimal pH value for action (for example, bacterial a-amylase has a pH optimum at 6, and microscopic fungal a-amylase has a pH optimum of 4.7). The optimal pH value is related to the amino acid composition of enzymes.

The bell-shaped shape of the curve can be explained by the amphoteric nature of the enzymes; the ascending and descending branches of this curve are typical titration curves and are determined by the pK values ​​of the ionic groups that are located in the active site of the enzymes.

To determine the functional groups included in the active center of an enzyme, it is necessary to determine the dependence of the activity of this enzyme on pH at different temperatures and determine the pK value. Knowing the ΔpK values ​​for the acidic and alkaline branches of the v = f(pH) relationship, functional groups that correspond to this value are found.

IV. All substances accompanying the enzyme during the reaction can be divided into activators, inhibitors and neutral compounds.

Activators– chemical compounds that increase the action of enzymes (for example, glutathione activates the action of proteases, NaCl increases the activity of amylases); inhibitors– compounds that suppress their activity (for example, the –CN group suppresses the activity of respiratory enzymes located in the cytochrome system) and neutral compounds have no effect on enzymes.

The inhibition process can be reversible And irreversible.

Reversible inhibitors are:

· Competitive action - the inhibitor interacts with the functional groups of the active site of enzymes. Inhibition in this case depends on the concentration of the substrate: if [S] is high, then the influence of the inhibitor [I] may not appear; if [S] is small, then the inhibitor can displace the substrate from the connection with the enzyme, the action of which is inhibited. The ESI ternary complex is never formed during competitive inhibition.

· Non-competitive inhibition is observed when the inhibitor is not able to attach to the enzyme, but it cannot bind to the enzyme-substrate complex, converting it into an inactive form.

· In mixed inhibition, the inhibitor acts on both the ES binding site and the catalytic site of the enzyme.