The study of the suppression of enzyme activity is one of the ways to decipher the mechanism of their action. An approach to solving the latter problem is to study the specificity of the action of enzymes. In turn, this requires the correct measurement of kinetic parameters in the presence of the studied substrate analog. Consider ways to determine the nature of the relationship substrates, their analogues and inhibitors of enzymatic activity by calculating a number of kinetic parameters.
Moreover, if the dissociation constant of the complex K s = K m is equal to:
Inhibitors Enzymes can be divided into two main groups: reversible and irreversible. After the removal of the first type inhibitor, the activity of the enzyme is restored; in the second case, the inhibitor cannot be removed or the activity of the enzyme is not restored even after the removal of the inhibitor. Irreversible inhibition is maximized when the entire enzyme is bound to the inhibitor. Reversible inhibition reaches an equilibrium state, the position of which is determined by inhibition constant characterizing the affinity of the enzyme for the inhibitor. The reversible inhibition scheme is shown below:
In competitive inhibition, the substrate and inhibitor bind to the same active site of the enzyme. In the presence of an inhibitor, the affinity of the enzyme for the substrate decreases. The value does not change, since at a “saturating” concentration the substrate displaces the inhibitor from the complex with the enzyme.
At non-competitive inhibition the substrate and inhibitor bind to different sites of the enzyme. In this case, the value of K ha does not change, and the value of V max decreases.
Intermediate or alternative cases are also possible, for example, when the inhibitor binds not to the enzyme, but to the enzyme-substrate complex, as in the case uncompetitive inhibition, in which both kinetic parameters change.
To determine the type of inhibition, a Lineweaver-Burk plot is usually used, obtained for a given substrate in the absence and presence of an inhibitor.
In competitive inhibition, if the value of Kt is determined in the presence of an inhibitor, the inhibition constant can be calculated using the following formula:
With non-competitive inhibition, by determining the changed value of V, K can be calculated using the following formula:
All biochemical processes in the cell are interrelated and interdependent, however, some of them primarily perform the function of building cellular material, and some of them provide energy sources for these “construction works”. Therefore, it is customary to divide biochemical processes into two main types: assimilation, called anabolism, including the synthesis of low molecular weight precursors and the construction of biopolymer molecules from them, and dissimilation, called catabolism consisting in providing a source of energy, an "energy drive" that drives anabolism.
Let us consider the main mechanisms of energy transformation processes in the cell, i.e. mechanisms of catabolic processes.
Distinguish between reversible and irreversible inhibition. If the inhibitor causes persistent changes in the spatial tertiary structure of the enzyme molecule or modification of the functional groups of the enzyme, then this type of inhibition is called irreversible. More often, however, there is a reversible inhibition that can be quantitatively studied on the basis of the Michaelis-Menten equation. Reversible inhibition, in turn, is divided into competitive and non-competitive, depending on whether or not it is possible to overcome the inhibition of the enzymatic reaction by increasing the concentration of the substrate.
Competitive inhibition can be caused by substances that have a structure similar to the structure of the substrate, but slightly different from the structure of the true substrate. Such inhibition is based on the binding of the inhibitor to the substrate-binding (active) site. A classic example of this type of inhibition is the inhibition of succinate dehydrogenase (SDH) by malonic acid. This enzyme catalyses oxidation by dehydrogenating succinic acid (succinate) to fumaric acid:
If malonate (inhibitor) is added to the medium, then as a result of its structural similarity with the true substrate succinate (the presence of two of the same ionized carboxyl groups), it will interact with the active site to form an enzyme-inhibitor complex, however, the transfer of a hydrogen atom from malonate is completely excluded. . The structures of the substrate (succinate) and inhibitor (malonate) are somewhat different. Therefore, they compete for binding to the active site, and the degree of inhibition will be determined by the ratio of the concentrations of malonate and succinate, and not by the absolute concentration of the inhibitor. Thus, the inhibitor can be reversibly bound by the spherment, forming an enzyme-inhibitor complex. This type of inhibition is sometimes referred to as metabolic antagonism inhibition (Figure 4.20).
AT general form The interaction reaction of an inhibitor with an enzyme can be represented by the following equation:
The resulting complex, called the enzyme-inhibitor complex EI, unlike the enzyme-substrate complex ES, does not decompose with the formation of reaction products. The dissociation constant of the EI complex, or the inhibitory constant K i , can, following the Michaelis–Menten theory, be defined as the ratio of the reverse and direct reaction constants:
The method of competitive inhibition has found wide application in medical practice. It is known, for example, that sulfanilamide preparations are used to treat certain infectious diseases caused by bacteria. It turned out that these drugs have a structural similarity to para-aminobenzoic acid, which bacterial cell uses for the synthesis of folic acid, which is an integral part
Rice. 4.20. The action of a competitive inhibitor (scheme according to V.L. Kretovich). E - enzyme; S - substrate; R 1 and R 2 - reaction products; I - inhibitor.
bacterial enzymes. Due to this structural similarity, sulfanilamide blocks the action of the enzyme by displacing para-aminobenzoic acid from the complex with the enzyme that synthesizes folic acid, which leads to inhibition of bacterial growth.
Non-competitive inhibition is caused by substances that do not have structural similarities with substrates and often bind not to the active site, but to another place in the enzyme molecule. The degree of inhibition in many cases is determined by the duration of the action of the inhibitor on the enzyme. With this type of inhibition, due to the formation of a stable covalent bond the enzyme often undergoes complete inactivation, and then the inhibition becomes irreversible. An example of irreversible inhibition is the action of iodoacetate, DPP, as well as diethyl-p-nitrophenyl phosphate and hydrocyanic acid salts. This action consists in binding and switching off the functional groups or metal ions and the enzyme molecule.
limited proteolysis.
Regulation of activity by hormones.
Hormonal regulation is carried out at the genetic level by reversible phosphorylation. For example, under the action of adrenaline, the process of glycogen breakdown is activated. During this process, a non-protein compound, y-AMP, is formed. γ-AMP is an intracellular hormone (second messenger) that is an allosteric regulator of a large number of protein lipases. γ-AMP is formed from ATP by the action of adenylate cyclases.
Regulation of activity by chemical modification.
Chemical modification- the attachment of any functional groups to the enzyme, with a subsequent change in its activity. Chemical modification is reversible. For example, the key enzymes of energy metabolism - phosphorylase, glycogen synthase are controlled by phosphorylation and dephosphorylation, carried out by specific enzymes - proteinase and phosphotase. And the level of activity of key enzymes will be determined by the ratio of phosphorylated and dephosphorylated forms of these enzymes.
All enzymes of the gastrointestinal tract and pancreas are synthesized in an inactive form in the form of proenzymes. Regulation in this case is reduced to their transformation into an active form. For example, trypsinogen activation occurs under the action of enterokinase and leads to the cleavage of an excess amino acid sequence. In this case, the formation of the active center and the tertiary structure of trypsin occurs. This phenomenon has been named limited proteolysis . Its biological significance lies in the fact that it eliminates the self-digestion of the organ (autocatalysis), which, for example, occurs when trypsin is activated in the pancreas itself. Secondly, a finer regulation of the amount of the enzyme is provided.
Limited proteolysis is under the control of environmental factors, pH, in the cell - under the control of Ca.
The rate of an enzymatic reaction is determined by the presence of effectors in the medium: activators and inhibitors. Activators increase the rate of the reaction and sometimes modify it, while inhibitors slow it down.
Activators: coenzymes, Me ions, SH-reagents. The activating effect is associated with the optimization of the structure of the protein molecule and the active center of the enzyme. This improves the interaction between the enzyme and the substrate.
Pancreatic lipase activator - bile acids.
Trypsinogen activator - enterokinase.
Hematrypsinogen activator - trypsin.
Pepsin and amylase activator - Ca ions.
Me can also act as activators:
Zn is an activator of carbonic anhydrase.
Inhibitors It is customary to call substances that cause partial or complete inhibition of the reaction.
Any agents that cause enzyme denaturation are inhibitors. However, such inhibition is nonspecific because it is not related to the mechanism of enzyme action. There are many more specific inhibitors that act on one particular enzyme or on a group of related enzymes. Such inhibitors can provide valuable information about the nature of the enzyme's active site. The mechanism of action of many toxins and poisons on the body is based on the inhibition of enzymes. So, in case of hydrocyanic acid poisoning, spasm occurs due to complete inhibition of respiratory enzymes (cytochrome oxidase).
Types of inhibition:
1) Reversible
2) Irreversible
If an inhibitor molecule causes persistent changes or modification of the active site of the enzyme, then such type of inhibition called irreversible .
Reversible inhibition is more common and is divided into competitive and non-competitive, depending on whether or not it is possible to overcome the inhibition of the enzymatic reaction by increasing (S). Competitive inhibition is possible if there is a structural similarity between the substrate and the inhibitor. For example, inhibition of succinate dehydrogenase activity by malonic acid:
NOOS - 2N NOOS
CH -------- CH
CH SDG CH
NOOS NOOS
succinate fumarate
If malonate is introduced into the medium instead of succinate, then due to its structural similarity to succinate, it will react with the active center of SDH. However, in this case, 2H transfer from malonate does not occur, since the structures of malonate and succinate are still somewhat different and they will compete for binding to the SDH active site, and the degree of inhibition will be determined by the ratio of malonate and succinate concentrations. A feature of this inhibition is reversibility due to an increase in (S).
I(+) E + I ------ EI
Partially non-competitive inhibition often occurs, in which a decrease in Vmax is combined with an increase in Km. In rare cases, the degree of inhibition of enzyme activity may increase with increasing (S). This so-called uncompetitive inhibition . In this case, it is possible to combine the inhibitor with the ES complex, therefore, an inactive or slowly reacting complex is formed.
ES+I------ESI
The action of many drugs is based on all these methods of inhibition. For example, sulfa drugs are used to treat certain infections that are structurally similar to PABA, which the bacterial cell uses as a substrate for the synthesis of folic acid. Due to the similarity, sulfanilamide blocks the action of the enzyme by displacing PABA from the ES complex, which leads to a decrease in bacterial growth. This is competitive inhibition .
All biochemical reactions occurring in the body are subject to specific control, which is carried out through an activating or inhibitory effect on regulatory enzymes. The latter are usually at the beginning of chains of metabolic transformations and either start a multi-stage process or slow it down. Some single reactions are also subject to regulation. Competitive inhibition is one of the main mechanisms for controlling the catalytic activity of enzymes.
The mechanism of enzymatic catalysis is based on the binding of the active site of the enzyme to the substrate molecule (ES complex), resulting in chemical reaction with the formation and release of the product (E+S = ES = EP = E+P).
Enzyme inhibition is a reduction in the rate or complete stop of the catalysis process. In a narrower sense, this term means a decrease in the affinity of the active center for the substrate, which is achieved by binding enzyme molecules to inhibitor substances. The latter can act in various ways, on the basis of which they are divided into several types, which correspond to the inhibition mechanisms of the same name.
Main types of inhibition
According to the nature of the process, inhibition can be of two types:
- Irreversible - causes persistent changes in the enzyme molecule, depriving it of functional activity (the latter cannot be restored). It can be either specific or non-specific. The inhibitor binds strongly to the enzyme by covalent interaction.
- Reversible is the main type of negative regulation of enzymes. It is carried out due to the reversible specific attachment of the inhibitor to the protein-enzyme by weak non-covalent bonds, amenable to kinetic description according to the Michaelis-Menten equation (with the exception of allosteric regulation).
There are two main types of reversible enzyme inhibition: competitive (can be weakened by increasing the concentration of the substrate) and non-competitive. In the latter case, the maximum possible rate of catalysis decreases.
The main difference between competitive and non-competitive inhibition lies in the site of attachment of the regulatory substance to the enzyme. In the first case, the inhibitor binds directly to the active site, and in the second, to another site of the enzyme, or to the enzyme-substrate complex.
There is also a mixed type of inhibition, in which binding to an inhibitor does not prevent the formation of ES, but slows down catalysis. In this case, the regulator substance is in the composition of double or triple complexes (EI and EIS). In the uncompetitive type, the enzyme only binds to ES.
Features of reversible competitive inhibition of enzymes
The competitive mechanism of inhibition is based on the structural similarity of the regulatory substance with the substrate. As a result, a complex of the active site with the inhibitor is formed, conventionally designated as EI.
Reversible competitive inhibition has the following features:
- binding to the inhibitor occurs at the active site;
- inactivation of the enzyme molecule is reversible;
- the inhibitory effect can be reduced by increasing the concentration of the substrate;
- the inhibitor does not affect the maximum rate of enzymatic catalysis;
- the EI complex can decompose, which is characterized by a corresponding dissociation constant.
With this type of regulation, the inhibitor and the substrate seem to compete (compete) with each other for a place in the active center, from where the name of the process comes from.
As a result, competitive inhibition can be defined as a reversible process of inhibition of enzymatic catalysis based on the specific affinity of the active site for the inhibitor substance.
Mechanism of action
The binding of the inhibitor to the active site prevents the formation of the enzyme-substrate complex required for catalysis. As a result, the enzyme molecule becomes inactive. Nevertheless, the catalytic center can bind not only to the inhibitor, but also to the substrate. The probability of formation of one or another complex depends on the ratio of concentrations. If there are significantly more substrate molecules, then the enzyme will react with them more often than with the inhibitor.
Influence on the rate of a chemical reaction
The degree of inhibition of catalysis during competitive inhibition is determined by the amount of enzyme that will form EI complexes. In this case, it is possible to increase the concentration of the substrate to such an extent that the role of the inhibitor will be replaced, and the catalysis rate will reach the maximum possible value corresponding to the value of V max according to the Michaelis-Menten equation.
This phenomenon is explained by a strong dilution of the inhibitor. As a result, the probability of enzyme molecules binding to it is reduced to zero, and active centers react only with the substrate.
Kinetic dependences of the enzymatic reaction with the participation of a competitive inhibitor
Competitive inhibition increases the Michaelis constant (Km), which is equal to the substrate concentration required to achieve ½ of the maximum rate of catalysis at the start of the reaction. The amount of the enzyme hypothetically able to bind to the substrate remains constant, while the number of actually formed ES complexes depends only on the concentration of the latter (EI complexes are not constant and can be displaced by the substrate).
Competitive inhibition of enzymes can be easily determined from kinetic dependence plots plotted for different substrate concentrations. In this case, the value of K m will change, and V max will remain constant.
In noncompetitive inhibition, the opposite is true: the inhibitor binds outside the active site, and the presence of the substrate cannot affect this in any way. As a result, some of the enzyme molecules are “turned off” from catalysis, and the maximum possible rate decreases. Nevertheless, active enzyme molecules can easily bind to the substrate both at low and at high concentrations of the latter. Therefore, the Michaelis constant remains constant.
Graphs of competitive inhibition in the system of double inverse coordinates are several straight lines intersecting the y-axis at the point 1/V max . Each straight line corresponds to a certain concentration of the substrate. Different points of intersection with the abscissa axis (1/[S]) indicate a change in the Michaelis constant.
The action of a competitive inhibitor on the example of malonate
A typical example of competitive inhibition is the process of reducing the activity of succinate dehydrogenase, an enzyme that catalyzes the oxidation of succinic acid (succinate) to fumaric acid. The inhibitor here is malonate, which is structurally similar to succinate.
The addition of an inhibitor to the medium causes the formation of complexes of malonate with succinate dehydrogenase. Such a bond does not cause damage to the active site, but blocks its accessibility to succinic acid. Increasing the concentration of succinate reduces the inhibitory effect.
Use in medicine
The mechanism of competitive inhibition is the basis for the action of many drugs, which are structural analogues of the substrates of some metabolic pathways, the inhibition of which is a necessary part of the treatment of diseases.
For example, to improve the conduction of nerve impulses in muscular dystrophies, it is required to increase the level of acetylcholine. This is achieved by inhibiting the activity of its hydrolyzing acetylcholinesterase. Quaternary ammonium bases that are part of drugs (proresin, endrophonium, etc.) act as inhibitors.
In a special group, antimetabolites are distinguished, which, in addition to the inhibitory effect, exhibit the properties of a pseudosubstrate. In this case, the formation of the EI complex leads to the formation of a biologically inert anomalous product. Antimetabolites include sulfonamides (used in the treatment of bacterial infections), nucleotide analogs (used to stop the cell growth of a cancerous tumor), etc.
Competitive Inhibition: Definition, Features, and Examples - All About Site Travel
Under certain conditions, the inhibitor can be easily separated from the enzyme.
Competitive reversible inhibition
In this case, a substance that is similar in structure to the usual substrate of the enzyme combines with the active site of the enzyme, but cannot react with it. Being here, it blocks access to the active center of any molecule of the real substrate. Since in this case the inhibitor and substrate compete for a place on the active site of the enzyme, this form of inhibition is called competitive inhibition. It is reversible, since the reaction rate increases with increasing substrate concentration.
6.4. Why does the reaction rate increase under these conditions?
Rice. 6.15 illustrates one example of competitive inhibition.
The phenomenon of competitive inhibition is used in chemotherapy. The goal of chemotherapy is to destroy the causative agent of the disease with the help of certain chemicals without damaging the tissues of the host organism. During the Second World War, they were widely used to combat infectious diseases. sulfa drugs, or sulfonamides, - derivatives of sulfanilic acid. Sulfonamides in their chemical structure are close to para-aminobenzoic acid (PABA) - an essential growth factor for many pathogenic bacteria. PABA is required by bacteria for the synthesis of folic acid, which serves as their enzyme cofactor. The action of sulfonamides is associated with a violation of the synthesis of folic acid from PABA.
Animal cells are not sensitive to sulfonamides, although they require folic acid for some reactions. This is explained by the fact that they use converted folic acid; the metabolic pathway that would provide its synthesis is absent in animals.
Noncompetitive reversible inhibition
Inhibitors of this genus are not structurally related to the substrate of this enzyme; in this case, it is not the active center of the enzyme that participates in the formation of the complex with the inhibitor, but some other part of its molecule (Fig. 6.16). The formation of the complex entails a change in the globular structure of the enzyme, and although the real substrate is nevertheless attached to the enzyme, catalysis nevertheless turns out to be impossible. An example is cyanide. It binds to metal ions, which act as a prosthetic group in some enzymes (in particular, to copper ions of cytochrome oxidase), and inhibits the activity of these enzymes. As the concentration of the inhibitor increases, the rate of the enzymatic reaction decreases more and more. By the moment of saturation with the inhibitor, it turns out to be practically equal to zero.
