Levels of regulation of enzyme activity. Regulation of enzyme activity and their methods Molecular mechanisms of regulation of enzyme activity
Being a unit of living matter, functioning as a complex of open biological systems, the cell constantly exchanges substances and energy with the external environment. To maintain homeostasis, there is a group of special protein substances - enzymes. The structure, functions, and regulation of enzyme activity are studied by a special branch of biochemistry called enzymology. In this article, using specific examples, we will consider various mechanisms and methods of regulating enzyme activity inherent in higher mammals and humans.
Conditions required for optimal enzyme activity
Biologically active substances that selectively influence both assimilation and degradation reactions exhibit their catalytic properties in cells under certain conditions. For example, it is important to find out in which part of the cell the chemical process in which enzymes participate occurs. Thanks to compartmentation (division of the cytoplasm into sections), antagonistic reactions occur in its various parts and organelles.
Thus, protein synthesis occurs in ribosomes, and their breakdown occurs in the hyaloplasm. Cellular regulation of the activity of enzymes that catalyze opposite biochemical reactions ensures not only the optimal rate of metabolism, but also prevents the formation of energetically useless metabolic pathways.
Multienzyme complex
The structural and functional organization of enzymes forms the enzymatic apparatus of the cell. Most of the chemical reactions occurring in it are interconnected. If in a multistage reaction the product of the first reaction is a reagent for the subsequent one, in this case the spatial arrangement of enzymes in the cell is especially pronounced.
It must be remembered that enzymes are either simple or complex proteins in nature. And their sensitivity to the cellular substrate is explained primarily by changes in the intrinsic spatial configuration of the tertiary or quaternary structure of the peptide. Enzymes also respond to changes not only within cellular parameters, such as the chemical composition of the hyaloplasm, the concentration of reagents and reaction products, temperature, but also to changes occurring in neighboring cells or in the intercellular fluid.
Why is the cell divided into compartments?
The rationality and logic of the structure of living nature is simply amazing. This fully applies to the vital manifestations characteristic of the cell. For a chemical scientist, it is completely clear that multidirectional enzymatic chemical reactions, for example, glucose synthesis and glycolysis, cannot occur in the same test tube. How then do opposite reactions occur in the hyaloplasm of one cell, which is the substrate for their implementation? It turns out that the cellular contents - the cytosol - in which antagonistic chemical processes take place, are spatially separated and form isolated loci - compartments. Thanks to them, the metabolic reactions of higher mammals and humans are regulated especially precisely, and metabolic products are converted into forms that freely penetrate through the partitions of cellular areas. Then they restore their original structure. In addition to the cytosol, enzymes are contained in organelles: ribosomes, mitochondria, nucleus, lysosomes.
The role of enzymes in energy metabolism
Let us consider the oxidative decarboxylation of pyruvate. The regulation of the catalytic activity of enzymes in it has been well studied by enzymology. This biochemical process occurs in mitochondria - double-membrane organelles of eukaryotic cells - and is an intermediate process between the oxygen-free breakdown of glucose and the pyruvate dehydrogenase complex - PDH - contains three enzymes. In higher mammals and humans, its decrease occurs with an increase in the concentration of Acetyl-CoA and NATH, that is, in the case of the appearance of alternative possibilities for the formation of Acetyl-CoA molecules. If the cell needs an additional portion of energy and requires new acceptor molecules to enhance the reactions of the tricarboxylic acid cycle, then the enzymes are activated.
What is allosteric inhibition
Regulation of enzyme activity can be carried out by special substances - catalytic inhibitors. They can covalently bind to certain loci of the enzyme, bypassing its active center. This leads to deformation of the spatial structure of the catalyst and automatically entails a decrease in its enzymatic properties. In other words, allosteric regulation of enzyme activity occurs. Let us also add that this form of catalytic action is inherent in oligomeric enzymes, that is, those whose molecules consist of two or more polymeric protein subunits. The PDH complex discussed in the previous heading contains precisely three oligomeric enzymes: pyruvate dehydrogenase, dehydrolipoyl dehydrogenase and hydrolipoyl transacetylase.
Regulatory enzymes
Research in enzymology has established the fact that it depends on both the concentration and the activity of the catalyst. Most often, metabolic pathways contain main enzymes that regulate all of their sections.
They are called regulatory and usually affect the initial reactions of the complex, and can also participate in the slowest chemical processes in irreversible reactions, or join reagents at branch points in the metabolic pathway.
How does peptide interaction occur?
One of the ways in which the activity of enzymes in cells is regulated is through protein-protein interaction. What are we talking about? Regulatory proteins are attached to the enzyme molecule, resulting in their activation. For example, the enzyme adenylate cyclase is located on the inner surface of the cell membrane and can interact with structures such as the hormone receptor, as well as with a peptide located between it and the enzyme. Since, as a result of the connection of the hormone and the receptor, the intermediate protein changes its spatial confirmation, this method of enhancing the catalytic properties of adenylate cyclase in biochemistry is called “activation due to the attachment of regulatory proteins.”
Protomers and their role in biochemistry
This group of substances, otherwise called protein kinases, accelerate the transfer of the PO 4 3- anion to the hydroxo groups of amino acids included in the peptide macromolecule. We will consider the regulation of the activity of protomer enzymes using the example of protein kinase A. Its molecule, a tetramer, consists of two catalytic and two regulatory peptide subunits and does not function as a catalyst until four cAMP molecules are attached to the regulatory regions of the protomer. This causes a transformation of the spatial structure of the regulatory proteins, which leads to the release of two activated catalytic protein particles, that is, dissociation of protomers occurs. If cAMP molecules are separated from the regulatory subunits, then the inactive protein kinase complex is again restored to a tetramer, as the association of catalytic and regulatory peptide particles occurs. Thus, the pathways discussed above for regulating enzyme activity ensure their reversible nature.
Chemical regulation of enzyme activity
Biochemistry has also studied mechanisms for regulating enzyme activity, such as phosphorylation and dephosphorylation. The mechanism for regulating enzyme activity in this case is as follows: amino acid residues of the enzyme containing OH - groups change their chemical modification due to the influence of phosphoprotein phosphatases on them. In this case, correction can be done, and for some enzymes this is the reason that activates them, and for others it inhibits them. In turn, the catalytic properties of phosphoprotein phosphatases themselves are regulated by the hormone. For example, animal starch - glycogen - and fat in the intervals between meals are broken down in the gastrointestinal tract, more precisely, in the duodenum under the influence of glucagon - a pancreatic enzyme.
This process is enhanced by phosphorylation of trophic enzymes in the gastrointestinal tract. During the period of active digestion, when food enters the duodenum from the stomach, glucagon synthesis increases. Insulin, another pancreatic enzyme produced by the alpha cells of the islets of Langerhans, interacts with the receptor, including the mechanism of phosphorylation of the same digestive enzymes.
Partial proteolysis
As we can see, the levels of regulation of enzyme activity in the cell are varied. For enzymes located outside the cytosol or organelles (in the blood plasma or in the gastrointestinal tract), the method of their activation is the process of hydrolysis of CO-NH peptide bonds. It is necessary because such enzymes are synthesized in an inactive form. The peptide part is split off from the enzyme molecule, and the active center in the remaining structure is modified. This leads to the fact that the enzyme “enters a working state,” that is, it becomes capable of influencing the course of a chemical process. For example, the inactive pancreatic enzyme trypsinogen does not break down food proteins entering the duodenum. Proteolysis occurs in it under the action of enteropeptidase. After which the enzyme is activated and is now called trypsin. Partial proteolysis is a reversible process. It occurs in cases such as the activation of enzymes that break down polypeptides in blood clotting processes.
The role of the concentration of starting substances in cell metabolism
The regulation of enzyme activity by substrate availability was partially discussed by us in the subtitle “Multi-enzyme complex”. The rate at which it occurs in several stages strongly depends on how many molecules of the original substance are in the hyaloplasm or organelles of the cell. This is due to the fact that the speed of the metabolic pathway is directly proportional to the concentration of the starting substance. The more reagent molecules are in the cytosol, the higher the rate of all subsequent chemical reactions.
Allosteric regulation
Enzymes, the activity of which is controlled not only by the concentration of initial reagent substances, but also by effector substances, are characterized by the so-called. Most often, such enzymes are represented by intermediate metabolic products in the cell. Thanks to effectors, enzyme activity is regulated. Biochemistry has proven that such compounds, called allosteric enzymes, are very important for cell metabolism, as they are extremely sensitive to changes in its homeostasis. If an enzyme inhibits a chemical reaction, that is, reduces its speed, it is called a negative effector (inhibitor). In the opposite case, when an increase in the reaction rate is observed, we are talking about an activator - a positive effector. In most cases, starting substances, that is, reagents that enter into chemical interactions, play the role of activators. The final products formed as a result of multi-stage reactions behave as inhibitors. This type of regulation, based on the relationship between the concentrations of reagents and products, is called heterotrophic.
1. The ability to regulate makes enzymes importantsignificant participants and original organizerscellular processes in the human body. Regulation of the rate of enzymatic reactions in the cell is the main mechanism not only for the control and coordination of metabolic pathways, but also for the growth and development of the cell, as well as its response to environmental changes.
2. There are two main ways to control the rate of enzymatic reactions:
— Controlling the amount of enzyme.
The amount of enzyme in the cell is determined the ratio of the rates of its synthesis and decay. This method of regulating the rate of an enzymatic reaction is a slower process (appears after a few hours) than regulating enzyme activity (an almost instantaneous response).
— Enzyme activity control.
Enzyme activity can be regulated by interaction with certain substances that change the conformation of the active center.
3. Enzymes that regulate the rate of metabolic pathways:
— usually act in the early stages of metabolic pathways, in places of key branches of metabolic pathways;
- catalyze practically irreversible reactions under cellular conditions that proceed most slowly (key ones).
Example 1. Feedback regulation: in multi-stage metabolic pathways, the final product inhibits the regulatory (key) enzyme of the process.
The first enzyme (Ej) in the sequential pathway for converting substance A into substance Z is usually inhibited by the end product of this metabolic pathway.
Change in key enzyme activity E 1 occurs as a result of a change in conformation after binding of substance Z in allostericskom center- area remote from the active center. EnzymeE 1 allosteric.
Feedback regulation occurs relatively quickly, and is often the cell's first response to changing conditions.
On the other hand, the enzyme E x will be active when the concentration of the substance decreasesZ.
4. The main types of regulation of the catalytic activity of enzymes in the cell and structural changes in enzymes during their activation are presented in Table. 2.3.
5. Impaired enzyme synthesis can lead toenzymopathies, in which the lack of one enzyme in the metabolic pathway can cause a disruption in the formation of the final product. Due to the interdependence of metabolic pathways, a defect in one enzyme often leads to a number of metabolic disorders:
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There is a possibility that the excess accumulated substrate may pass into a side metabolic pathway with the formation of an unusual and often toxic substance Bj.
6. Individual examples of enzymopathies (disaccharidoses, glycogenoses, aglycogenoses, phenylpyruvic oligophrenia) will be considered when studying the following sections.
Regulation of enzyme activity.
Activation mechanisms.
1) Covalent modification (that is, the covalent bonds of enzymes change)
a) partial proteolysis (pepsinogen and trypsinogen can be affected not only by hydrochloric acid and enterokinase, but also by active enzymes - pepsin and trypsin, respectively, that is, autocatalysis occurs).
b) phosphorylation and dephosphorylation. Phosphorylation is carried out by protein kinases.
2) Completion of the active center (most often these are metal ions, especially manganese, but in some cases the metal combines with sulfur, and then the sulfur interacts more easily with the active center).
3) Allosteric activation. As a rule, the effect occurs on the subunit where there is no active center (that is, this is more often typical for oligomers), but this subunit has a regulatory site that can be affected by some metabolite (for example, ADP), and the subunit changes its structure , at the same time changing the structure of the subunit containing the active center, thereby making it more accessible to the substrate. As a rule, allosteric activation and inhibition are self-regulatory processes, when intermediate or final metabolites regulate the reaction rate.
Structural organization of the enzyme in the cell .
Each cellular structure has a specific set of enzymes that allows it to perform a specific function. For example, mitochondria are equipped with enzymes that can oxidize certain substrates and utilize the resulting energy. Nuclei (they contain the synthesis of DNA and RNA, which are capable of storing and transmitting hereditary information) and also have a specific set of enzymes (RNA and DNA polymerases, etc.). Lysosomes (they destroy various complex compounds) also have a corresponding set of enzymes (hydrolases, lyases, etc.).
All these sets of enzymes are strictly structured, that is, they are built, for example, into the mitochondrial membrane (respiratory chain) in a certain order and are in a complex (for example, a complex that ensures the synthesis of fatty acids; a complex that promotes the conversion of pyruvic acid) sometimes they even talk about indicator (marker) enzymes for cellular structures (succinate dehydrogenase for mitochondria, RNA polymerase for the nucleus, acid phosphatase for lysosomes).
During the metabolic process, enzyme activity is constantly regulated, that is, the enzyme never works monotonously. There are different ways to regulate enzyme activity:
1) the amount of the enzyme may change (that is, either the synthesis of the enzyme increases or decreases). This occurs due to changes in gene expression.
2) The chemical modification of the enzyme may change (under the influence of activators, inhibitors, or changes in pH). These are partial proteolysis, phosphorylation and dephosphorylation, sulfonation, etc.
3) The activity of enzymes changes under the action of hormones (various mechanisms).
4) The activity of the enzyme can be influenced by the substrate itself or the reaction product (being either an activator or an inhibitor).
5) The phenomenon of compartmentalization is also observed in cells, that is, with the help of biological membranes, enzymes and those substrates that these enzymes could destroy, but the cell does not need this (for example, enzymes of lysosome proteinases, phosphatases, etc.) are separated. dens from substances located in the cytoplasm) Or metabolic processes that are mutually incompatible at the same time are separated using membranes (for example, the synthesis of fatty acids occurs in the cytoplasm, and the breakdown of fatty acids in mitochondria). Not all enzymes are subject to regulation. But in the chain of enzymatic reactions there are key enzymes that are activated or inhibited.
Principles of enzyme isolation .
To detect enzymes, their specificity property is used. They take a certain (specific) substrate, select optimal conditions (pH, temperature) and add an enzyme to see if the reaction occurs, while the concentration of the substrate decreases and the formation of the product increases. A quantitative assessment of enzymes is given by their activity (since enzymes are contained in negligible quantities), that is, the rate of the enzymatic reaction is determined. Enzyme activity is determined at a constant temperature (25 or 37 degrees Celsius), creating an optimum pH. In this case, the substrate concentration must be quite high. Under these conditions, the reaction rate directly depends on the enzyme concentration \/ = K[F]. A unit of enzyme activity is taken to be the minimum amount of enzyme that, under optimal conditions, causes the conversion of one micromole of substrate in one minute.
Specific activity is enzymatic activity per mg of protein. According to the recommendations of the Commission of the International Biochemical Union on the nomenclature of enzymes, it is proposed to use katal to express enzymatic activity. 1 catal - ϶ᴛᴏ catalytic activity, capable of carrying out a reaction at a rate equal to one mole per second.
Regulation of enzyme activity. - concept and types. Classification and features of the category "Regulation of enzyme activity." 2017, 2018.
Regulation of enzymatic activity is a process no less important for the successful functioning of a cell than the regulation of gene expression at the transcription level. The existence of these mechanisms allows cells and the entire body to clearly coordinate the implementation of numerous branched metabolic reactions, ensuring the highest and most economical level of metabolism, as well as rapid adaptation to changing environmental conditions. In this case, the regulation of enzyme synthesis is a slower mechanism, acting over many minutes or even hours, while the change in enzymatic activity occurs instantly and acts within a few minutes or seconds. Regulation of enzyme activity can be called “fine tuning” of cellular metabolism.
Regulation of enzymatic activity can be carried out in several ways, among which the most common are allosteric regulation And covalent modification.
Not all enzymes are subject to allosteric regulation, but only those that have an allosteric (from the Greek allos - other and stereos - body, space) center in the molecule - a site that differs from the active center, characterized by high affinity for regulatory molecules.
Such enzymes are called allosteric. Their activity is regulated with the participation of low molecular weight substances ( effectors), the common property of which is the ability to interact with the allosteric center, which leads to distortion of the conformation of the protein molecule. This distortion is transmitted to the active site, resulting in changes in the activity of the enzyme and the rate of the corresponding reaction.
Effectors can act as both inhibitors of enzyme activity and their activators. Example inhibition Enzymatic activity may be due to a decrease in the activity of the first enzyme in the tryptophan biosynthesis pathway in E. coli, anthranilate synthetase, when there is an excess of tryptophan in the cell. In this case, tryptophan, as the final product of the named biosynthetic pathway, serves as an inhibitor of the activity of the key enzyme, which coordinates the rate of synthesis of this amino acid and allows the cell to save its resources. Indeed, with an excess of tryptophan, for example, when it is present in the growth medium, the cell does not need to spend building blocks and energy on its synthesis; it can use an exogenous amino acid. Indeed, it has been experimentally proven that during the growth process, bacteria preferentially use amino acids, purines and pyrimidines added to the growth medium and that these compounds have an inhibitory effect on their own synthesis from precursor molecules. Since in this case tryptophan is the end product of the biosynthetic pathway, the rate of which decreases when the key enzyme is inhibited, this type of regulation is called “ retroinhibition».
An increase in the activity of an allosteric enzyme upon binding to an effector (activator) can be considered using the example of aspartate transcarbamoylase (ATKase), which catalyzes the first reaction of pyrimidine biosynthesis. This enzyme is activated by adenosine triphosphate (ATP), a purine nucleotide. It should be noted that at the same time ATCase is inhibited by one of the end products of the named biosynthetic pathway - cytidine triphosphate (CTP), and the activator and inhibitor bind to the same allosteric center. Thus, by regulating the activity of one enzyme, coordination of the synthesis of purine and pyrimidine nucleotides is ensured.
Mutational damage to the allosteric center can cause the enzyme to lose its ability to bind effector molecules and change its activity in response to this. This phenomenon is used in the selection of microorganisms to obtain mutants with desensitized enzymes. Such microorganisms are often producers of biologically active substances, and analogues of metabolites are used for their selection. For example, 5-methyltryptophan, like tryptophan, is capable of inhibiting the activity of anthranilate synthetase, but does not replace tryptophan in protein. Therefore, E. coli bacteria are not able to form colonies on a synthetic medium with this substance. However, E. coli mutants are known that grow on a medium with 5-methyltryptophan. These bacteria contain in their cells anthranilate synthetase that is insensitive to retroinhibition (desensitized) and synthesize tryptophan in excess quantities, releasing it into the external environment.
Another common way to regulate enzyme activity is covalent modification - the addition or removal of a small chemical group from the enzyme. With the help of such modifications, usually either a completely inactive form of the enzyme becomes active, or, conversely, a completely active enzyme is inactivated. The phenomenon of covalent modification includes: limited proteolysis (shortening of polypeptide chains), phosphorylation - dephosphorylation, adenylation - deadenylation, acetylation - deacetylation, etc. For example, glycogen synthetase of mammalian cells, which catalyzes the conversion of glucose into glycogen, is inactivated after the covalent attachment of a phosphate group to the side chain of one from serine residues and is reactivated upon elimination of phosphate. Other examples of covalent modification of enzymes are described in Chapter 3.
A special case of regulation of enzyme activity is represented by protein-protein interactions, in which special proteins play the role of enzyme inhibitors. With such interactions, the active center of the enzyme is blocked. Inhibition by proteins is of particular importance for regulating the activity of proteinases involved in post-translational modification of proteins. This contributes to a change in the rate of maturation of many proteins important for the cell, and, consequently, the intensity of the processes in which the latter take part.
Chapter 7. COFACTORS
In some cases, enzymes require special intermediaries - cofactors - to carry out catalysis. Cofactors are non-protein substances that function at intermediate stages of an enzymatic reaction (or reaction cycle), but are not consumed during catalysis. In the vast majority of cases, cofactors are regenerated unchanged upon completion of the catalytic act.
Cofactors of various chemical natures can be divided into two main groups: coenzymes(weakly bound to the enzyme and separated from it during catalysis) and prosthetic groups(strongly bound to the enzyme molecule).
The main mechanisms according to which cofactors take part in catalysis are as follows:
Act as carriers between enzymes. Interacting with one enzyme, the transporter accepts part of the substrate, migrates to another enzyme and transfers the transferred part to the substrate of the second enzyme, after which it is released. This mechanism is typical for most coenzymes;
They act as an “intraenzyme” carrier, which is typical, first of all, for prosthetic groups. The prosthetic group attaches part of the substrate molecule and transfers it to a second substrate bound in the active site of the same enzyme. In this case, the prosthetic group can be considered as part of the catalytic site of the enzyme;
They change the conformation of the enzyme molecule by interacting with it outside the active center, which can induce the transition of the active center to a catalytically active configuration;
Stabilize the conformation of the enzyme, promoting a catalytically active state;
Performs the function of a matrix. For example, nucleic acid polymerases need a “program” - a matrix on which a new molecule is built;
They play the role of intermediate compounds. Sometimes an enzyme can use a cofactor molecule in a reaction, forming a product from it, but at the same time form a new cofactor molecule at the expense of the substrate.
Among currently known enzymes, approximately 40% are capable of catalysis only through cofactors. The most common cofactors are those that transfer reducing equivalents, phosphate, acyl and carboxyl groups.
The main concept of enzyme kinetics is the concept of enzyme-substrate complexes (ES). As with inorganic catalysts, the enzyme ensures that the reaction proceeds along a more efficient pathway, with a lower activation energy. The higher catalytic activity of the enzymatic reaction is due to the fact that the process proceeds through the stage of ES formation. The rate of enzymatic reactions is 10 3 - 10 13 times higher than the rate of a non-catalytic reaction. This sharp increase in speed is due to two reasons - the convergence effect, which is also observed in non-enzymatic reactions, and the orientation effect, which is carried out extremely effectively in enzymatic reactions.
Enzyme molecules, unlike other catalysts, have a very complex structure. This makes it possible to implement mechanisms for increasing reaction rates that are impossible with non-biological catalysts. Here, interactions of a special kind are possible that are absent in conventional catalysis. If we assume that the binding of the substrate on the enzyme molecule occurs not at one, but at three points, then this alone sharply increases the probability of the required orientations and increases the reaction rate by several orders of magnitude.
Covalent, ionic, hydrogen bonds, and hydrophobic interactions can take part in the formation of enzyme-substrate complexes. The catalytic activity of the enzyme is associated with its spatial structure, in which rigid sections of helices alternate with flexible elastic linear sections.
In explaining the mechanism of action of enzymes, Koshland's hypothesis of “induced” or “forced” compliance has become widely accepted. In accordance with this hypothesis, the necessary arrangement of functional groups in the active center of the enzyme occurs under the influence of the substrate. The reactive conformation of the entire enzyme molecule and its active center arises as a result of the deforming effect of the substrate. It should be borne in mind that induced compliance is created not only by a change in the conformation of the enzyme, but also by a rearrangement of the substrate molecule.
The “forced fit” hypothesis was experimentally confirmed when a change in the arrangement of functional groups of the active site during the process of substrate attachment was proven. The specificity of the enzyme is probably due to the possibility of conformational rearrangements of the active center. If the possibilities of rearrangement are great, then the enzyme can interact with several substrates that are similar in structure and exhibit group specificity; if the possibility is sharply limited, then the enzyme is highly specific.
The induced correspondence hypothesis assumes the presence between the enzyme and the substrate not only of spatial complementarity, but also of electrostatic interaction due to the oppositely charged groups of the substrate and enzyme.
The body simultaneously implements a huge number of biochemical reactions important for vital processes, which must be strictly regulated in accordance with the needs of the body. This regulation should ensure the supply of the necessary components in a given period of time with the least energy consumption. Since virtually every biologically important reaction is an enzymatic reaction, it is clear that such regulation occurs primarily through the control of enzymes that catalyze key metabolic reactions.
The rate of formation of the final product of a metabolic pathway can be regulated either by changing the activity of the corresponding enzymes or by increasing or decreasing the number of enzyme molecules (induction or repression).
Regulation of enzyme activities in cells occurs in various ways. For most enzymes that obey the Michaelis-Menten equation, substrate concentration is an important regulatory factor. The value K m was introduced, representing the concentration of the substrate at which the reaction rate is 50% of the maximum. Since the concentration of substrates in the cell is close to Km or slightly lower, minor changes in the concentration of substrates lead to relatively large changes in reaction rates.
Regulation of enzyme activity can be carried out through direct effects on substrate binding centers, for example, inhibition of the enzyme by substrate analogues.
Inhibitors of protein nature bind tightly to the active center of the enzyme. For example, a trypsin inhibitor is a protein with a molecular weight of 6000. It has a strong inhibitory effect, since it is strictly complementary to the structure of the active center of the enzyme.
However, the allosteric (non-covalent) type of regulation of enzyme activity is much more common.
Allosteric regulation characteristic of enzymes consisting of 2 or more subunits and having more than one substrate-binding center. These enzymes contain allosteric centers (different from substrate-binding centers) that are capable of binding certain substances called allosteric effectors. If the binding of an effector reduces the rate of an enzymatic reaction, then it is called an allosteric inhibitor; if it increases, it is called an allosteric activator. Various metabolites, hormones, and coenzymes act as allosteric effectors of enzymes. One of the ways of regulating allosteric enzymes is inhibition through “negative feedback” or “retroinhibition”, i.e. inhibition by the end product of the reaction. Some enzyme molecules have several allosteric centers, some of which are specific to positive, others to negative, effectors. Allosteric centers of enzymes, like active centers, can exhibit pronounced specificity, when they can bind only one specific effector, or relative specificity, when binding of effectors similar in structure can occur.
The mechanism of action of the allosteric effector is associated with a change in the conformation of the subunits from which the enzyme is built, which affects the catalytic activity of the enzyme.
Allosteric regulation is one of the most subtle and highly specific mechanisms of “quick response” to certain processes in the environment and is used to fine-tune metabolic systems. An effector can act only in one or several tissues of the body and be associated with a strictly defined part of metabolism.
Allosteric enzymes are characterized by the phenomenon of cooperativity. It manifests itself in the fact that the catalytic centers of the subunits interact not autonomously, but interconnectedly. Interaction with the substrate or effector of one of these centers enhances the ability of other active centers to interact (positive cooperativity). In some cases, the binding of one active center of the substrate reduces the ability to bind other centers (negative cooperativity).
Positive cooperativity has been most well studied using the example of the hemoglobin molecule, which has four 0 2 binding sites (heme groups). The binding of an oxygen molecule by one center leads to increased interaction with oxygen in other areas. The affinity of hemoglobin for 0 2 to the last (fourth) group is more than 100 times greater than to the first. Since the oxygen-binding regions are separated in the molecule by large distances, they cannot interact directly. Obviously, upon oxygenation, the conformation of the molecule as a whole changes, which leads to a change in the affinity of the binding sites.
Cooperation is also one of the ways to regulate enzyme activity.
Enzyme activity can also change as a result of the so-called covalent (post-translational) modification, in which either a part of the molecule is split off or small groups are attached to the enzyme. In both cases, these modifications of the enzyme molecule involve the breaking or formation of covalent bonds.
It is known that proteolytic enzymes of the gastrointestinal tract (pepsin, trypsin, chymotrypsin) are synthesized in the form of inactive precursors - proenzymes. The regulation of enzyme activity in this case is that under the action of specific substances (enzymes) the inactive form is converted into an active one. For example, trypsin is synthesized in the pancreas in the form of trypsinogen, which, upon entering the small intestine, is converted into trypsin under the action of the enzyme enterokinase. In this case, a hexapeptide is cleaved from trypsinogen. Trypsin, in turn, breaks one peptide bond in chymotrypsinogen, which leads to structural changes in the active center and converts it into active chymotrypsin.
The conversion of pepsinogen into the active form of pepsin is also associated with the cleavage of the peptide from the inactive pepsinogen molecule. The synthesis of proteolytic enzymes in the form of proenzymes is important in the process of regulating the digestion process in the gastrointestinal tract.
Regulation of the activity of proteolytic enzymes in the gastrointestinal tract occurs not only by converting the proenzyme into an active enzyme, but also by binding enzymes to natural inhibitors. Low molecular weight proteins that inhibit the action of pepsin and trypsin were found in the mucous membrane of the stomach and intestines. A very active pepsin inhibitor was isolated from the pig stomach, and a trypsin inhibitor from the pancreas.
Covalent modification of an enzyme with a change in its activity can occur not only as a result of breaking peptide bonds, but by attaching a specific group to the enzyme molecule. For example, regulation of the activity of the enzyme glycogen synthetase, which plays a major role in the fine regulation of glycogen synthesis, is carried out by phosphorylation and dephosphorylation.
Phosphorylation by protein kinases is a common form of regulation of enzyme activity by covalent modification. The activity of a large number of enzymes and the intensity of the corresponding metabolic processes are determined by the ratio of phosphorylated and dephosphorylated forms of these enzymes.
Regulation of enzymatic activity can be carried out by enhancing the synthesis of existing enzymes or even new enzymes in response to changed living conditions (the emergence of new food factors, chemicals).
When exposed to specific substances, “inducers” or “repressors,” the transcription process is initiated or suppressed, respectively. This regulation, carried out during the biosynthesis of the enzyme, can lead to changes in the concentration of the enzyme, changes in the types of enzymes present in the cell and the isoenzyme composition.
This regulatory pathway is slower, as it is associated with changes in protein biosynthesis. Therefore, a certain amount of time will pass between the signal about the need to change the enzyme concentration and the establishment of its new content - from several hours to several days. Consequently, by changing the concentration of the enzyme, rapid regulation of reaction rates cannot be achieved. However, in cases where not a rapid change in metabolism is needed, but long-term regulation of the metabolic process, this pathway becomes important.
For example, in cases where it is necessary to stimulate gluconeogenesis, the concentration of enzymes such as glucose-6-phosphatase, fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxylase increases. The need for increased amounts of these enzymes is due to the fact that they catalyze reactions that bypass the physiologically irreversible stages of the direct cycle.
It has been shown that during metabolic acidosis in animals, the synthesis of glutaminase increases. This is due to the need to neutralize acidic products accumulating in the body with ammonia.
Evidence in the literature suggests that enzyme induction or repression may be caused by dietary factors.
Administration of glucose to rats that had previously fasted for 5 days caused a sharp increase in glucokinase activity. Since injection of puromycin or actimicin D suppressed this activation, it was concluded that the reason for the increase in enzyme activity was due to an increase in its synthesis (puromycin inhibits protein synthesis, and actinomycin inhibits mRNA synthesis).
The relationship between the activity of urea cycle enzymes and the amount of protein in the diet is well known. An increase in protein content in the diet is accompanied by an increase in the activity of these enzymes, and this increase is proportional to the intensity of urea synthesis. There were no changes in the kinetic properties of enzymatic molecules, or the presence of any inhibitors or activators, which led to the conclusion that the increase in activity is associated with increased synthesis of the corresponding enzymes.
The induction of enzymes in pathology is of great importance. Enzyme induction is often associated with the development of protective processes in the event of pathological conditions in the body. At the same time, it should be borne in mind that in some cases, increased synthesis of enzymes in response to changes in external environmental conditions can lead to the development of a pathological process.
In some cases, when medicinal or other foreign substances enter the body, enzyme induction also occurs. However, this does not always contribute to the body’s adaptation to a new substance and does not always provide more favorable conditions for the life of the body, since the product of enzymatic transformation can be more toxic than the original substance. In this case, the effect will be negative.
It has been established that many medicinal substances have the ability to induce the formation of enzymes - barbiturates, volatile anesthetics, hypoglycemic substances, analgesics, insecticides, etc. This phenomenon can explain the often observed addiction to some medicinal substances with their long-term use.
For example, when experimentally administering phenylbutazone to dogs, its content in the blood increased and the phenomenon of intoxication was observed. Repeated administration of this drug no longer caused such a pronounced increase in its blood levels and no toxic effect.
Enzyme induction by pharmacological substances is often not highly specific. Enzymes are often formed that promote the conversion of not only this substance - the inducer, but some other medicinal substances. For example, the introduction of pentabarbiturate into the body leads to increased metabolism not only of this substance, but also causes increased oxidation of hexabarbiturate and even substances that do not belong to the group of barbiturates.
When talking about the metabolism of medicinal substances in the body, it must be borne in mind that it can be carried out not only through the induction of enzymes, but also through allosteric and covalent modification of enzymes.
Principles of clinical enzyme diagnostics
