Metabolism. Basic processes of cellular metabolism

13.4.1. The Krebs cycle reactions are the third stage of nutrient catabolism and occur in the mitochondria of the cell. These reactions belong to the general pathway of catabolism and are characteristic of the breakdown of all classes of nutrients (proteins, lipids and carbohydrates).

The main function of the cycle is the oxidation of the acetyl residue with the formation of four molecules of reduced coenzymes (three molecules of NADH and one molecule of FADH2), as well as the formation of a GTP molecule by substrate phosphorylation. The carbon atoms of the acetyl residue are released as two CO2 molecules.

13.4.2. The Krebs cycle includes 8 successive stages, paying particular attention to the dehydrogenation reactions of substrates:

Figure 13.6. Krebs cycle reactions, including the formation of α-ketoglutarate

a) condensation of acetyl-CoA with oxaloacetate, as a result of which citrate is formed (Fig. 13.6, reaction 1); so the Krebs cycle is also called citrate cycle. In this reaction, the methyl carbon of the acetyl group interacts with the keto group of oxaloacetate; cleavage of the thioether bond occurs simultaneously. The reaction releases CoA-SH, which can take part in the oxidative decarboxylation of the next pyruvate molecule. The reaction is catalyzed citrate synthase, it is a regulatory enzyme, it is inhibited by high concentrations of NADH, succinyl-CoA, citrate.

b) conversion of citrate to isocitrate through the intermediate formation of cis-aconitate. The citrate formed in the first reaction of the cycle contains a tertiary hydroxyl group and is not capable of being oxidized under cell conditions. Under the action of an enzyme aconitase there is a splitting off of a water molecule (dehydration), and then its addition (hydration), but in a different way (Fig. 13.6, reactions 2-3). As a result of these transformations, the hydroxyl group moves to a position that favors its subsequent oxidation.

in) isocitrate dehydrogenation followed by the release of a CO2 molecule (decarboxylation) and the formation of α-ketoglutarate (Fig. 13.6, reaction 4). This is the first redox reaction in the Krebs cycle, resulting in the formation of NADH. isocitrate dehydrogenase, which catalyzes the reaction, is a regulatory enzyme, activated by ADP. Excess NADH inhibits the enzyme.

Figure 13.7. Krebs cycle reactions starting with α-ketoglutarate.

G) oxidative decarboxylation of α-ketoglutarate, catalyzed by a multienzyme complex (Fig. 13.7, reaction 5), accompanied by the release of CO2 and the formation of a second NADH molecule. This reaction is similar to the pyruvate dehydrogenase reaction. The inhibitor is the reaction product, succinyl-CoA.

e) substrate phosphorylation at the level of succinyl-CoA, during which the energy released during the hydrolysis of the thioether bond is stored in the form of a GTP molecule. Unlike oxidative phosphorylation, this process proceeds without the formation of the electrochemical potential of the mitochondrial membrane (Fig. 13.7, reaction 6).

e) succinate dehydrogenation with the formation of fumarate and the FADH2 molecule (Fig. 13.7, reaction 7). The enzyme succinate dehydrogenase is tightly bound to the inner mitochondrial membrane.

and) fumarate hydration, as a result of which an easily oxidized hydroxyl group appears in the molecule of the reaction product (Fig. 13.7, reaction 8).

h) malate dehydrogenation, leading to the formation of oxaloacetate and the third NADH molecule (Fig. 13.7, reaction 9). The oxaloacetate formed in the reaction can be reused in the condensation reaction with the next acetyl-CoA molecule (Fig. 13.6, reaction 1). Therefore, this process is cyclical.

13.4.3. Thus, as a result of the described reactions, the acetyl residue undergoes complete oxidation CH3 -CO-. The number of acetyl-CoA molecules converted in mitochondria per unit time depends on the concentration of oxaloacetate. The main ways to increase the concentration of oxaloacetate in mitochondria (relevant reactions will be discussed later):

13.4.4. Some metabolites of the Krebs cycle can be used to synthesis building blocks for building complex molecules. Thus, oxaloacetate can be converted to the amino acid aspartate, and α-ketoglutarate can be converted to the amino acid glutamate. Succinyl-CoA is involved in the synthesis of heme, the prosthetic group of hemoglobin. Thus, the reactions of the Krebs cycle can participate both in the processes of catabolism and anabolism, that is, the Krebs cycle performs amphibolic function(see 13.1).

Metabolism, inextricably linked with the exchange of energy, is a natural order of the transformation of matter and energy in living systems, aimed at their conservation and self-reproduction. F. Engels noted metabolism as the most important property of life, with the termination of which life itself ceases. He emphasized the dialectical nature of this process and pointed out that

I. M. Sechenov, the founder of Russian physiology, considered the role of metabolism in the life of organisms from a consistently materialistic position. K. A. Timiryazev consistently pursued the idea that the main property that characterizes living organisms is the constant active exchange between the substance that makes up the body and the substance of the environment, which the body constantly perceives, assimilates, turns it into a similar one, again changes and highlights in the process of dissimilation. IP Pavlov considered metabolism as the basis for the manifestation of vital activity, as the basis for the physiological functions of the body. A significant contribution to the knowledge of the chemistry of life processes was made by AI Oparin, who studied the basic patterns of the evolution of metabolism in the course of the emergence and development of life on Earth.

BASIC CONCEPTS AND TERMS

Or metabolism is a set of chemical reactions in the body that provide it with the substances and energy necessary for life: self-preservation and self-reproduction. Self-reproduction is understood as the transformation of a substance coming from outside into the substances and structures of the organism itself, as a result of which there is a continuous renewal of tissues, growth and reproduction.

In the metabolism secrete:

• external exchange- includes extracellular transformation of substances on the way of their entry into the body and excretion of metabolic products from it [show] .

  The intake of substances into the body and the release of metabolic products together constitute the exchange of substances between the environment and the body, and is defined as external exchange.

  External exchange of substances (and energy) is carried out constantly.

  Oxygen, water, mineral salts, nutrients, vitamins, necessary for building and updating the structural elements of cells and tissues, and generating energy, enter the human body from the external environment. All these substances can be called food products, some of which are of biological origin (vegetable and animal products) and a smaller part of non-biological (water and mineral salts dissolved in it).

  Nutrients supplied with food are degraded with the formation of amino acids, monosaccharides, fatty acids, nucleotides and other substances, which, mixing with the same substances formed in the process of continuous decay of the structural and functional components of the cell, constitute the general fund of the body's metabolites. This fund is spent in two directions: part is used to renew the decayed structural and functional components of the cell; the other part is converted into end products of metabolism, which are excreted from the body.

  During the decay of substances to the final products of metabolism, energy is released, in an adult 8,000-12,000 kJ (2000-3000 kcal) per day. This energy is used by the cells of the body to perform various kinds of work, as well as to maintain the body temperature at a constant level.

• intermediate exchange- includes the transformation of substances inside biological cells from the moment they enter to the formation of final products (for example, amino acid metabolism, carbohydrate metabolism, etc.)

Stages of metabolism. There are three successive stages.

More about

• intake (Nutrition is an integral part of metabolism (the intake of substances from the environment into the body))
• digestion (Biochemistry of digestion (digestion of nutrients))
• absorption (Biochemistry of digestion (absorption of nutrients))

Intermediate metabolism (or metabolism) - the transformation of substances in the body from the moment they enter the cells to the formation of end products of metabolism, i.e. the totality of chemical reactions that occur in living cells and provide the body with substances and energy for its vital activity, growth, reproduction. This is the most difficult part of the metabolism.

Once inside the cell, the nutrient is metabolized - undergoing a series of chemical changes catalyzed by enzymes. A certain sequence of such chemical changes is called a metabolic pathway, and the resulting intermediate products are called metabolites. Metabolic pathways can be represented in the form of a metabolic map.

As a result of metabolic activity in all parts of the body, harmful substances are formed that enter the bloodstream and must be removed. This function is performed by the kidneys, which separate harmful substances and direct them to the bladder, from where they are then excreted from the body. Other organs also take part in the process of metabolism: liver, pancreas, gallbladder, intestines, sweat glands.

A person excretes with urine, feces, sweat, exhaled air the main end products of metabolism - CO 2, H 2 O, urea H 2 N - CO - NH 2. In the form of H 2 O, hydrogen of organic substances is excreted, and the body releases more water than it consumes (see Table 24): approximately 400 g of water is formed per day in the body from the hydrogen of organic substances and the oxygen of the inhaled air (metabolic water). Carbon and oxygen of organic substances are removed in the form of CO 2, and nitrogen is removed in the form of urea.

In addition, a person releases many other substances, but in small quantities, so that their contribution to the overall balance of metabolism between the body and the environment is small. However, it should be noted that the physiological significance of the release of such substances can be significant. For example, a violation of the release of heme breakdown products or metabolic products of foreign compounds, including drugs, can cause severe metabolic disorders and body functions.

Substrates of metabolism- chemical compounds that come with food. Among them, two groups can be distinguished: the main nutritional substances (carbohydrates, proteins, lipids) and minor ones that come in small quantities (vitamins, mineral compounds).

It is customary to distinguish among nutrients interchangeable and irreplaceable. Indispensable are those nutrients that cannot be synthesized in the body and, therefore, must be supplied with food.

metabolic pathway- this is the nature and sequence of chemical transformations of a particular substance in the body. The intermediate products formed during the conversion process are called metabolites, and the last compound of the metabolic pathway is the final product.

Chemical transformations take place continuously in the body. As a result of the nutrition of the body, the initial substances undergo metabolic transformations; end products of metabolism are constantly excreted from the body. Thus, an organism is a thermodynamically open chemical system. The simplest example of a metabolic system is a single unbranched metabolic chain:

--> a --> b --> c --> d -->

With a constant flow of substances in such a system, a dynamic equilibrium is established, when the rate of formation of each metabolite is equal to the rate of its consumption. This means that the concentration of each metabolite is kept constant. Such a state of the system is called stationary, and the concentrations of substances in this state are called stationary concentrations.

A living organism at any given moment does not meet the given definition of a stationary state. However, considering the average value of its parameters over a relatively long period of time, one can note their relative constancy and thereby justify the application of the concept of a stationary system to living organisms. [show] .

On fig. 64 shows a hydrodynamic model of an unbranched metabolic chain. In this device, the height of the liquid column in the cylinders simulates the concentrations of metabolites a-d, respectively, and the throughput of the connecting tubes between the cylinders simulates the rate of the corresponding enzymatic reactions.

At a constant rate of liquid entering the system, the height of the liquid column in all cylinders remains constant: this is a stationary state.

If the rate of fluid inflow increases, then both the height of the fluid column in all cylinders and the rate of fluid flow through the entire system will increase: the system has passed into a new stationary state. Similar transitions occur in metabolic processes in a living cell.

Regulation of metabolite concentration

Usually there is a reaction in the metabolic chain that proceeds much more slowly than all other reactions - this is the rate-limiting step of the pathway. In the figure, such a stage is modeled by a narrow connecting tube between the first and second cylinders. The rate-limiting stage determines the overall rate of transformation of the starting substance into the final product of the metabolic chain. Often the enzyme catalyzing the limiting reaction is a regulatory enzyme: its activity can change under the action of cellular inhibitors and activators. In this way, the regulation of the metabolic pathway is ensured. On fig. 64 A transition tube with a damper between the first and second cylinders simulates a regulatory enzyme: by raising or lowering the damper, it is possible to transfer the system to a new stationary state, with a different overall fluid flow rate and different fluid levels in the cylinders.

In branched metabolic systems, regulatory enzymes usually catalyze the first reactions at the branching site, such as the reactions b --> c and b --> i in Fig. 65. This ensures the possibility of independent regulation of each branch of the metabolic system.

Many metabolic reactions are reversible; the direction of their flow in a living cell is determined by the consumption of the product in the subsequent reaction or the removal of the product from the sphere of the reaction, for example, by excretion (Fig. 65).

With changes in the state of the body (eating, transition from rest to motor activity, etc.), the concentration of metabolites in the body changes, i.e., a new stationary state is established. However, under the same conditions, for example, after a night's sleep (before breakfast), they are approximately the same in all healthy people; due to the action of regulatory mechanisms, the concentration of each metabolite is maintained at its characteristic level. The average values ​​of these concentrations (with indication of the limits of fluctuations) serve as one of the characteristics of the norm. In diseases, stationary concentrations of metabolites change, and these changes are often specific to a particular disease. Many biochemical methods of laboratory diagnostics of diseases are based on this.

There are two directions in the metabolic pathway - anabolism and catabolism (Fig. 1).

• Anabolic reactions are aimed at converting simpler substances into more complex ones, forming structural and functional components of the cell, such as coenzymes, hormones, proteins, nucleic acids, etc. These reactions are predominantly reductive, accompanied by the expenditure of free chemical energy (endergonic reactions). The source of energy for them is the process of catabolism. In addition, the energy of catabolism is used to ensure the functional activity of the cell (motor and others).
• Catabolic transformations are the processes of splitting complex molecules, both those that come with food and that are part of the cell, to simple components (carbon dioxide and water); these reactions are usually oxidative, accompanied by the release of free energy (exergonic reactions).

Amphibolic way(dual) - a path in which catabolic and anabolic transformations are combined, i.e. along with the destruction of one compound, another is synthesized.

Amphibolic pathways are associated with the terminal, or final, oxidation system of substances, where they burn to final products (CO 2 and H 2 O) with the formation of a large amount of energy. In addition to them, the end products of metabolism are urea and uric acid, which are formed in special reactions of the exchange of amino acids and nucleotides. Schematically, the relationship of metabolism through the ATP-ADP system and the amphibolic cycle of metabolites is shown in Fig. 2.

ATP-ADP system(ATP-ADP cycle) - a cycle in which the continuous formation of ATP molecules occurs, the hydrolysis energy of which is used by the body in various types of work.

This is such a metabolic pathway, one of the end products of which is identical to one of the compounds involved in this process (Fig. 3).

Anaplerotic path- metabolic, the end product of which is identical to one of the intermediate products of any cyclic pathway. The anaplerotic path in the example of fig. 3 replenishes the cycle with product X (anaplerosis - replenishment).

Let's use this example. Buses of brands X, Y, Z run in the city. Their routes are shown in the diagram (Fig. 4).

Based on this example, we define the following.

• A private metabolic pathway is a set of transformations that is characteristic only of a particular compound (for example, carbohydrates, lipids or amino acids).
• A common metabolic pathway is a set of transformations that involve two or more types of compounds (for example, carbohydrates and lipids or carbohydrates, lipids and amino acids).

Localization of metabolic pathways

Catabolic and anabolic pathways in eukaryotic individuals differ in their localization in the cell (Table 22.).

This division is due to the confinement of enzyme systems to certain areas of the cell (compartmentalization), which provides both segregation and integration of intracellular functions, as well as appropriate control.

At present, thanks to electron microscopic and histochemical studies, as well as the method of differential centrifugation, significant progress has been made in determining the intracellular localization of enzymes. As can be seen from fig. 74, in a cell, one can find a cellular or plasma membrane, a nucleus, mitochondria, lysosomes, ribosomes, a system of tubules and vesicles - an endoplasmic reticulum, a lamellar complex, various vacuoles, intracellular inclusions, etc. The main mass undifferentiated part of the cell cytoplasm is hyaloplasm ( or cytosol).

It has been established that RNA polymerases, i.e., enzymes that catalyze the formation of mRNA, are localized in the nucleus (more precisely, in the nucleolus). The nucleus contains enzymes involved in the process of DNA replication, and some others (Table 23).

Relationship of enzymes with cell structures:

• Mitochondria. Mitochondria are associated with enzymes of the biological oxidation chain (tissue respiration) and oxidative phosphorylation, as well as enzymes of the pyruvate dehydrogenase complex, the tricarboxylic acid cycle, urea synthesis, fatty acid oxidation, etc.
• Lysosomes. Lysosomes contain mainly hydrolytic enzymes with an optimum pH in the region of 5. It is because of the hydrolytic affiliation of enzymes that these particles are called lysosomes.
• Ribosomes. The enzymes of protein synthesis are localized in ribosomes, in these particles mRNA is translated and amino acids are bound into polypeptide chains with the formation of protein molecules.
• Endoplasmic reticulum. The endoplasmic reticulum contains lipid synthesis enzymes, as well as enzymes involved in hydroxylation reactions.
• Plasma membrane. ATP-ase transporting Na + and K +, adenylate cyclase and a number of other enzymes are primarily associated with the plasma membrane.
• Cytosol. Enzymes of glycolysis, pentose cycle, synthesis of fatty acids and mononucleotides, activation of amino acids, as well as many enzymes of gluconeogenesis are localized in the cytosol (hyaloplasm).

In table. 23 summarizes data on the localization of the most important enzymes and individual metabolic steps in various subcellular structures.

Multienzyme systems are localized in the structure of organelles in such a way that each enzyme is located in close proximity to the next enzyme in a given sequence of reactions. Due to this, the time required for the diffusion of intermediate reaction products is reduced, and the entire sequence of reactions is strictly coordinated in time and space. This is true, for example, for enzymes involved in the oxidation of pyruvic acid and fatty acids, in protein synthesis, as well as for electron transfer and oxidative phosphorylation enzymes.

Compartmentalization also ensures that chemically incompatible reactions occur at the same time, i.e. independence of catabolism and anabolism pathways. So, in the cell, the oxidation of long-chain fatty acids to the stage of acetyl-CoA and the oppositely directed process - the synthesis of fatty acids from acetyl-CoA can occur simultaneously. These chemically incompatible processes occur in different parts of the cell: fatty acid oxidation occurs in mitochondria, and their synthesis outside mitochondria occurs in hyaloplasm. If these paths coincided and differed only in the direction of the process, then so-called useless or futile cycles would arise in the exchange. Such cycles take place in pathology, when useless circulation of metabolites is possible.

Elucidation of the individual links of metabolism in different classes of plants, animals and microorganisms reveals a fundamental commonality of the paths of biochemical transformations in living nature.

BASIC PROVISIONS OF THE REGULATION OF METABOLISM

Regulation of metabolism at the cellular and subcellular levels is carried out

1. by regulating the synthesis and catalytic activity of enzymes.

   These regulatory mechanisms are

   • suppression of the synthesis of enzymes by the end products of the metabolic pathway,
   • induction of synthesis of one or more enzymes by substrates,
   • modulation of the activity of already present enzyme molecules,
   • regulation of the rate of entry of metabolites into the cell. Here the leading role belongs to the biological membranes surrounding the protoplasm and the nucleus, mitochondria, lysosomes and other subcellular organelles located in it.
2. by regulating the synthesis and activity of hormones. So, the protein metabolism is influenced by the thyroid hormone - thyroxin, the fat metabolism is influenced by the hormones of the pancreas and thyroid glands, adrenal glands and pituitary gland, the carbohydrate metabolism is influenced by the hormones of the pancreas (insulin) and adrenal glands (adrenaline). A special role in the mechanism of action of hormones belongs to cyclic nucleotides (cAMP and cGMP).

   In animals and humans, the hormonal regulation of metabolism is closely related to the coordinating activity of the nervous system. An example of the influence of the nervous system on carbohydrate metabolism is the so-called sugar injection by Claude Bernard, which leads to hyperglycemia and glucosuria.

3. The most important role in the processes of integration of metabolism belongs to the cerebral cortex. As I. P. Pavlov pointed out: “The more perfect the nervous system of an animal organism, the more centralized it is, the higher its department is more and more the manager and distributor of all the activities of the organism ... This higher department contains in its jurisdiction all the phenomena that occur in body".

Thus, a special combination, strict consistency and the rate of metabolic reactions in the aggregate form a system that reveals the properties of the feedback mechanism (positive or negative).

METHODS FOR STUDYING INTERMEDIATE METABOLISM

Two approaches are used to study metabolism:

• whole body studies (in vivo experiments) [show]

  A classic example of research on the whole organism, carried out at the beginning of our century, is the experiments of Knoop. He studied the way fatty acids break down in the body. To do this, Knoop fed dogs various fatty acids with an even (I) and odd (II) number of carbon atoms, in which one hydrogen atom in the methyl group was replaced by a phenyl radical C 6 H 5:

  In the first case, phenylacetic acid C 6 H 5 -CH 2 -COOH was always excreted in the urine of dogs, and in the second case, benzoic acid C 6 H 5 -COOH. Based on these results, Knoop concluded that the breakdown of fatty acids in the body occurs through the sequential elimination of two-carbon fragments, starting from the carboxyl end:

  CH 3 -CH 2 -|-CH 2 -CH 2 -|-CH 2 -CH 2 -|-CH 2 -CH 2 -|-CH 2 -COOH

  This conclusion was later confirmed by other methods.

  In essence, in these studies, Knoop applied the method of labeling molecules: he used as a label a phenyl radical that does not undergo changes in the body. Starting around the 40s of the XX century. the use of substances whose molecules contain radioactive or heavy isotopes of elements has become widespread. For example, by feeding various compounds containing radioactive carbon (14 C) to experimental animals, it was found that all carbon atoms in the cholesterol molecule come from carbon atoms of acetate:

  

  Usually, either stable isotopes of elements that differ in mass from elements widely distributed in the body (usually heavy isotopes) or radioactive isotopes are used. Of the stable isotopes, hydrogen isotopes with a mass of 2 (deuterium, 2 N), nitrogen with a mass of 15 (15 N), carbon with a mass of 13 (13 C) and oxygen with a mass of 18 (18 C) are most often used. Of the radioactive isotopes, hydrogen isotopes (tritium, 3 H), phosphorus (32 P and 33 P), carbon (14 C), sulfur (35 S), iodine (131 I), iron (59 Fe), sodium (54 Na ) and etc.

  Having marked with the help of a stable or radioactive isotope a molecule of the studied compound and introduced it into the body, then the labeled atoms or chemical groups containing them are determined and, having discovered them in certain compounds, a conclusion is made about the ways of transformation of the labeled substance in the body. With the help of an isotope label, one can also establish the residence time of a substance in the body, which, with a known approximation, characterizes the biological half-life, i.e., the time during which the amount of an isotope or labeled compound is halved, or to obtain accurate information about the permeability of the membranes of individual cells. Isotopes are also used to determine whether a given substance is a precursor or decay product of another compound, and to determine the rate of tissue renewal. Finally, if there are several metabolic pathways, it is possible to determine which of them prevails.

  In studies on whole organisms, the body's need for nutrients is also studied: if the elimination of a substance from the diet leads to a violation of the growth and development or physiological functions of the body, then this substance is an indispensable nutritional factor. The required amounts of nutrients are determined in a similar way.

• and studies on isolated parts of the body - analytical-disintegrating methods (in vitro experiments, that is, outside the body, in a test tube or other laboratory vessels). The principle of these methods is the gradual simplification, or rather disintegration, of a complex biological system in order to isolate individual processes. If we consider these methods in descending order, i.e. from more complex to simpler systems, then they can be arranged in the following order:
  • removal of individual organs [show]

    When organs are removed, there are two objects of study: an organism without a removed organ and an isolated organ.

    isolated organs. If a solution of a substance is injected into the artery of an isolated organ and the substances are analyzed in the fluid flowing from the vein, then it is possible to establish what transformations this substance undergoes in the organ. For example, in this way it has been found that the liver is the main site for the formation of ketone bodies and urea.

    Similar experiments can be carried out on organs without their isolation from the body (arterio-venous difference method): in these cases, blood is taken for analysis using cannulas inserted into the artery and vein of the organ, or with a syringe. In this way, for example, it can be established that in the blood flowing from working muscles, the concentration of lactic acid is increased, and flowing through the liver, the blood is freed from lactic acid.

  • tissue section method [show]

    Sections are thin pieces of tissue that are made using a microtome or simply a razor blade. Sections are incubated in a solution containing nutrients (glucose or others) and a substance, the transformation of which in cells of this type is to be determined. After incubation, analyze the metabolic products of the test substance in the incubation fluid.

    The tissue sectioning method was first proposed by Warburg in the early 1920s. Using this technique, it is possible to study tissue respiration (oxygen consumption and carbon dioxide release by tissues). A significant limitation in the study of metabolism in the case of the use of tissue sections are cell membranes, which - more often act as barriers between the contents of the cell and the "nutrient" solution.

  • homogenates and subcellular fractions [show]

    

    Homogenates are cell-free preparations. They are obtained by destroying cell membranes by rubbing tissue with sand or in special devices - homogenizers (Fig. 66). In homogenates, there is no impermeability barrier between added substrates and enzymes.

    Destruction of cell membranes allows direct contact between the contents of the cell and the added compounds. This makes it possible to establish which enzymes, coenzymes and substrates are important for the process under study.

    Fractionation of homogenates. Subcellular particles can be isolated from the homogenate, both supramolecular (cellular organelles) and individual compounds (enzymes and other proteins, nucleic acids, metabolites). For example, using differential centrifugation, fractions of nuclei, mitochondria, and microsomes can be obtained (microsomes are fragments of the endoplasmic reticulum). These organelles differ in size and density and therefore precipitate at different centrifugation speeds. The use of isolated organelles makes it possible to study the metabolic processes associated with them. For example, isolated ribosomes are used to study the pathways and mechanisms of protein synthesis, and mitochondria are used to study the oxidative reactions of the Krebs cycle or a chain of respiratory enzymes.

    After sedimentation of microsomes, soluble components of the cell remain in the supernatant liquid - soluble proteins, metabolites. Each of these fractions can be further fractionated by different methods, isolating their constituent components. It is possible to reconstruct biochemical systems from the isolated components, for example, a simple "enzyme + substrate" system and such complex ones as systems for the synthesis of proteins and nucleic acids.

  • partial or complete reconstruction of the enzyme system in vitro using enzymes, coenzymes and other reaction components [show]

    Use to integrate highly purified enzymes and coenzymes. For example, with the help of this method, it became possible to completely reproduce a fermentation system that has all the essential features of yeast fermentation.

Of course, these methods are of value only as a step necessary to achieve the ultimate goal - understanding the functioning of the whole organism.

FEATURES OF STUDYING HUMAN BIOCHEMISTRY

There are far-reaching similarities in the molecular processes of the various organisms inhabiting the Earth. Such fundamental processes as matrix biosynthesis, mechanisms of energy transformation, the main ways of metabolic transformations of substances are approximately the same in organisms from bacteria to higher animals. Therefore, many of the results of studies conducted with E. coli are applicable to humans. The greater the phylogenetic relationship of species, the more common in their molecular processes.

The vast majority of knowledge about human biochemistry is obtained in this way: based on the known biochemical processes in other animals, a hypothesis is built about the most probable variant of this process in the human body, and then the hypothesis is tested by direct studies of human cells and tissues. This approach makes it possible to conduct research on a small amount of biological material obtained from humans. Most often, tissues removed during surgical operations, blood cells (erythrocytes and leukocytes), as well as human tissue cells grown in vitro culture are used.

The study of human hereditary diseases, which is necessary for the development of effective methods for their treatment, simultaneously provides a lot of information about the biochemical processes in the human body. In particular, the congenital defect of the enzyme leads to the accumulation of its substrate in the body; in the study of such metabolic disorders, new enzymes and reactions are sometimes discovered, quantitatively insignificant (which is why they were not noticed when studying the norm), which, however, are of vital importance.

A large number of various chemical reactions constantly occur in the cell, which form metabolic pathways - the sequential transformation of one compound into another. Metabolism- the totality of all metabolic pathways that occur in the cells of the body.

Among all metabolic pathways occurring in the body, oppositely directed processes are distinguished: catabolism and anabolism. catabolism- the breakdown of complex substances into simple ones with the release of energy.

Anabolism- synthesis from simpler more complex substances. Metabolic pathways are coordinated with each other in place, time and intensity of flow. This consistency in the course of all processes is ensured by complex and diverse regulatory mechanisms.

Organization of chemical reactions into metabolic pathways

The optimal activity of enzymes that catalyze the reactions of one metabolic pathway is achieved due to a certain spatial organization in the cell.

1. Spatial localization of enzymes

Most enzymes are localized intracellularly and are unevenly distributed in the body. All enzymes of the same metabolic pathway, as a rule, are located in one part of the cell. Especially the separation of metabolic pathways is important for oppositely directed catabolic and anabolic processes. For example, the synthesis of fatty acids occurs in the cytoplasm, and their breakdown in the mitochondria. If such a separation did not exist, paths that would be useless from a functional and energetic point of view would be formed.

In metabolic pathways, the product of the first enzymatic reaction serves as a substrate for the second, and so on until the final product is formed. Metabolic pathway intermediates can be released from the reaction sequence and used in other metabolic pathways, ie. metabolic pathways are interconnected by intermediate products.

In a number of cases, the spatial organization of enzymes is so pronounced that the reaction product under no circumstances can be isolated from the metabolic pathway and necessarily serves as a substrate for the next reaction. This organization of the metabolic pathway is called multienzyme complex and arises as a result of the structural and functional organization of enzymes. Typically, such complexes are associated with membranes. Examples of multienzyme complexes include the pyruvate dehydrogenase complex, under the action of which the oxidative decarboxylation of pyruvic acid (pyruvate) occurs (see Section 6), fatty acid synthase, which catalyzes the synthesis of palmitic acid (see Section 8).

1. Structure of metabolic pathways

The structure of metabolic pathways in the cell is extremely diverse.

In the case when the substrate is converted into one product as a result of a series of enzymatic processes, this pathway is called linear metabolic pathway. Often there are branched metabolic pathways leading to the synthesis of various end products depending on the needs of the cell.

The enzyme composition of different cells is not the same. Enzymes that perform the function of cell life support are found in all cells of the body. In the process of cell differentiation, a change in the enzymatic composition of cells occurs. Thus, the enzyme arginase, which is involved in the synthesis of urea, is found only in liver cells, and acid phosphatase, which is involved in the hydrolysis of orthophosphoric acid monoesters, is found in prostate cells. These are the so-called organ-specific enzymes.

A cell is a complex functional system that regulates its life support. The diversity of cell functions is provided by spatial and temporal (primarily, depending on the rhythm of nutrition) regulation of certain metabolic pathways. Spatial regulation is associated with the strict localization of certain enzymes in various organelles. So, in the nucleus there are enzymes associated with the synthesis of DNA and RNA molecules, in the cytoplasm - glycolysis enzymes, in lysosomes - hydrolytic enzymes, in the mitochondrial matrix - TCA enzymes, in the inner membrane of mitochondria - enzymes of the electron transport chain, etc.

Principles of regulation of metabolic pathways

All chemical reactions in the cell proceed with the participation of enzymes. Therefore, in order to influence the rate of the metabolic pathway, it is sufficient to regulate the amount or activity of enzymes. There are usually key enzymes in metabolic pathways that regulate the rate of the entire pathway. These enzymes (one or more in the metabolic pathway) are called regulatory enzymes; they catalyze, as a rule, the initial reactions of the metabolic pathway, irreversible reactions, rate-limiting reactions (the slowest ones), or reactions at the point of switching of the metabolic pathway (branch points).

Regulation of the rate of enzymatic reactions is carried out at 3 independent levels:

• change in the number of enzyme molecules;
• availability of substrate and coenzyme molecules;
• change in the catalytic activity of the enzyme molecule.

1. Regulation of the number of enzyme molecules
   in a cage.

It is known that proteins in the cell are constantly updated. The number of enzyme molecules in a cell is determined by the ratio of 2 processes - the synthesis and decay of the enzyme protein molecule:

Protein synthesis and folding is a multi-step process. Regulation of protein synthesis can occur at any stage of the formation of a protein molecule. The mechanism of regulation of the synthesis of a protein molecule at the level of transcription, which is carried out by certain metabolites, hormones, and a number of biologically active molecules, is the most studied.

As for the breakdown of enzymes, the regulation of this process is less studied. One can only assume that this is not just a process of proteolysis (destruction of a protein molecule), but a complex mechanism, possibly determined at the genetic level.

1. Rate regulation of enzymatic
   reactions by the availability of substrate molecules
   and coenzymes.

An important parameter that controls the flow of the metabolic pathway is the presence of substrates, and mainly the presence of the first substrate. The higher the concentration of the initial substrate, the higher the rate of the metabolic pathway.

Another parameter that limits the course of the metabolic pathway is the presence of regenerated coenzymes. For example, in dehydrogenation reactions, the coenzyme of dehydrogenases is the oxidized forms of NAD+, FAD, FMN, which are reduced during the reaction. In order for the coenzymes to participate in the reaction again, their regeneration is necessary, i.e. conversion to the oxidized form.

1. Regulation of catalytic activity
   enzymes

The regulation of the catalytic activity of one or more key enzymes of a given metabolic pathway plays a crucial role in changing the rate of metabolic pathways. This is a highly effective and fast way to regulate metabolism.

The main ways to regulate the activity of enzymes:

• allosteric regulation;
• regulation by protein-protein interactions;
• regulation by phosphorylation/dephosphorylation of the enzyme molecule;
• regulation by partial (limited) proteolysis.

Allosteric regulation. Allosteric enzymes are enzymes whose activity is regulated not only by the number of substrate molecules, but also by other substances called effectors. The effectors involved in allosteric regulation are often cellular metabolites of the very pathway they regulate.

Thus, the formation of glycogen from lactic acid in the liver seems to provide an important link between muscle and liver metabolism. With the participation of the liver, glycogen from the muscles is converted into available blood sugar, and this sugar, in turn, is converted into muscle glycogen. Consequently, there is a closed cycle of glucose molecule transformations in the body... Adrenaline has been shown to accelerate these reactions in the direction from muscle glycogen to liver glycogen... At the same time, insulin accelerates reactions in the direction from blood glucose to muscle glycogen.

C. F. Corey and G. T. Corey, from an article in Biological Chemistry, 1929

15. PRINCIPLES OF REGULATION OF METABOLISM

The regulation of metabolic reactions is the main content of research in biochemistry, and this is one of the most remarkable abilities of the living cell. Among the thousands of enzymatic reactions that occur in the cell, perhaps there is not one that would not be regulated in one form or another. Although it is common (and useful) in textbooks to subdivide the metabolic process into separate "pathways" that perform certain functions in the life support of the cell, such a division does not exist in the cell itself. Moreover, every pathway discussed in this book is inextricably linked to every other cellular process, as illustrated by the multidimensional reaction network (Figure 15-1). For example, in ch. 14, we discussed three possible pathways for the conversion of glucose-6-phosphate in liver cells: participation in glycolysis to store ATP, participation in the pentose phosphate pathway to produce NADPH and pentose phosphates, and hydrolysis to glucose and phosphate to replenish blood glucose stores. But in fact there are a number of other possible ways of transforming glucose-6-phosphate; it can, for example, be used to synthesize other sugars such as glucosamine, galactose, galactosamine, fucose, and neuraminic acid, participate in protein glycosylation, or be partially degraded to provide acetyl-CoA for the synthesis of fatty acids and sterols. For example, the bacterium Escherichia coli uses glucose to synthesize the carbon skeletons of absolutely all of its molecules. When a cell directs glucose-6-phosphate along one of the pathways, it affects all other pathways in which this substance is a precursor or intermediate. Any change in the distribution of glucose-6-phosphate in one metabolic pathway directly or indirectly affects its participation in all other pathways.

Similar changes in the distribution of metabolites often occur during the life of a cell. Louis Pasteur was the first to describe a significant increase in glucose consumption (more than 10 times) by a yeast culture during the transition from aerobic to anaerobic conditions. This phenomenon, called the Pasteur effect, is not accompanied by any noticeable fluctuations in the concentration of ATP or any other substance from hundreds of intermediates and products of glucose metabolism. Similar changes are observed in the skeletal muscle cells of a sprinter. Cells have an amazing ability to simultaneously and economically carry out all these interconnected metabolic transformations and receive each product in a strictly defined amount and at a strictly defined moment in time under changing environmental conditions.

Rice. 15-1. 3D network of metabolic reactions. A typical eukaryotic cell is capable of synthesizing about 30,000 different proteins, catalyzing thousands of reactions that produce hundreds of metabolites, many involved in multiple metabolic pathways. Illustration taken from the database KEGG PATHWAY (Kyoto Encyclopedia of Genes and Genomes www.genome.ad.jp/kegg/pathway/map/map0ll00.html). Each area can be considered in more detail, down to the level of individual enzymes and intermediates.

In this chapter, we will illustrate the basic principles of metabolic regulation using the example of glucose metabolism. We begin by looking at the general role of regulation in achieving metabolic homeostasis and introduce a theory of metabolic control that can be used to quantify complex metabolic processes. Next, we will dwell on the features of the regulation of individual enzymes of glucose metabolism and consider the catalytic activity of the enzymes involved in glycolysis and gluconeogenesis, described in Chap. 14. We will also discuss the catalytic and regulatory properties of enzymes involved in the synthesis and destruction of glycogen, one of the most studied examples of metabolic regulation. Choosing carbohydrate metabolism to illustrate the principles of metabolic regulation, we artificially separated it from fatty acid metabolism. In fact, these two processes in the cell are very closely related, as we will see in Chap. 23.

15.1. Regulation of metabolic pathways

Catabolism reactions in glycogen metabolism provide the energy needed to overcome the "forces" of entropy, and anabolism reactions lead to the formation of initial molecules for biosynthesis and the storage of metabolic energy. These processes are so important for the life of cells that in the course of evolution very complex regulatory mechanisms have arisen that ensure the movement of metabolites along the right paths, in the right direction and at the right speed in order to fully satisfy the current needs of the cell or organism; when external conditions change, the rate of transformations of metabolites in the corresponding metabolic pathways is corrected.

External conditions do change, sometimes quite strongly. With heavy physical exertion, the need for muscles in ATP can increase hundreds of times in a matter of seconds. Oxygen availability may decrease due to hypoxia (impaired oxygen delivery to tissues) or ischemia (reduced blood flow to tissues). The ratio of carbohydrates, fats and proteins in food varies, and energy-rich nutrients enter the body irregularly, as a result of which, between meals and during fasting, it becomes necessary to correct the ongoing metabolic processes. Enormous amounts of energy and molecules are required for biosynthesis, such as wound healing.

Cells and organisms exist in a dynamic stationary state

Energy-rich molecules, such as glucose, are absorbed by the cell, and metabolic waste products, such as CO 2, leave it, but the mass and composition of the cell, individual organ, or adult animal practically do not change over time; cells and organisms exist in a dynamic stationary state, but not in equilibrium with the environment. The substrate for each reaction of the metabolic pathway comes from the previous reaction at the same rate as it is further converted into a product. In other words, although the speed (v) of the flow of matter (or simply - flow) at this stage of metabolism can be high and vary greatly, the concentration of the substrate remains constant. For a two step reaction

at v 1 = v 2 the concentration is constant. For example, a change in the rate of entry of glucose from various sources into the blood is compensated by a change in v 2 absorption of glucose from the blood into tissues, thus, the concentration of glucose in the blood is maintained at about 5 mm. it homeostasis at the molecular level. In humans, a violation of the mechanisms of homeostasis is often the cause of diseases. For example, in diabetes mellitus, the regulation of blood glucose concentration is impaired due to a lack of insulin or insensitivity to it, which leads to detrimental health consequences.

When external influences are not limited to a mere temporary influence, or when a cell of one type is transformed into a cell of another type, the regulation of cell composition and metabolism may be more significant and require marked and lasting changes in the distribution of energy and starting materials for synthesis in order to accurately effect this transition. Imagine, for example, the process of differentiation of a bone marrow stem cell into an erythrocyte. The original cell contains a nucleus, mitochondria and little or no hemoglobin, while in a fully differentiated erythrocyte there is a huge amount of hemoglobin, but no nucleus or mitochondria. The composition of this cell constantly changed in response to signals coming from outside, and the metabolism changed accordingly. Cell differentiation requires precise regulation of cellular protein concentrations.

In the course of evolution, a remarkable set of regulatory mechanisms has emerged to maintain homeostasis at the level of molecules, cells, and whole organisms. The significance of metabolic regulation for an organism is reflected in the relative number of genes encoding elements of the regulatory apparatus: in humans, about 4000 genes (about 12% of all genes) encode regulatory proteins, including various receptors, gene expression regulators, and about 500 different protein kinases! Regulatory mechanisms operate in different time ranges (from seconds to days) and differ in sensitivity to changes in the external environment. In many cases, these mechanisms overlap: the same enzyme can be the object of regulation in several regulatory mechanisms.

Not only the number of enzymes is regulated, but also their catalytic activity

The intensity of the enzymatic process can be regulated both by changing the amount of enzymes and by modulating the catalytic activity of the enzyme molecules present. Such transformations occur in the time range from several milliseconds to several hours and serve as a response to an intracellular or external signal. Very rapid allosteric changes in enzymatic activity are usually initiated in situ by altering the local concentration of small molecules of a given metabolic pathway's substrate (in glycolysis-glucose reactions), a pathway product (ATP in glycolysis), or a key metabolite or cofactor (such as NADH) that is associated with metabolic capacity of the cell. Second messengers (such as cyclic AMP and Ca 2+), which are formed inside cells in response to extracellular signals (hormones, cytokines, etc.), also mediate allosteric regulation, but somewhat more slowly influencing the signal transduction mechanisms (see Ch. . 12).

Extracellular signals (Figure 15-2, F) can be hormonal (insulin or adrenaline), neuronal (acetylcholine), or mediated by growth factors or cytokines. The amount of this enzyme in the cell is determined by the ratio between the rates of its synthesis and degradation. The rate of synthesis is regulated by the activation (in response to some external signal) of the transcription factor (Fig. 15-2, (D ; see Chapter 28 for details). Transcription factors - are nuclear proteins that, after activation, bind to specific regions of DNA (responsive elements) near the gene promoter region (transcription start point) and activate or suppress the transcription of this gene, which leads to an increase or decrease in the production of the corresponding protein. Activation of a transcription factor often occurs as a result of its binding to a specific ligand, and sometimes is caused by its phosphorylation or dephosphorylation. Each gene is controlled by one or more response elements that are recognized by specific transcription factors. Some genes contain several response elements and are therefore controlled by several different transcription factors that respond to several different signals. Groups of genes encoding proteins whose actions are interrelated, as in the case of the enzymes of glycolysis or gluconeogenesis, often contain response elements with the same sequence, so that the same signal acting through a certain transcription factor turns on or off the entire group of genes at the same time. In sec. 15.3 discusses the regulation of carbohydrate metabolism by specific transcription factors.

The resistance of mRNA molecules to ribonucleases (Fig. 15-2, (D) can be different, so that the amount of mRNA of a given type in a cell is a function of the rate of its synthesis and degradation (Chapter 26). Finally, the rate of translation of mRNA on ribosomes ( Fig. 15-2, (4)) is also regulated and depends on several factors, detailed in chapter 27.

Rice. 15-2. Factors affecting the activity of enzymes. The overall activity of an enzyme can change due to a change in the number of molecules of a given (quantity) enzyme in a cell, its effective activity in a particular cellular compartment ((1)-(6)) or modulation of the activity of existing enzyme molecules, as detailed in the text. The activity of a particular enzyme is determined by a combination of these factors.

Note that an n-fold increase in mRNA production does not always mean an n-fold increase in the corresponding protein synthesis.

The resulting protein molecule exists for a limited time, namely, from several minutes to many days (Table 15-1). The rate of degradation of enzymes (Fig. 15-2, (5)) is also different and is determined by intracellular conditions. Some proteins undergo degradation in the proteasome (see Chapter 28) as a result of covalent binding to ubiquitin (remember the cyclin protein; see Figure 12-46). Quick turnover(synthesis with subsequent degradation) is associated with high energy costs, however, proteins with a shorter half-life (the time during which half of the original amount of a substance remains) can reach a new steady state in their content faster than proteins with a long half-life, and the gain from such a fast reactions must balance or be greater than the energy costs of the cell.

Table 15-1. Approximate half-life of proteins in mammalian organs

Another factor affecting the effective activity of an enzyme is the availability of its substrate (Fig. 15-2, (6)). Muscle hexokinase cannot act on glucose until that sugar is taken from the blood to muscle cells, and the rate at which glucose enters the cells depends on carrier molecules (see Table 11-3) in the plasma membrane. Inside the cell, some enzymes and enzyme systems are contained in various membrane-bound compartments; the delivery of substrates to these compartments may be a limiting factor for the enzyme.

Due to the presence of these several mechanisms of regulation of enzymatic activity, cells are able to significantly change the set of enzymes in response to changing metabolic conditions. In vertebrates, the most adaptable organ is the liver; for example, replacing a carbohydrate-rich meal with a high-lipid meal affects the transcription of hundreds of genes and hence the synthesis of hundreds of proteins. Such global changes in gene expression can be quantified using DNA microarrays (see Fig. 9-22), which allow analysis of the entire set of mRNA of a given cell or organ type. (transcriptome), or using 2D gel electrophoresis (see Figure 3-21), a method for examining all proteins in a given cell type or organ (proteome). Both of these methods are very useful in studies of metabolic regulation. Changes in the proteome often entail changes in the entire ensemble of low molecular weight metabolites. - metabolome.

After a certain amount of each enzyme is formed in the cell as a result of the action of regulatory mechanisms that control protein synthesis and degradation, the activity of these enzymes is further subject to regulation: by changing the concentration of substrates; by exposure to allosteric effectors; by covalent modification; or by binding regulatory proteins. All these processes can change the activity of individual enzyme molecules (Fig. 15-2, (7)-(10)).

All enzymes are sensitive to the concentration of their substrates (Fig. 15-2, (7)). Recall that in the simplest case (under the conditions of Michaelis-Menten kinetics), the initial reaction rate is half the maximum rate at a substrate concentration equal to the Km value (i.e., when the enzyme is half-saturated with the substrate). With a decrease in the concentration of the substrate, the reaction rate also decreases, and at "K m, the reaction rate depends linearly on . This is important to remember because the intracellular substrate concentration is often close to or below Km. For example, hexokinase activity is dependent on glucose concentration, and intracellular glucose concentration varies with blood glucose concentration. As we will see below, different forms (isoforms) of hexokinase correspond to different Km, and, consequently, the presence of different isoforms of hexokinase depends on the intracellular concentration of glucose, which has a certain physiological significance.

Example 15-1. Glucose transporter activity

If for the glucose transporter in the liver (GLU T2) Kt (equivalent to Km) = 40 mM, determine the change in the rate of entry (flow) of glucose into hepatocytes with an increase in blood glucose concentration from 3 to 10 mM.

Solution. To determine the initial rate of glucose intake, use equation 11-1 (vol. 1, p. 555).

At 3 mM glucose

V 0 \u003d V m ah (3 mM) / (40 mM + 3 mM) \u003d V m ah (3 mM / 43 mM) \u003d 0.07 V m ah At 10 mM glucose

V 0 \u003d V m ah (10 mM) / (40 mM + 10 mM) \u003d V m ah (10 mM / 50 mM) \u003d 0.20 V m ah

Thus, if the concentration of glucose in the blood increased from 3 to 10 mM, this means that the rate of glucose flow into hepatocytes increased by almost 3 times (0.20/0.07).

Enzymatic activity can be increased or decreased by allosteric effectors (Fig. 15-2, (8); see also Fig. 6-34). Under the influence of allosteric effectors, the reaction kinetics usually becomes S-shaped instead of hyperbolic, or vice versa (for example, see Fig. 15-14, b). In the steepest part of the S-curve, small changes in the concentration of the substrate or allosteric effector can significantly affect the reaction rate. As we discussed in Chap. 5 (p. 239, v. 1), to describe the behavior of allosteric enzymes, the Hill cooperativity coefficient is used, and a large value of this coefficient means a higher cooperativity. For an allosteric enzyme with a Hill coefficient of 4, a threefold increase in the substrate concentration leads to an increase in the reaction rate from 0.1 Vm ax to 0.9 Vm ax, while for an enzyme that does not have the property of cooperativity (Hill coefficient 1; see Table. 15-2), for the same change in enzymatic activity, an increase in the concentration of the substrate by 81 times is required!

Covalent modifications of an already existing enzyme or other protein (Fig. 15-2, (9)) occur within seconds or minutes from the moment a signal, usually an extracellular one, arrives. The most common modification is phosphorylation-dephosphorylation (Fig. 15-3); up to half of all proteins in a eukaryotic cell undergo phosphorylation under certain conditions. Phosphorylation can change the electrostatic properties of the active center of the enzyme, shift the inhibitory site of the protein away from the active center, affect the interaction of this protein with other molecules, or cause conformational changes leading to changes in Vm ax and K m. could return the protein to its original state. A family of phosphoprotein phosphatases, some members of which are themselves under control, catalyze the dephosphorylation of proteins that have been phosphorylated by protein kinases.

Table 15-2. Relationship between the Hill coefficient and the effect of substrate concentration on the reaction rate for allosteric enzymes

Rice. 15-3. Phosphorylation-dephosphorylation of a protein. Protein kinases transfer a phosphoryl group from ATP to Ser, Thr, or Tyr residues in the protein. Protein phosphatases remove the phosphoryl group as P i .

Finally, the regulation of many enzymes is achieved by binding to regulatory proteins (Fig. 15-2, (10)). For example, cAMP-dependent protein kinase (PKA; see Fig. 12-6) remains inactive until cAMP binding separates the catalytic and regulatory subunits of the enzyme.

The considered mechanisms of influence on the rate of a certain reaction of the metabolic pathway do not exclude each other. Quite often, the same enzyme is regulated at the level of transcription, as well as through allosteric mechanisms and covalent binding. The combination of these mechanisms provides fast and efficient regulation in response to a wide variety of changes in the cell and incoming signals.

For the discussion that follows, it is useful to consider the changes in enzymatic activity when performing two different, but nonetheless complementary, functions. term metabolic regulation we will denote a process aimed at maintaining homeostasis at the molecular level, i.e., maintaining certain cellular parameters (such as metabolite concentrations) even when the metabolite flux in a given metabolic pathway changes. term metabolic control we will call the processes leading to a change in the result of the metabolic pathway in time in response to some external signals or changing conditions. It should be said, however, that it is not always easy to draw a clear line between these two concepts.

Usually, reactions far from equilibrium are regulated in the cell.

At some stages of the metabolic pathway, the reactions approach an equilibrium state (Fig. 15-4). The total flow of metabolites in such reactions is determined by a small difference between the rates of the forward and reverse reactions, which, when approaching the equilibrium state, have close values. Small changes in the concentration of the substrate or reaction product can greatly change the overall rate of the process and even its direction. We can identify these almost equilibrium reactions in the cell if we compare the ratio of the acting masses Q with the equilibrium constant of the reaction K "eq. Recall that for the reaction A + B -> C + D Q \u003d [C] / [A] [B] . It is believed that when Q and K "eq differ only by 1-2 orders of magnitude, the reaction is close to equilibrium. For example, this is observed for six out of 10 glycolysis reactions (Table 15-3).

Rice. 15-4. Equilibrium and non-equilibrium stages of metabolism. In the cell, stages (2) and (3) of this pathway are almost in equilibrium; the rates of their direct reactions are only slightly higher than the rates of the reverse reactions, so that the overall rate (10) is rather low, and the change in free energy ∆G' for each of these stages is close to zero. Increasing the intracellular concentration of metabolites C or D can change the direction of these steps. Stage (1) in the cell is far from equilibrium - the rate of the forward reaction far exceeds the rate of the reverse reaction. The total rate of stage (1) (10) is much higher than the rate of the reverse reaction (0.01) and in the stationary state is equal to the rates of stages (2) and (3). Stage (1) is characterized by a large negative value of ∆G'.

Many reactions in the cell, however, are far from equilibrium. For example, for the glycolysis reaction catalyzed by phosphofructokinase-1 (PPK-1), K "eq ≈ 1000, and for a typical cell in a stationary state, Q \u003d [fructose-1,6-bisphosphate] [AD P] / [fructose-6- phosphate][ATP]) ≈ 0.1 (Table 15-3). Precisely because this reaction is so far from equilibrium, under intracellular conditions, this process is exergonic and proceeds in the forward direction. This reaction is far from equilibrium, since at At normal intracellular concentrations of the substrate, product, and effector, the rate of conversion of fructose-6-phosphate to fructose-1,6-bisphosphate is limited by the activity of PFK-1, which is regulated by the number of PFK-1 molecules and the action of effectors.Thus, the rate of the direct process coincides with the rate of the general the flow of glycolysis intermediates in other reactions of this pathway, and the reverse flow rate in the reaction involving PFK-1 is almost zero.

Table 15-3. Equilibrium constants, mass action ratios, and changes in the free energy of enzymatic reactions during carbohydrate metabolism

* For simplicity, we assume that all reactions for which ∆G′<6, близки к равновесию.

The cell cannot allow reactions with large equilibrium constants to approach equilibrium. If at normal cellular concentrations of fructose-6-phosphate, ATP and ADP (a few millimoles) the reaction catalyzed by PFK-1 could reach equilibrium, then the concentration of fructose-1,6-bisphosphate would be in the molar range, which would lead to cell death from due to high osmotic pressure.

Let's consider another example. If in a cell the reaction ATP —> ADP + Pi could approach equilibrium, for this reaction the change in free energy ∆G′ —> 0 (∆Gp ; see example 13-2, p. 31); as a result, ATP would lose its high potential as a carrier of phosphate groups, which is so necessary for the cell. Therefore, it is very important that the enzymes that catalyze the decomposition of ATP and other exergonic reactions in the cell are subject to regulation, i.e., when metabolic processes change as a result of external influences, the reactions involving these enzymes are corrected so that the ATP concentration remains much higher than the equilibrium level . With such changes in metabolism, the activities of enzymes in all interconnected metabolic pathways are adjusted, which does not allow critical stages to reach equilibrium. Therefore, it is not surprising that many enzymes (such as PFK-1) that catalyze reactions with large negative free energy changes are finely regulated in many different ways. This regulation occurs in such a complex way that when studying the properties of only one enzyme of the metabolic pathway, it is impossible to determine how much influence this enzyme has on the course of the process as a whole; for this, it is necessary to involve the theory of metabolic control, which we will address in Sec. 15.2.

Adenine nucleotides play a special role in the regulation of metabolism

Perhaps the second most important task of the cell (after protecting against DNA damage) is to maintain a constant supply of ATP. Many ATP-dependent enzymes have Km between 0.1 and 1 mM, and the normal concentration of ATP in the cell is 5 mM. If the concentration of ATP were much lower, these enzymes would not be able to achieve saturation with their substrate (ATP), resulting in a decrease in the rate of hundreds of reactions that occur with the participation of ATP (Fig. 15-5). The cell probably could not survive such a kinetic impact on such a large number of reactions.

In addition, the decrease in ATP concentration has important thermodynamic consequences. Because ATP is converted to ADP or AMP when doing work in the cell, the ratio / has a profound effect on the course of all reactions in which these cofactors are involved. The same applies to other cofactors - NADH /NAD + and NADPH /NADP +.

Rice. 15-5. Effect of ATP concentration on the initial rate of a reaction catalyzed by a typical ATP-dependent enzyme. Based on these experimental data for ATP, K m ≈ 5 mM. In animal tissues, the [ATP] concentration is ≈ 5 mM.

For example, consider the reaction catalyzed by hexokinase:

Note that this expression is only true when the reactants and reaction products are in equilibrium concentrations at which ∆G' = 0. For any other concentration, ∆G' ≠ 0. Recall (ch. 13) that the concentration ratio reaction products to substrate concentrations (the ratio of the effective masses Q determines the magnitude and sign of ∆G' and, consequently, the driving force (∆G') of the reaction:

Since a change in this driving force affects all reactions involving ATP, in the process of evolution, organisms have developed regulatory mechanisms responsible for maintaining the / ratio.

The concentration of AMP is much more sensitive to the energy state of the cell than the concentration of ATP. Usually, in cells, the concentration of ATP (5-10 mM) is much higher than the concentration of AMP (<0,1 мМ). При расходовании АТР, например при мышечном сокращении, АМР образуется в результате двустадийного процесса. Сначала при гидролизе АТР образуется ADP , а затем в результате действия adenylate kinase- AMP:

2ADP AMP + ATP

With a decrease in the concentration of ATP by 10%, the relative increase in the concentration of AMP is more significant than for ADP (Table 15-4). Therefore, it is not surprising that many regulatory processes are associated precisely with the concentration of AMP. plays an important role as a mediator of regulation AMP-dependent protein kinase, which, with an increase in AMP concentration, begins to phosphorylate key proteins, thereby regulating their activity. An increase in [AMP] may be due to insufficient intake of nutrients or to a large physical load. The action of AMP-dependent protein kinase (not to be confused with cAMP-dependent protein kinase, see section 15.5) enhances glucose transport, activates glycolysis and fatty acid oxidation, but at the same time suppresses such energy-consuming processes as the synthesis of fatty acids, cholesterol and proteins ( Fig. 15-6). In ch. 23 we will discuss this enzyme and its mechanism of action in these processes in more detail.

Table 15-4. Relative changes in ATP and AMP concentrations upon consumption of ATP or functional groups

Rice. 15-6. The role of AMP-dependent protein kinase (AMPK) in the metabolism of fats and carbohydrates. During exercise, AMPK is activated in response to an increase in AMP or a decrease in ATP by signals from the sympathetic nervous system (SNS) or adipose tissue hormones (leptin and adiponectin, see Chapter 23 for details). Activated AMPK phosphorylates key proteins and thereby regulates metabolism in many tissues, inhibiting such energy-consuming processes as the synthesis of glycogen, fatty acids and cholesterol; directs metabolism outside the liver to use fatty acids as fuel molecules; and in the liver it triggers gluconeogenesis to supply the brain with glucose. In the hypothalamus, AMPK stimulates eating behavior so that the body receives more nutrients.

Along with ATP, hundreds of metabolic intermediates must be present in the cell in the required concentrations. For example, glycolysis intermediates dihydroxyacetone phosphate and 3-phosphoglycerate serve as precursors for triacylglycerols and serine, respectively. If necessary, the rate of glycolysis should be adjusted so as to provide the required amount of these substances without reducing the level of ATP formation. The same pattern is true for other important cofactors such as NADH and NADPH: changing the ratio of their effective masses (i.e. the ratio of the concentration of the reduced form of the cofactor to the concentration of its oxidized form) has a very strong effect on metabolism.

Of course, the evolutionary development of regulatory mechanisms was also influenced by the priorities that arise in the life of the whole organism. In the brain of mammals, energy reserves are practically absent, so brain activity is completely dependent on the supply of glucose through the bloodstream. When the blood glucose level decreases by 2 times compared to the norm (4-5 mM), brain activity is disturbed, and a 5-fold decrease in blood glucose levels leads to a state of coma and death. The hormones insulin and glucagon, which are released during high and low glucose levels, respectively, help maintain normal blood glucose levels; these hormones trigger a series of metabolic reactions aimed at normalizing glucose levels.

In addition, in the course of evolution, another selective influence should have been carried out, which led to the selection of regulatory mechanisms aimed at solving quite specific problems.

1. Ensuring maximum energy efficiency by preventing the simultaneous occurrence of reactions of oppositely directed metabolic pathways (for example, glycolysis and gluconeogenesis).

2. Distribution of metabolites between alternative metabolic pathways (such as glycolysis and the pentose phosphate pathway).

3. Choosing the most suitable energy source for solving the current tasks of the body (glucose, fatty acids, glycogen or amino acids).

4. Stopping the ways of biosynthesis during the accumulation of its products.

The following chapters provide many examples of each type of regulatory mechanism.

Summary of section 15.1. Regulation of metabolic pathways

■ In a cell with an active metabolism, which is in a stationary state, metabolic intermediates are formed and consumed at the same rate. If, as a result of any influences, the rate of formation or consumption of a metabolite changes, a compensatory change in the activities of enzymes occurs in the cell, leading to the restoration of a stationary state.

■ Cells regulate their metabolism using various mechanisms in the time range from milliseconds to several days, changing the activity of already existing enzymes or the number of synthesized molecules of a specific enzyme.

■ Various signals can activate or inactivate transcription factors that regulate gene expression in the cell nucleus. Changes in the transcriptome lead to changes in the proteome and, ultimately, in the metabolome of the cell or tissue.

■ In multi-step processes such as glycolysis, some steady state reactions are close to equilibrium; the rates of these reactions are controlled by the substrate concentration and decrease and increase with its change. Other reactions are far from equilibrium; they usually control the entire flow of substances in a given metabolic pathway.

■ Regulatory mechanisms are aimed at maintaining in cells an almost constant level of key metabolites, such as ATP and NADH, or blood glucose; when the needs of the body change, glycogen stores are used.

Chemical reactions in cells are catalyzed by enzymes. It is not surprising, therefore, that most methods of regulating metabolism are based on two leading processes: changes in the concentration of enzymes and their activity. These methods of metabolic regulation are characteristic of all cells and are carried out using a variety of mechanisms in response to signals of various kinds. In addition, cells have additional ways of regulating metabolism, the variety of which is convenient to consider in accordance with several levels of organization.

Transcriptional regulation. This type of regulation is discussed in Chapter 3 with several examples of positive and negative transcriptional control of prokaryotic genes. This mechanism is characteristic, first of all, for the regulation of the amount of mRNA that determines the structure of enzymes, and besides this, histone proteins, ribosomal, and transport proteins. The group of the latter, not possessing catalytic activity, also takes a great part in changing the rate of the corresponding processes (the formation of chromosomes and ribosomes, the transport of substances through membranes), and hence the metabolism as a whole.

Regulatory proteins are involved in the regulation of gene transcription, the structure of which is determined by specific genes (regulators), their complexes with ligands(for example, lactose during transcription induction or tryptophan during repression), cAMP-CAP complexes, guanosine tetraphosphate, and in some cases, proteins - products of expression of their own genes have this effect. Such important signaling molecules as cAMP and guanosine tetraphosphate are of particular importance in these processes. We can say that cAMP signals the cell about energy hunger - the absence of glucose. In response to this, the frequency of transcription of structural genes responsible for the catabolism of other sources of carbon and energy increases (activation of catabolite operons, catabolite repression, Chapter 3). Guanosine tetraphosphate (guanosine-5'-diphosphate-3'-diphosphate) is an amino acid starvation signal. This nucleotide binds to RNA polymerase and changes its affinity for various gene promoters. As a result, the expression of genes responsible for the biosynthesis of carbohydrates, lipids, nucleotides, etc. decreases, while the expression of other genes, in particular those determining the processes of protein proteolysis, on the contrary, increases.

The transcription process is more often regulated by changing the frequency of transcription initiation events, but, in addition, the rate of transcription elongation and the frequency of its premature termination can be regulated. The events of elongation and termination are primarily influenced by the conformational state of DNA or mRNA itself (presence of "stop signals", hairpin structures).

Allosteric regulation of enzyme activity. This type of regulation is one of the fastest and most flexible, and is carried out with the help of effector molecules interacting with the allosteric center of the enzyme (Chapter 6). Allosteric regulation, as well as operon, are subject to key enzymes certain metabolic pathways. Thus, the rate of the entire biosynthetic or catabolic process depends on one or more rarely several reactions catalyzed by key enzymes.

Regulation is of particular importance for the biosynthesis of proteinogenic amino acids. Since there are 20 of them, and each of them is presented in a certain ratio in the total cellular protein in different organisms, very clear regulation is required, coordinating the processes of synthesis of individual amino acids. Such control eliminates the overproduction of amino acids, and their release from the cell is possible only in deregulated microorganisms.

An example of the regulation of the biosynthesis of amino acids of the aspartate family in enterobacteria is shown in Fig. 19.3. Four amino acids share a common precursor, aspartic acid. Its conversion to aspartyl phosphate in E. coli bacteria is catalyzed by three isozyme forms of aspartokinase, each of which is repressed and/or inhibited by different end products of this branched metabolic pathway. The synthesis of homoserine dehydrogenase is regulated in a similar way.

Note the existence of a mechanism feedback, which lies in the fact that the end products of metabolic processes regulate the level of synthesis and / or activity of enzymes that catalyze the first steps in the formation of these metabolites.

Allosteric effectors can be a variety of substances: substrates and end products of metabolic pathways, sometimes intermediate metabolites; in catabolic processes, nucleoside diphosphates and nucleoside triphosphates, as well as carriers of reducing equivalents; in cascade reactions - cAMP and cGMP, which regulate the activity of enzymes (for example, protein kinases) involved in the covalent modification of proteins; metal ions and many other compounds. Examples of allosteric regulation of enzymes are given in Chapter 6 and other sections.

Covalent modification of enzymes. This type of regulation of enzyme activity is otherwise called enzyme interconversions, since the essence of this process is the transformation of active forms of enzymes into inactive ones and vice versa. Features and examples of covalent modification are described in Chapter 6. These processes are under various control, including hormonal. A classic example of enzyme interconversions is the regulation of glycogen metabolism in the liver.

The rate of synthesis of this reserve polysaccharide is controlled by glycogen synthase, and cleavage is catalyzed by glycogen phosphorylase. Both enzymes can be in active and inactive forms. During starvation or in stressful situations, hormones are released into the blood - adrenaline and glucagon, which bind to receptors on the plasma membranes of cells and activate the enzyme adenylate cyclase (catalyses the synthesis of cAMP) through G-proteins. cAMP binds to protein kinase A and activates it, which leads to phosphorylation of glycogen synthase and its conversion to an inactive form. Glycogen stops being synthesized. In addition, protein kinase A in the course of cascade reactions causes phosphorylation of glycogen phosphorylase, which as a result is activated and begins to break down glycogen. Another hormone, insulin, also acts on the processes of glycogen synthesis and breakdown. In this example, the signaling molecules are hormones and the messengers are G-protein and cAMP. Interconversions of enzymes are carried out during phosphorylation-dephosphorylation.

Hormonal regulation. This type of regulation of metabolism involves the participation of hormones - signaling substances formed in the cells of the endocrine glands, so hormonal regulation is characteristic only of higher organisms. The action of hormones on the process of glycogen metabolism, in which the activity of enzymes is regulated at the level of covalent modification, has been described above. In addition, hormones can affect the rate of transcription (operon regulation).

From specialized cells where hormones are synthesized, the latter enter the bloodstream and are transferred to target cells that have receptors that can bind hormones and thereby perceive a hormonal signal. Binding of a hormone to a receptor triggers a cascade of reactions involving mediator molecules that culminate in a cellular response. Lipophilic hormones bind to an intracellular receptor (protein) and regulate the transcription of certain genes. Hydrophilic hormones act on target cells by binding to receptors on the plasma membrane.

In addition to hormones, other signaling substances have a similar effect: mediators, neurotransmitters, growth factors. There is no clear boundary to distinguish hormones from the listed substances. Mediators are called signaling substances that are produced not by endocrine glands, but by various types of cells. Mediators include histamine, prostaglandins, which have a hormone-like effect.

Neurotransmitters are considered signaling substances produced by the cells of the central nervous system.

Change in the concentration of metabolites . An important condition that ensures a high rate of one or another metabolic pathway is the concentration of substrates. It may depend on the intensity of other processes that also consume these substrates (competition), or on the rate of transport of these substances through membranes (plasma or organelles). In particular, in eukaryotic cells, it becomes possible to regulate metabolism by redistributing metabolites in separate compartments.

In addition, the rate of metabolic processes is determined by the concentration of cofactors. For example, glycolysis and TCA are regulated by the availability of ADP (chapter 10, 11) at the level of changes in the activity of key allosteric enzymes.

Post-transcriptional and post-translational modification of macromolecules. These processes are also described in the relevant sections (Chapter 3). Modification and/or processing of primary RNA transcripts are carried out at different rates, which determines the concentration of mature RNA molecules capable of translation, and hence the intensity of protein synthesis. In turn, peptides, before turning into a mature protein, must also be modified, and if this concerns enzymes, then we are talking about their covalent modification.