TRICARBOXYLIC ACIDS CYCLE (KREBS CYCLE)

Glycolysis converts glucose into pyruvate and produces two ATP molecules from a glucose molecule—a small fraction of that molecule's potential energy.

Under aerobic conditions, pyruvate is converted from glycolysis to acetyl-CoA and oxidized to CO2 in the tricarboxylic acid cycle (citric acid cycle). In this case, the electrons released in the reactions of this cycle pass through NADH and FADH 2 to 0 2 - the final acceptor. Electron transport is associated with the creation of a proton gradient in the mitochondrial membrane, the energy of which is then used for the synthesis of ATP as a result of oxidative phosphorylation. Let's look at these reactions.

Under aerobic conditions, pyruvic acid (1st stage) undergoes oxidative decarboxylation, more efficient than transformation into lactic acid, with the formation of acetyl-CoA (2nd stage), which can be oxidized to the final products of glucose breakdown - CO 2 and H 2 0 (3rd stage). G. Krebs (1900-1981), a German biochemist, having studied the oxidation of individual organic acids, combined their reactions into a single cycle. Therefore, the tricarboxylic acid cycle is often called the Krebs cycle in his honor.

The oxidation of pyruvic acid to acetyl-CoA occurs in mitochondria with the participation of three enzymes (pyruvate dehydrogenase, lipoamide dehydrogenase, lipoyl acetyltransferase) and five coenzymes (NAD, FAD, thiamine pyrophosphate, lipoic acid amide, coenzyme A). These four coenzymes contain B vitamins (B x, B 2, B 3, B 5), which indicates the need for these vitamins for the normal oxidation of carbohydrates. Under the influence of this complex enzyme system, pyruvate is converted in an oxidative decarboxylation reaction into the active form of acetic acid - acetyl coenzyme A:

Under physiological conditions, pyruvate dehydrogenase is an exclusively irreversible enzyme, which explains the impossibility of converting fatty acids into carbohydrates.

The presence of a high-energy bond in the acetyl-CoA molecule indicates the high reactivity of this compound. In particular, acetyl-CoA can act in mitochondria to generate energy; in the liver, excess acetyl-CoA is used for the synthesis of ketone bodies; in the cytosol it participates in the synthesis of complex molecules such as steroids and fatty acids.

Acetyl-CoA obtained in the reaction of oxidative decarboxylation of pyruvic acid enters the tricarboxylic acid cycle (Krebs cycle). The Krebs cycle, the final catabolic pathway for the oxidation of carbohydrates, fats, and amino acids, is essentially a “metabolic cauldron.” The reactions of the Krebs cycle, which occur exclusively in mitochondria, are also called the citric acid cycle or the tricarboxylic acid cycle (TCA cycle).

One of the most important functions of the tricarboxylic acid cycle is the generation of reduced coenzymes (3 molecules of NADH + H + and 1 molecule of FADH 2) followed by the transfer of hydrogen atoms or their electrons to the final acceptor - molecular oxygen. This transport is accompanied by a large decrease in free energy, part of which is used in the process of oxidative phosphorylation for storage in the form of ATP. It is clear that the tricarboxylic acid cycle is aerobic, oxygen dependent.

1. The initial reaction of the tricarboxylic acid cycle is the condensation of acetyl-CoA and oxaloacetic acid with the participation of the mitochondrial matrix enzyme citrate synthase to form citric acid.

2. Under the influence of the enzyme aconitase, which catalyzes the removal of a water molecule from citrate, the latter turns

to cis-aconitic acid. Water combines with cis-aconitic acid, turning into isocitric acid.

3. The enzyme isocitrate dehydrogenase then catalyzes the first dehydrogenase reaction of the citric acid cycle, when isocitric acid is converted by oxidative decarboxylation to α-ketoglutaric acid:

In this reaction, the first molecule of CO 2 and the first molecule of NADH 4- H + cycle are formed.

4. Further conversion of α-ketoglutaric acid to succinyl-CoA is catalyzed by the multienzyme complex of α-ketoglutaric dehydrogenase. This reaction is chemically analogous to the pyruvate dehydrogenase reaction. It involves lipoic acid, thiamine pyrophosphate, HS-KoA, NAD +, FAD.

As a result of this reaction, a molecule of NADH + H + and CO 2 is again formed.

5. The succinyl-CoA molecule has a high-energy bond, the energy of which is stored in the next reaction in the form of GTP. Under the influence of the enzyme succinyl-CoA synthetase, succinyl-CoA is converted into free succinic acid. Note that succinic acid can also be obtained from methylmalonyl-CoA by oxidation of fatty acids with an odd number of carbon atoms.

This reaction is an example of substrate phosphorylation, since the high-energy GTP molecule in this case is formed without the participation of the electron and oxygen transport chain.

6. Succinic acid is oxidized to fumaric acid in the succinate dehydrogenase reaction. Succinate dehydrogenase, a typical iron-sulfur-containing enzyme, the coenzyme of which is FAD. Succinate dehydrogenase is the only enzyme anchored to the inner mitochondrial membrane, while all other cycle enzymes are located in the mitochondrial matrix.

7. This is followed by the hydration of fumaric acid into malic acid under the influence of the fumarase enzyme in a reversible reaction under physiological conditions:

8. The final reaction of the tricarboxylic acid cycle is the malate dehydrogenase reaction with the participation of the active enzyme mitochondrial NAD~-dependent malate dehydrogenase, in which the third molecule of reduced NADH + H + is formed:

The formation of oxaloacetic acid (oxaloacetate) completes one revolution of the tricarboxylic acid cycle. Oxalacetic acid can be used in the oxidation of a second molecule of acetyl-CoA, and this cycle of reactions can be repeated many times, constantly leading to the production of oxaloacetic acid.

Thus, the oxidation of one molecule of acetyl-CoA in the TCA cycle as a substrate of the cycle leads to the production of one molecule of GTP, three molecules of NADP + H + and one molecule of FADH 2. Oxidation of these reducing agents in the biological oxidation chain

lenition leads to the synthesis of 12 ATP molecules. This calculation is clear from the topic “Biological oxidation”: the inclusion of one NAD + molecule in the electron transport system is ultimately accompanied by the formation of 3 ATP molecules, the inclusion of a FADH 2 molecule ensures the formation of 2 ATP molecules, and one GTP molecule is equivalent to 1 ATP molecule.

Note that two carbon atoms of adetyl-CoA enter the tricarboxylic acid cycle and two carbon atoms leave the cycle as CO 2 in decarboxylation reactions catalyzed by isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase.

With the complete oxidation of a glucose molecule under aerobic conditions to C0 2 and H 2 0, the formation of energy in the form of ATP is:

• 4 molecules of ATP during the conversion of a glucose molecule into 2 molecules of pyruvic acid (glycolysis);
• 6 ATP molecules formed in the 3-phosphoglyceraldehyde dehydrogenase reaction (glycolysis);
• 30 ATP molecules formed during the oxidation of two molecules of pyruvic acid in the pyruvate dehydrogenase reaction and in the subsequent transformations of two molecules of acetyl-CoA to CO 2 and H 2 0 in the tricarboxylic acid cycle. Therefore, the total energy output from complete oxidation of a glucose molecule can be 40 ATP molecules. However, it should be taken into account that during the oxidation of glucose, two ATP molecules are consumed at the stage of converting glucose into glucose-6-phosphate and at the stage of converting fructose-6-phosphate into fructose-1,6-diphosphate. Therefore, the “net” energy output from the oxidation of a glucose molecule is 38 ATP molecules.

You can compare the energetics of anaerobic glycolysis and aerobic catabolism of glucose. Of the 688 kcal of energy theoretically contained in 1 gram molecule of glucose (180 g), 20 kcal is in two molecules of ATP formed in the reactions of anaerobic glycolysis, and 628 kcal theoretically remains in the form of lactic acid.

Under aerobic conditions, from 688 kcal of a gram molecule of glucose in 38 ATP molecules, 380 kcal are obtained. Thus, the efficiency of glucose use under aerobic conditions is approximately 19 times higher than in anaerobic glycolysis.

It should be noted that all oxidation reactions (oxidation of triose phosphate, pyruvic acid, four oxidation reactions of the tricarboxylic acid cycle) compete in the synthesis of ATP from ADP and phosphorus (Pasteur effect). This means that the resulting molecule NADH + H + in oxidation reactions has a choice between the reactions of the respiratory system, transferring hydrogen to oxygen, and the enzyme LDH, transferring hydrogen to pyruvic acid.

In the early stages of the tricarboxylic acid cycle, its acids can leave the cycle to participate in the synthesis of other cell compounds without disrupting the functioning of the cycle itself. Various factors are involved in the regulation of tricarboxylic acid cycle activity. Among them, primarily the supply of acetyl-CoA molecules, the activity of the pyruvate dehydrogenase complex, the activity of the components of the respiratory chain and associated oxidative phosphorylation, as well as the level of oxaloacetic acid should be mentioned.

Molecular oxygen is not directly involved in the tricarboxylic acid cycle, but its reactions are carried out only under aerobic conditions, since NAD ~ and FAD can be regenerated in mitochondria only by transferring electrons to molecular oxygen. It should be emphasized that glycolysis, in contrast to the tricarboxylic acid cycle, is also possible under anaerobic conditions, since NAD~ is regenerated during the transition of pyruvic acid to lactic acid.

In addition to the formation of ATP, the tricarboxylic acid cycle has another important meaning: the cycle provides intermediary structures for various biosyntheses of the body. For example, most of the atoms of porphyrins come from succinyl-CoA, many amino acids are derivatives of α-ketoglutaric and oxaloacetic acids, and fumaric acid occurs in the process of urea synthesis. This demonstrates the integrity of the tricarboxylic acid cycle in the metabolism of carbohydrates, fats, and proteins.

As the reactions of glycolysis show, the ability of most cells to generate energy lies in their mitochondria. The number of mitochondria in various tissues is associated with the physiological functions of the tissues and reflects their ability to participate in aerobic conditions. For example, red blood cells do not have mitochondria and therefore do not have the ability to generate energy using oxygen as the final electron acceptor. However, in cardiac muscle functioning under aerobic conditions, half the volume of the cell cytoplasm is represented by mitochondria. The liver also depends on aerobic conditions for its various functions, and mammalian hepatocytes contain up to 2 thousand mitochondria per cell.

Mitochondria include two membranes - outer and inner. The outer membrane is simpler, consisting of 50% fats and 50% proteins, and has relatively few functions. The inner membrane is structurally and functionally more complex. Approximately 80% of its volume is proteins. It contains most of the enzymes involved in electron transport and oxidative phosphorylation, metabolic intermediaries and adenine nucleotides between the cytosol and the mitochondrial matrix.

Various nucleotides involved in redox reactions, such as NAD +, NADH, NADP +, FAD and FADH 2, do not penetrate the inner mitochondrial membrane. Acetyl-CoA cannot move from the mitochondrial compartment to the cytosol, where it is required for the synthesis of fatty acids or sterols. Therefore, intramitochondrial acetyl-CoA is converted into the citrate synthase reaction of the tricarboxylic acid cycle and enters the cytosol in this form.

Krebs cycle

Tricarboxylic acid cycle (Krebs cycle, citrate cycle) - the central part of the general path of catabolism, a cyclic biochemical aerobic process during which the conversion of two- and three-carbon compounds formed as intermediate products in living organisms during the breakdown of carbohydrates, fats and proteins occurs to CO 2. In this case, the released hydrogen is sent to the tissue respiration chain, where it is further oxidized to water, directly participating in the synthesis of a universal energy source - ATP.

The Krebs cycle is a key stage in the respiration of all cells that use oxygen, the intersection of many metabolic pathways in the body. In addition to the significant energy role, the cycle also has a significant plastic function, that is, it is an important source of precursor molecules, from which, during other biochemical transformations, compounds important for the life of the cell are synthesized, such as amino acids, carbohydrates, fatty acids, etc.

The cycle of conversion of citric acid in living cells was discovered and studied by the German biochemist Hans Krebs, for this work he (together with F. Lipman) was awarded the Nobel Prize (1953).

Stages of the Krebs cycle

	Substrates	Products	Enzyme	Reaction type	A comment
1	Oxaloacetate + Acetyl-CoA+ H2O	Citrate + CoA-SH	Citrate synthase	Aldol condensation	limiting stage converts C4 oxaloacetate to C6
2	Citrate	cis-aconiat + H2O	aconitase	Dehydration	reversible isomerization
3	cis-aconiat + H2O	isocitrate		hydration
4	Isocitrate +	isocitrate dehydrogenase	Oxidation	NADH is formed (equivalent to 2.5 ATP)
5	Oxalosuccinate		α-ketoglutarate + CO2	decarboxylation	reversible stage C5 is formed
6	α-ketoglutarate + NAD++ CoA-SH	succinyl-CoA+ NADH+H++ CO2	alpha-ketoglutarate dehydrogenase	Oxidative decarboxylation	NADH is formed (equivalent to 2.5 ATP), regeneration of C 4 pathway (released by CoA)
7	succinyl-CoA+ GDP + Pi	succinate + CoA-SH+ GTP	succinyl coenzyme A synthetase	substrate phosphorylation	or ADP ->ATP, 1 ATP is formed
8	succinate + ubiquinone (Q)	fumarate + ubiquinol (QH 2)	succinate dehydrogenase	Oxidation	FAD is used as a prosthetic group (FAD->FADH 2 in the first stage of the reaction) in the enzyme, the equivalent of 1.5 ATP is formed
9	fumarate + H2O	L-malate	fumarase	H 2 O-addition (hydration)
10	L-malate + NAD+	oxaloacetate + NADH+H+	malate dehydrogenase	oxidation	NADH is formed (equivalent to 2.5 ATP)

The general equation for one revolution of the Krebs cycle is:

Acetyl-CoA → 2CO 2 + CoA + 8e −

Notes

Links

Wikimedia Foundation. 2010.

See what the “Krebs Cycle” is in other dictionaries:

- (citric and tricarboxylic acid cycle), a system of biochemical reactions through which most EUKARYOTIC organisms obtain their main energy as a result of the oxidation of food. Occurs in MITOCHONDRIA CELLS. Includes several chemical... ... Scientific and technical encyclopedic dictionary

Krebs cycle- Tricarboxylic acid cycle, a cycle of sequential reactions in the cells of aerobic organisms, as a result of which the synthesis of ATP molecules occurs Biotechnology topics EN Krebs cycle ... Technical Translator's Guide

Krebs cycle- - metabolic pathway leading to the complete destruction of acetyl CoA to the final products - CO2 and H2O ... A brief dictionary of biochemical terms

Krebs cycle- trikarboksirūgščių ciklas statusas T sritis chemija apibrėžtis Baltymų, riebalų ir angliavandenių oksidacinio skaidymo organizme ciklas. atitikmenys: engl. citric acid cycle; Krebs cycle; tricarboxylic acid cycle rus. Krebs cycle; lemon cycle... ... Chemijos terminų aiškinamasis žodynas

Tricarboxylic acid (Krebs, citric acid) cycle The most important cyclic sequence of metabolic reactions in aerobic organisms (eu and prokaryotes), as a result of which a sequential... ... Molecular biology and genetics. Dictionary.

Same as tricarboxylic acid cycle... Natural science. encyclopedic Dictionary

A complex cycle of reactions where enzymes act as catalysts; these reactions take place in the cells of all animals and consist in the decomposition of acetate in the presence of oxygen with the release of energy in the form of ATP (via the electron transfer chain) and... ... Medical terms

KREBS CYCLE, CITRIC ACID CYCLE- (citric acid cycle) a complex cycle of reactions where enzymes act as catalysts; these reactions take place in the cells of all animals and consist in the decomposition of acetate in the presence of oxygen with the release of energy in the form of ATP (via the transmission chain... ... Explanatory dictionary of medicine

KREBS CYCLE (tricarboxylic acid cycle- citric acid cycle) is a complex cyclic enzymatic process in which pyruvic acid is oxidized in the body to produce carbon dioxide, water and energy in the form of ATP; occupies a central position in the overall system... ... Dictionary of botanical terms

Cycle ... Wikipedia

Krebs cycle

Tricarboxylic acid cycle (Krebs cycle, citrate cycle) - the central part of the general path of catabolism, a cyclic biochemical aerobic process during which the conversion of two- and three-carbon compounds formed as intermediate products in living organisms during the breakdown of carbohydrates, fats and proteins occurs to CO 2. In this case, the released hydrogen is sent to the tissue respiration chain, where it is further oxidized to water, directly participating in the synthesis of a universal energy source - ATP.

The Krebs cycle is a key stage in the respiration of all cells that use oxygen, the intersection of many metabolic pathways in the body. In addition to the significant energy role, the cycle also has a significant plastic function, that is, it is an important source of precursor molecules, from which, during other biochemical transformations, compounds important for the life of the cell are synthesized, such as amino acids, carbohydrates, fatty acids, etc.

The cycle of conversion of citric acid in living cells was discovered and studied by the German biochemist Hans Krebs, for this work he (together with F. Lipman) was awarded the Nobel Prize (1953).

Stages of the Krebs cycle

	Substrates	Products	Enzyme	Reaction type	A comment
1	Oxaloacetate + Acetyl-CoA+ H2O	Citrate + CoA-SH	Citrate synthase	Aldol condensation	limiting stage converts C4 oxaloacetate to C6
2	Citrate	cis-aconiat + H2O	aconitase	Dehydration	reversible isomerization
3	cis-aconiat + H2O	isocitrate		hydration
4	Isocitrate +	isocitrate dehydrogenase	Oxidation	NADH is formed (equivalent to 2.5 ATP)
5	Oxalosuccinate		α-ketoglutarate + CO2	decarboxylation	reversible stage C5 is formed
6	α-ketoglutarate + NAD++ CoA-SH	succinyl-CoA+ NADH+H++ CO2	alpha-ketoglutarate dehydrogenase	Oxidative decarboxylation	NADH is formed (equivalent to 2.5 ATP), regeneration of C 4 pathway (released by CoA)
7	succinyl-CoA+ GDP + Pi	succinate + CoA-SH+ GTP	succinyl coenzyme A synthetase	substrate phosphorylation	or ADP ->ATP, 1 ATP is formed
8	succinate + ubiquinone (Q)	fumarate + ubiquinol (QH 2)	succinate dehydrogenase	Oxidation	FAD is used as a prosthetic group (FAD->FADH 2 in the first stage of the reaction) in the enzyme, the equivalent of 1.5 ATP is formed
9	fumarate + H2O	L-malate	fumarase	H 2 O-addition (hydration)
10	L-malate + NAD+	oxaloacetate + NADH+H+	malate dehydrogenase	oxidation	NADH is formed (equivalent to 2.5 ATP)

The general equation for one revolution of the Krebs cycle is:

Acetyl-CoA → 2CO 2 + CoA + 8e −

Notes

Links

Wikimedia Foundation. 2010.

• Calvin cycle
• Humphrey cycle

See what the “Krebs Cycle” is in other dictionaries:

KREBS CYCLE- (citric and tricarboxylic acid cycle), a system of biochemical reactions through which most EUKARYOTIC organisms obtain their main energy as a result of the oxidation of food. Occurs in MITOCHONDRIA CELLS. Includes several chemical... ... Scientific and technical encyclopedic dictionary

Krebs cycle- Tricarboxylic acid cycle, a cycle of sequential reactions in the cells of aerobic organisms, as a result of which the synthesis of ATP molecules occurs Biotechnology topics EN Krebs cycle ... Technical Translator's Guide

Krebs cycle- - metabolic pathway leading to the complete destruction of acetyl CoA to the final products - CO2 and H2O ... A brief dictionary of biochemical terms

Krebs cycle- trikarboksirūgščių ciklas statusas T sritis chemija apibrėžtis Baltymų, riebalų ir angliavandenių oksidacinio skaidymo organizme ciklas. atitikmenys: engl. citric acid cycle; Krebs cycle; tricarboxylic acid cycle rus. Krebs cycle; lemon cycle... ... Chemijos terminų aiškinamasis žodynas

Krebs cycle- tricarboxylic acid (Krebs, citric acid) cycle tricarboxylic acid cycle, Krebs cycle. The most important cyclic sequence of metabolic reactions in aerobic organisms (eu and prokaryotes), as a result of which a sequential... ... Molecular biology and genetics. Dictionary.

KREBS CYCLE- the same as the tricarboxylic acid cycle... Natural science. encyclopedic Dictionary

Krebs Cycle, Citric Acid Cycle- a complex cycle of reactions where enzymes act as catalysts; these reactions take place in the cells of all animals and consist in the decomposition of acetate in the presence of oxygen with the release of energy in the form of ATP (via the electron transfer chain) and... ... Medical terms

KREBS CYCLE, CITRIC ACID CYCLE- (citric acid cycle) a complex cycle of reactions where enzymes act as catalysts; these reactions take place in the cells of all animals and consist in the decomposition of acetate in the presence of oxygen with the release of energy in the form of ATP (via the transmission chain... ... Explanatory dictionary of medicine

KREBS CYCLE (tricarboxylic acid cycle- citric acid cycle) is a complex cyclic enzymatic process in which pyruvic acid is oxidized in the body to produce carbon dioxide, water and energy in the form of ATP; occupies a central position in the overall system... ... Dictionary of botanical terms

Tricarboxylic acid cycle- Cycle... Wikipedia

Hello! Summer is coming, which means that all second-year medical students will take biochemistry. A difficult subject, indeed. To help a little those who are repeating material for exams, I decided to make an article in which I will tell you about the “golden ring” of biochemistry - the Krebs cycle. It is also called the tricarboxylic acid cycle and the citric acid cycle, these are all synonyms.

I will write out the reactions themselves in . Now I will talk about why the Krebs cycle is needed, where it takes place and what its features are. I hope it turns out clear and accessible.

First, let's look at what metabolism is. This is the basis without which understanding the Krebs Cycle is impossible.

Metabolism

One of the most important properties of living things (remember) is the exchange of substances with the environment. Indeed, only a living being can absorb something from the environment and then release something into it.

In biochemistry, metabolism is usually called “metabolism”. Metabolism, the exchange of energy with the environment is metabolism.

When we, say, ate a chicken sandwich, we received proteins (chicken) and carbohydrates (bread). During the digestion process, proteins are broken down into amino acids, and carbohydrates into monosaccharides. What I have described now is called catabolism, that is, the breakdown of complex substances into simpler ones. The first part of metabolism is catabolism.

One more example. The tissues in our body are constantly renewed. When old tissue dies, its fragments are taken away by macrophages, and they are replaced by new tissue. New tissue is created through the process of protein synthesis from amino acids. Protein synthesis occurs in ribosomes. Creating a new protein (complex substance) from amino acids (simple substance) is anabolism.

So, anabolism is the opposite of catabolism. Catabolism is the destruction of substances, anabolism is the creation of substances. By the way, so as not to confuse them, remember the association: “Anabolics. Blood and sweat". This is a Hollywood movie (quite boring, in my opinion) about athletes using anabolic steroids to grow muscles. Anabolics - growth, synthesis. Catabolism is the reverse process.

The intersection point of decay and synthesis.

The Krebs cycle as a stage of catabolism.

How are metabolism and the Krebs cycle related? The fact is that the Krebs cycle is one of the most important points at which the paths of anabolism and catabolism converge. This is precisely its meaning.

Let's look at this in diagrams. Catabolism can be roughly thought of as the breakdown of proteins, fats and carbohydrates in our digestive system. So, we ate food made from proteins, fats, and carbohydrates, what next?

• Fats - into glycerol and fatty acids (there may be other components, I decided to take the simplest example);
• Proteins - into amino acids;
• Polysaccharide molecules of carbohydrates are divided into single monosaccharides.

Further, in the cytoplasm of the cell, these simple substances will be converted into pyruvic acid(aka pyruvate). From the cytoplasm, pyruvic acid enters the mitochondrion, where it is converted into acetyl coenzyme A. Please remember these two substances - pyruvate and acetyl CoA, they are very important.

Let's now see how the stage that we have now described occurs:

An important detail: amino acids can be converted to acetyl CoA directly, bypassing the pyruvic acid stage. Fatty acids are immediately converted to acetyl CoA. Let's take this into account and edit our diagram to get it right:

The transformation of simple substances into pyruvate occurs in the cytoplasm of cells. After this, pyruvate enters the mitochondria, where it is successfully converted into acetyl CoA.

Why is pyruvate converted to acetyl CoA? Precisely in order to start our Krebs cycle. Thus, we can make one more inscription in the diagram, and we will get the correct sequence:

As a result of the reactions of the Krebs cycle, substances important for life are formed, the main of which are:

• NADH(Nicotine Amide Adenine DiNucleotide + hydrogen cation) and FADH 2(Flavin Adenine DiNucleotide + hydrogen molecule). I specifically highlighted the constituent parts of the terms in capital letters to make it easier to read; normally they are written as one word. NADH and FADH 2 are released during the Krebs cycle to then take part in the transfer of electrons into the cell's respiratory chain. In other words, these two substances play a critical role in cellular respiration.
• ATP, that is, adenosine triphosphate. This substance has two bonds, the rupture of which provides a large amount of energy. Many vital reactions are supplied with this energy;

Water and carbon dioxide are also released. Let's reflect this in our diagram:

By the way, the entire Krebs cycle occurs in mitochondria. This is where the preparatory stage takes place, that is, the conversion of pyruvate into acetyl CoA. It’s not for nothing that mitochondria are called the “energy station of the cell.”

The Krebs cycle as the beginning of synthesis

The Krebs cycle is amazing because it not only provides us with valuable ATP (energy) and coenzymes for cellular respiration. If you look at the previous diagram, you will understand that the Krebs cycle is a continuation of catabolic processes. But at the same time, it is also the first step of anabolism. How is this possible? How can the same cycle both destroy and create?

It turns out that individual reaction products of the Krebs cycle can be partially used for the synthesis of new complex substances, depending on the needs of the body. For example, gluconeogenesis is the synthesis of glucose from simple substances that are not carbohydrates.

• The reactions of the Krebs cycle are cascading. They occur one after another, and each previous reaction triggers the next one;
• The reaction products of the Krebs cycle are partly used to start the subsequent reaction, and partly to the synthesis of new complex substances.

Let's try to reflect this on the diagram so that the Krebs cycle is designated precisely as the point of intersection of decay and synthesis.

I marked with blue arrows the paths of anabolism, that is, the creation of new substances. As you can see, the Krebs cycle is truly the intersection point of many processes, both destruction and creation.

The most important

• The Krebs cycle is a cross-point of metabolic pathways. It ends catabolism (breakdown), it begins anabolism (synthesis);
• The reaction products of the Krebs Cycle are partly used to launch the next reaction of the cycle, and partly are sent to create new complex substances;
• The Krebs cycle produces the coenzymes NADH and FADH 2, which transport electrons for cellular respiration, as well as energy in the form of ATP;
• The Krebs cycle occurs in the mitochondria of cells.

We continue to analyze the Krebs cycle. In the last article, I talked about what it is, why the Krebs cycle is needed and what place it occupies in metabolism. Now let's get down to the reactions of this cycle themselves.

I’ll make a reservation right away - for me personally, memorizing reactions was a completely pointless exercise until I sorted out the above questions. But if you have already understood the theory, I suggest moving on to practice.

You can see many ways to write the Krebs cycle. The most common options are something like this:

But what seemed most convenient to me was the method of writing reactions from the good old textbook on biochemistry from the authors T.T. Berezov. and Korovkina B.V.

The already familiar Acetyl-CoA and Oxaloacetate combine and turn into citrate, that is, into citric acid.

Second reaction

Now we take citric acid and turn it isocitric acid. Another name for this substance is isocitrate.

In fact, this reaction is somewhat more complicated, through an intermediate stage - the formation of cis-aconitic acid. But I decided to simplify it so that you remember it better. If necessary, you can add the missing step here if you remember everything else.

In essence, the two functional groups simply swapped places.

Third reaction

So, we have isocitric acid. Now it needs to be decarboxylated (that is, COOH is removed) and dehydrogenated (that is, H is removed). The resulting substance is a-ketoglutarate.

This reaction is notable for the formation of the HADH2 complex. This means that the NAD transporter picks up hydrogen to start the respiratory chain.

I like the version of the Krebs Cycle reactions in the textbook by Berezov and Korovkin precisely because the atoms and functional groups that participate in the reactions are immediately clearly visible.

Fourth reaction

Again, nicotine Amide Adenine Dinucleotide works like clockwork, that is ABOVE. This nice carrier comes here, just like in the last step, to grab the hydrogen and carry it into the respiratory chain.

By the way, the resulting substance is succinyl-CoA, should not scare you. Succinate is another name for succinic acid, which is familiar to you from the days of bioorganic chemistry. Succinyl-Coa is a compound of succinic acid with coenzyme-A. We can say that this is an ester of succinic acid.

Fifth reaction

In the previous step, we said that succinyl-CoA is an ester of succinic acid. And now we get the sama succinic acid, that is, succinate, from succinyl-CoA. An extremely important point: it is in this reaction that substrate phosphorylation.

Phosphorylation in general (it can be oxidative and substrate) is the addition of a phosphorus group PO3 to HDP or ATP to obtain a complete GTF, or, respectively, ATP. The substrate differs in that this same phosphorus group is torn away from any substance containing it. Well, simply put, it is transferred from the SUBSTRATE to HDF or ADP. That is why it is called “substrate phosphorylation”.

Once again: at the beginning of substrate phosphorylation, we have a diphosphate molecule - guanosine diphosphate or adenosine diphosphate. Phosphorylation consists in the fact that a molecule with two phosphoric acid residues - HDP or ADP - is “completed” to a molecule with three phosphoric acid residues to produce guanosine TRIphosphate or adenosine TRIphosphate. This process occurs during the conversion of succinyl-CoA to succinate (i.e., succinic acid).

In the diagram you can see the letters F (n). It means "inorganic phosphate". Inorganic phosphate is transferred from the substrate to HDP so that the reaction products contain good, complete GTP. Now let's look at the reaction itself:

Sixth reaction

Next transformation. This time, the succinic acid that we obtained in the last step will turn into fumarate, note the new double bond.

The diagram clearly shows how it participates in the reaction FAD: This tireless carrier of protons and electrons picks up hydrogen and drags it directly into the respiratory chain.

Seventh reaction

We are already at the finish line.

TRICARBOXYLIC ACIDS CYCLE (KREBS CYCLE)

The penultimate stage of the Krebs Cycle is the reaction that converts fumarate to L-malate. L-malate is another name L-malic acid, familiar from the bioorganic chemistry course.

If you look at the reaction itself, you will see that, firstly, it goes both ways, and secondly, its essence is hydration. That is, fumarate simply attaches a water molecule to itself, resulting in L-malic acid.

Eighth reaction

The last reaction of the Krebs Cycle is the oxidation of L-malic acid to oxaloacetate, that is, to oxaloacetic acid. As you understand, “oxaloacetate” and “oxaloacetic acid” are synonyms. You probably remember that oxaloacetic acid is a component of the first reaction of the Krebs cycle.

Here we note the peculiarity of the reaction: formation of NADH2, which will carry electrons into the respiratory chain. Don’t forget also reactions 3,4 and 6, electron and proton carriers for the respiratory chain are also formed there.

As you can see, I specifically highlighted in red the reactions during which NADH and FADH2 are formed. These are very important substances for the respiratory chain. I highlighted in green the reaction in which substrate phosphorylation occurs and GTP is produced.

How to remember all this?

Actually, it's not that difficult. After reading my two articles in full, as well as your textbook and lectures, you just need to practice writing these reactions. I recommend remembering the Krebs cycle in blocks of 4 reactions. Write these 4 reactions several times, for each one choosing an association that suits your memory.

For example, I immediately very easily remembered the second reaction, in which isocitric acid is formed from citric acid (which, I think, is familiar to everyone from childhood).

You can also use mnemonics such as: " A Whole Pineapple and a Piece of Soufflé Is Actually My Lunch Today, which corresponds to the series - citrate, cis-aconitate, isocitrate, alpha-ketoglutarate, succinyl-CoA, succinate, fumarate, malate, oxaloacetate." There are a bunch more like them.

But, to be honest, I almost never liked such poems. In my opinion, it is easier to remember the sequence of reactions itself. It helped me a lot to divide the Krebs cycle into two parts, each of which I practiced writing several times an hour. As a rule, this happened in classes like psychology or bioethics. This is very convenient - without being distracted from the lecture, you can spend literally a minute writing the reactions as you remember them, and then check them with the correct option.

By the way, in some universities, during tests and exams in biochemistry, teachers do not require knowledge of the reactions themselves. You just need to know what the Krebs cycle is, where it occurs, what its features and significance are, and, of course, the chain of transformations itself. Only the chain can be named without formulas, using only the names of the substances. This approach is not without meaning, in my opinion.

I hope my guide to the TCA cycle has been helpful to you. And I want to remind you that these two articles are not a complete replacement for your lectures and textbooks. I wrote them only so that you roughly understand what the Krebs cycle is. If you suddenly see any error in my guide, please write about it in the comments. Thank you for your attention!

The tricarboxylic acid cycle was first discovered by the English biochemist Krebs. He was the first to postulate the importance of this cycle for the complete combustion of pyruvate, the main source of which is the glycolytic conversion of carbohydrates. It was subsequently shown that the tricarboxylic acid cycle is a “focus” at which almost all metabolic pathways converge.

So, acetyl-CoA formed as a result of oxidative decarboxylation of pyruvate enters the Krebs cycle. This cycle consists of eight consecutive reactions (Fig. 91). The cycle begins with the condensation of acetyl-CoA with oxaloacetate and the formation of citric acid. ( As will be seen below, in the cycle it is not acetyl-CoA itself that undergoes oxidation, but a more complex compound - citric acid (tricarboxylic acid).)

Then citric acid (a six-carbon compound), through a series of dehydrogenations (removal of hydrogen) and decarboxylation (elimination of CO2), loses two carbon atoms and again oxaloacetate (a four-carbon compound) appears in the Krebs cycle, i.e., as a result of a complete revolution of the cycle, the acetyl-CoA molecule burns to CO2 and H2O, and the oxaloacetate molecule is regenerated. Below are all eight sequential reactions (stages) of the Krebs cycle.

In the first reaction, catalyzed by the enzyme citrate synthase, acetyl-CoA is condensed with oxaloacetate. As a result, citric acid is formed:

Apparently, in this reaction, citril-CoA bound to the enzyme is formed as an intermediate product. The latter is then spontaneously and irreversibly hydrolyzed to form citrate and HS-CoA.

In the second reaction of the cycle, the resulting citric acid undergoes dehydration to form cis-aconitic acid, which, by adding a water molecule, becomes isocitric acid. These reversible hydration-dehydration reactions are catalyzed by the enzyme aconitate hydratase:

In the third reaction, which appears to be the rate-limiting reaction of the Krebs cycle, isocitric acid is dehydrogenated in the presence of NAD-dependent isocitrate dehydrogenase:

(There are two types of isocitrate dehydrogenases in tissues: NAD- and NADP-dependent. It has been established that NAD-dependent isocitrate dehydrogenase plays the role of the main catalyst for the oxidation of isocitric acid in the Krebs cycle.)

During the isocitrate dehydrogenase reaction, isocitric acid is decarboxylated. NAD-dependent isocitrate dehydrogenase is an allosteric enzyme that requires ADP as a specific activator. In addition, the enzyme requires Mg2+ or Mn2+ ions to exhibit its activity.

In the fourth reaction, α-ketoglutaric acid is oxidatively decarboxylated to succinyl-CoA. The mechanism of this reaction is similar to the reaction of oxidative decarboxylation of pyruvate to acetyl-CoA. The α-ketoglutarate dehydrogenase complex is similar in structure to the pyruvate dehydrogenase complex. In both cases, five coenzymes take part in the reaction: TDP, lipoic acid amide, HS-CoA, FAD and NAD. In total, this reaction can be written as follows:

The fifth reaction is catalyzed by the enzyme succinyl-CoA synthetase. During this reaction, succinyl-CoA, with the participation of GDP and inorganic phosphate, is converted into succinic acid (succinate). At the same time, the formation of a high-energy phosphate bond of GTP1 occurs due to the high-energy thioester bond of succinyl-CoA:

(The resulting GTP then donates its terminal phosphate group to ADP, resulting in the formation of ATP. The formation of a high-energy nucleoside triphosphate during the succinyl-CoA synthetase reaction is an example of phosphorylation at the substrate level.)

In the sixth reaction, succinate is dehydrogenated to fumaric acid. The oxidation of succinate is catalyzed by succinate dehydrogenase, in the molecule of which the coenzyme FAD is covalently bound to the protein:

In the seventh reaction, the resulting fumaric acid is hydrated under the influence of the enzyme fumarate hydratase. The product of this reaction is malic acid (malate). It should be noted that fumarate hydratase is stereospecific—during this reaction, L-malic acid is formed:

Finally, in the eighth reaction of the tricarboxylic acid cycle, under the influence of mitochondrial NAD-dependent malate dehydrogenase, L-malate is oxidized to oxaloacetate:

As you can see, in one turn of the cycle, consisting of eight enzymatic reactions, complete oxidation (“combustion”) of one molecule of acetyl-CoA occurs. For continuous operation of the cycle, a constant supply of acetyl-CoA into the system is necessary, and coenzymes (NAD and FAD), which have passed into a reduced state, must be oxidized again and again. This oxidation occurs in the electron transport system (or chain of respiratory enzymes) located in the mitochondria.

The energy released as a result of the oxidation of acetyl-CoA is largely concentrated in the high-energy phosphate bonds of ATP. Of the four pairs of hydrogen atoms, three pairs are transferred through NAD to the electron transport system; in this case, for each pair in the biological oxidation system, three ATP molecules are formed (in the process of conjugate oxidative phosphorylation), and therefore a total of nine ATP molecules. One pair of atoms enters the electron transport system through FAD, resulting in the formation of 2 ATP molecules. During the reactions of the Krebs cycle, 1 molecule of GTP is also synthesized, which is equivalent to 1 molecule of ATP. So, the oxidation of acetyl-CoA in the Krebs cycle produces 12 ATP molecules.

As already noted, 1 molecule of NADH2 (3 molecules of ATP) is formed during the oxidative decarboxylation of pyruvate into acetyl-CoA.

Krebs cycle reactions

Since the breakdown of one molecule of glucose produces two molecules of pyruvate, when they are oxidized to 2 molecules of acetyl-CoA and the subsequent two turns of the tricarboxylic acid cycle, 30 molecules of ATP are synthesized (hence, the oxidation of one molecule of pyruvate to CO2 and H2O produces 15 molecules of ATP).

To this we must add 2 ATP molecules formed during aerobic glycolysis, and 4 ATP molecules synthesized through the oxidation of 2 molecules of extramitochondrial NADH2, which are formed during the oxidation of 2 molecules of glyceraldehyde-3-phosphate in the dehydrogenase reaction. In total, we find that when 1 molecule of glucose is broken down in tissues according to the equation: C6H1206 + 602 -> 6CO2 + 6H2O, 36 ATP molecules are synthesized, which contributes to the accumulation of adenosine triphosphate in high-energy phosphate bonds 36 X 34.5 ~ 1240 kJ (or, according to other data, 36 X 38 ~ 1430 kJ) free energy. In other words, of all the free energy released during aerobic oxidation of glucose (about 2840 kJ), up to 50% of it is accumulated in mitochondria in a form that can be used to perform various physiological functions. There is no doubt that, energetically, the complete breakdown of glucose is a more efficient process than glycolysis. It should be noted that the NADH2 molecules formed during the conversion of glyceraldehyde-3-phosphate 2 subsequently, upon oxidation, produce not 6 ATP molecules, but only 4. The fact is that the extramitochondrial NADH2 molecules themselves are not able to penetrate through the membrane into the mitochondria. However, the electrons they donate can be included in the mitochondrial chain of biological oxidation using the so-called glycerophosphate shuttle mechanism (Fig. 92). As can be seen in the figure, cytoplasmic NADH2 first reacts with cytoplasmic dihydroxyacetone phosphate to form glycerol-3-phosphate. The reaction is catalyzed by NAD-dependent cytoplasmic glycerol-3-phosphate dehydrogenase:

Dihydroxyacetone phosphate + NADH2 glycerol-3-phosphate + NAD

The resulting glycerol-3-phosphate easily penetrates the mitochondrial membrane. Inside the mitochondria, another (mitochondrial) glycerol-3-phosphate dehydrogenase (flavin enzyme) again oxidizes glycerol-3-phosphate to dihydroxyacetone phosphate:

Glycerol-3-phosphate + FAD Dihydroxyacetone phosphate + fADN2

The reduced flavoprotein (enzyme - FADH2) introduces, at the level of KoQ, the electrons acquired by it into the chain of biological oxidation and associated oxidative phosphorylation, and dihydroxyacetone phosphate leaves the mitochondria into the cytoplasm and can again interact with cytoplasmic NADH2. Thus, a pair of electrons (from one molecule of cytoplasmic NADH2) introduced into the respiratory chain using the glycerophosphate shuttle mechanism produces not 3 ATP, but 2 ATP.

It is now clearly established that the glycerophosphate shuttle mechanism takes place in liver cells. Regarding other fabrics, this issue has not yet been clarified.

Krebs cycle also called tricarboxylic acid cycle, since they are formed in it as intermediate products. It is an enzymatic ring conveyor that “works” in the mitochondrial matrix.

The result of the Krebs cycle is the synthesis of a small amount of ATP and the formation of NAD H2, which is then sent to the next stage of cellular respiration - the respiratory chain (oxidative phosphorylation), located on the inner membrane of mitochondria.

The pyruvic acid (pyruvate) formed as a result of glycolysis enters the mitochondria, where it is ultimately completely oxidized, turning into carbon dioxide and water. This occurs first in the Krebs cycle, then during oxidative phosphorylation.

Before the Krebs cycle, pyruvate is decarboxylated and dehydrogenated. As a result of decarboxylation, a CO2 molecule is eliminated; dehydrogenation is the elimination of hydrogen atoms. They connect to NAD.

As a result, acetic acid is formed from pyruvic acid, which is added to coenzyme A. It turns out acetyl coenzyme A(acetyl-CoA) – CH3CO~S-CoA containing a high-energy bond.

The conversion of pyruvate to acetyl-CoA is accomplished by a large enzymatic complex consisting of dozens of polypeptides associated with electron carriers.

The Krebs cycle begins with the hydrolysis of acetyl-CoA, which removes an acetyl group containing two carbon atoms. Next, the acetyl group is included in the tricarboxylic acid cycle.

An acetyl group attaches to oxaloacetic acid, which has four carbon atoms. The result is citric acid, which contains six carbon atoms. The energy for this reaction is supplied by the high-energy acetyl-CoA bond.

What follows is a chain of reactions in which the acetyl group bound in the Krebs cycle is dehydrogenated, releasing four pairs of hydrogen atoms, and decarboxylated to form two molecules of CO2. In this case, oxygen is used for oxidation, split off from two water molecules, not molecular. The process is called oxidative decarboxylation. At the end of the cycle, oxaloacetic acid is regenerated.

Let's return to the citric acid stage. Its oxidation occurs through a series of enzymatic reactions in which isocitric, oxalosuccinic and other acids are formed.

As a result of these reactions, at different stages of the cycle, three molecules of NAD and one FAD are reduced, GTP (guanosine triphosphate) is formed, containing a high-energy phosphate bond, the energy of which is subsequently used to phosphorylate ADP. As a result, an ATP molecule is formed.

Citric acid loses two carbon atoms to form two CO2 molecules.

As a result of enzymatic reactions, citric acid is converted into oxaloacetic acid, which can again combine with acetyl-CoA. The cycle repeats.

In citric acid, the added acetyl-CoA residue burns to form carbon dioxide, hydrogen atoms and electrons. Hydrogen and electrons are transferred to NAD and FAD, which are acceptors for it.

The oxidation of one molecule of acetyl-CoA produces one molecule of ATP, four hydrogen atoms and two molecules of carbon dioxide. That is carbon dioxide released during aerobic respiration is formed during the Krebs cycle. In this case, molecular oxygen (O2) is not used here; it is necessary only at the stage of oxidative phosphorylation.

Hydrogen atoms attach to NAD or FAD, and in this form they then enter the respiratory chain.

One molecule of glucose produces two molecules of pyruvate and therefore two acetyl-CoA. Thus, for one molecule of glucose there are two turns of the tricarboxylic acid cycle. A total of two ATP molecules, four CO2, and eight H atoms are formed.

It should be noted that not only glucose and the pyruvate formed from it enter the Krebs cycle. As a result of the breakdown of fats by the lipase enzyme, fatty acids are formed, the oxidation of which also leads to the formation of acetyl-CoA, the reduction of NAD, as well as FAD (flavin adenine dinucleotide).

If a cell is deficient in carbohydrates and fats, then amino acids may undergo oxidation. In this case, acetyl-CoA and organic acids are formed, which further participate in the Krebs cycle.

Thus, it does not matter what the primary source of energy was. In any case, acetyl-CoA is formed, which is a compound universal for the cell.

Tricarboxylic acid (Krebs) cycle

(TCA cycle, citric acid cycle, Krebs cycle)

The TCA cycle, like mitochondrial oxidation reactions, occurs in mitochondria. It is a series of reactions closed in a cycle.

The resulting PCA molecules react with a new Acetyl-CoA molecule and the cycle repeats again from the formation of citrate to its conversion to PCA.

Four of the nine MtO substrates participate in the reactions of this cycle.

A series of dehydrogenase reactions occurs. Of these, the 3rd, 4th and 8th occur with the participation of NAD-dependent dehydrogenases, and each of these reactions produces 3 ATP molecules. At the 6th stage, a FAD-dependent dehydrogenase reaction occurs, which is associated with the formation of 2 ATP molecules (P/O = 2).

At the 5th stage, 1 ATP molecule is formed by substrate phosphorylation.

In total, 12 ATP molecules are formed during 1 turnover of the TCA cycle.

The point of the TCA cycle is to break down acetic acid residues to form a large amount of ATP. In addition, CO2 and H2O are formed from acetate residues as end products of metabolism.

CO2 is formed during the TTC cycle twice:

1. at the third stage (oxidation of isocitrate)

2. at the fourth stage (oxidation of alpha-ketoglutarate).

If we add one more molecule of CO2, which is formed before the start of the TCA cycle - during the conversion of PVK into Acetyl-CoA, then we can talk about three molecules of CO2 formed during the breakdown of PVK. In total, these molecules, formed during the breakdown of PVC, account for up to 90% of the carbon dioxide that is excreted from the body.

FINAL CTK EQUATION

BIOLOGICAL SIGNIFICANCE OF THE TCA cycle

THE MAIN ROLE OF THE TCA CYCLE IS THE FORMATION OF A LARGE AMOUNT OF ATP.

1. The TCA cycle is the main source of ATP. The energy for the formation of a large amount of ATP is provided by the complete breakdown of Acetyl-CoA to CO2 and H2O.

2. The TCA cycle is a universal terminal stage in the catabolism of substances of all classes.

3. The TCA cycle plays an important role in the processes of anabolism (intermediate products of the TCA cycle):

— from citrate → synthesis of fatty acids

— from alpha-ketoglutarate and PKA → synthesis of amino acids

— from PIKE → synthesis of carbohydrates

— from succinyl-CoA → synthesis of heme hemoglobin

AUTONOMOUS SELF-REGULATION OF CTC

There are two key enzymes in the TCA cycle:

1) citrate synthase (1st reaction)

2) isocitrate dehydrogenase (3rd reaction)

Both enzymes are allosterically inhibited by excess ATP and NADH2. Isocitrate dehydrogenase is strongly activated by ADP.

Tricarboxylic acid cycle

If there is no ADP, then this enzyme is inactive. Under conditions of energy rest, the concentration of ATP increases, and the rate of TCA cycle reactions is low—ATP synthesis decreases.

Isocitrate dehydrogenase is inhibited by ATP much more strongly than citrate synthase, therefore, under conditions of energy rest, the concentration of citrate increases, and it enters the cytoplasm along a concentration gradient by facilitated diffusion. In the cytoplasm, citrate is converted to Acetyl-CoA, which is involved in the synthesis of fatty acids.

Modern classifications of the cardiovascular system
Blood flow speed, heart development
Thrombocytopenic purpura
Transport of gases by blood, composition of plasma
Fibrinolysis and blood clotting
Composition and properties of blood plasma components
Coagglutination, compensation, Coombs, sedimentation, passive hemagglutination reaction

Lesson No. 12. “The tricarboxylic acid cycle”

Purpose of the lesson: study the mechanism of some reactions of the Krebs cycle. Master the method of quantitative determination of pyruvic acid in urine.

QUESTIONS FOR THE TEST:

1. Oxidative decarboxylation of pyruvate as a preliminary step in the citric acid cycle. List the vitamins and coenzymes involved in this process.

2. Reactions of the citric acid cycle. What determines the general direction of reactions in the cycle? In what part of the cell does this process take place? Why?

3. What coenzymes and vitamins are involved in the Krebs cycle? Explain how they work, including specific reactions.

4. Tell us about the reactions of the Krebs cycle, as a result of which NADH2 and FADH2 are formed. What is the future fate of these compounds?

5. Functions of the tricarboxylic acid cycle. Explain what significance the anaplerotic reaction has for the citric acid cycle?

6. Energy output of the tricarboxylic acid cycle. How many ATP molecules are produced during the turnover of one citric acid molecule through the cycle? Are all ATP molecules formed during the complete oxidation of active acetyl synthesized by oxidative phosphorylation? How is cycle speed controlled?

Experimental work.

One of the methods of quantitative analysis in biochemistry is photocalorimetry. The method is based on measuring the optical density of colored solutions, which are obtained by interacting the substrate with various chemical agents. The concentration of the substrate is proportional to the degree of coloration of the solution.

Before starting laboratory experiments, familiarize yourself with the FEC device and the rules for working on it.

Experiment 1. Determination of the concentration of pyruvic acid (PVA) in urine.

2. Set of pipettes.

3. Photocolorimeter.

4. Cuvettes, 0.5 cm.

Reagents. 1. Distilled water.

3. Sodium hydroxide, 10% solution.

4. 2,4-dinitrophenylhydrazine, solution.

Blood PVC condenses with 2,4-dinitrophinylhydrazine to form hydrazone, which in an alkaline environment gives a brown-red solution. The intensity of its color is used to judge the content of PVC.

1. Add reagents to three test tubes according to the following table:

2. Place the contents of the test tubes in a dark place at room temperature for 15 minutes.

Up to 10% of the energy in a cell comes from amino acids

Add 1 ml of 10% NaOH solution to each test tube and after five minutes measure the optical density at a wavelength of 620 nm of the test sample against the control (O) and the calibration sample against the control (K).

4. Perform the calculation using the prepared calibration schedule.

= mg/day

To convert the PVC content (in mg) into units of the amount of substance (μmol), it is necessary to multiply the corresponding values ​​by 11.4 (conversion factor).

Normal for humans: 10-25 mg/day or 114-284 µmol/day of pyruvic acid.

Compare the obtained values ​​with normal values. What are the causes of increased levels of pyruvic acid in blood serum and urine?

Experiment 2. Determination of muscle succinate dehydrogenase activity.

Devices. 1. Rack with test tubes.

2. Set of pipettes.

3. Mortar and pestle.

4. Water bath.

Reagents. 1. Chicken or rabbit muscle tissue.

2. Succinic acid, 5% solution.

3. Methylene blue, 0.01% solution.

4. Vegetable oil.

5. Glass sand.

1. Weigh 10 g of muscle tissue and grind in a mortar with glass sand.

2. Rinse the resulting homogenate several times on gauze with saline solution to remove soluble substances.

3. Pour 5 ml of the resulting mixture into three numbered test tubes.

4. Immerse the first test tube in a boiling water bath for 5 minutes, then cool it to room temperature.

5. Add 3 ml of 5% succinic acid and 3 drops of methylene blue solution to test tubes No. 1 and No. 2 (until a blue color appears).

6. Add 0.5 ml of distilled water and 3 drops of methylene blue solution to test tube No. 3 (until a blue color appears).

7. Then pour a little oil into all test tubes to isolate the mixture from air oxygen.

8. Incubate all test tubes in a water bath (40°C) for 10 minutes.

Give an explanation for the observed phenomena. What is the function of methylene blue in this experiment? What compound is responsible for this function in a living cell?

Completion date ________ Point ____ Teacher signature ____________

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