Tricarboxylic acid cycle (Krebs cycle)

Glycolysis converts glucose to pyruvate and produces two ATP molecules from a glucose molecule - this is a small fraction of the potential energy of this molecule.

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

Under aerobic conditions, pyruvic acid (stage 1) undergoes oxidative decarboxylation, which is more efficient than transformation into lactic acid, with the formation of acetyl-CoA (stage 2), which can be oxidized to the end 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, lipoylacetyltransferase) 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 in the oxidative decarboxylation reaction is converted 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 macroergic 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 is involved in the synthesis of complex molecules such as sterides 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, amino acids, is essentially a "metabolic boiler". The reactions of the Krebs cycle, which take place exclusively in the mitochondria, are also called the citric acid cycle or the tricarboxylic acid cycle (TCA).

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 understood that the tricarboxylic acid cycle is aerobic, dependent on oxygen.

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

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

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

3. Then the enzyme isocitrate dehydrogenase catalyzes the first dehydrogenase reaction of the citric acid cycle, when isocitric acid is converted into α-ketoglutaric acid in oxidative decarboxylation reactions:

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, the molecule of NADH + H + and CO 2 is again formed.

5. The succinyl-CoA molecule has a macroergic 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-sulphur-containing enzyme whose coenzyme is FAD. Succinate dehydrogenase is the only enzyme fixed on 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 to 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 involving the active enzyme of mitochondrial NAD~-dependent malate dehydrogenase, in which the third molecule of reduced NADH + H + is formed:

The formation of oxaloacetic acid (oxaloacetate) completes one turn of the tricarboxylic acid cycle. Oxaloacetic acid can be used in the oxidation of the second acetyl-CoA molecule, 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 as a cycle substrate in the TCA cycle leads to the production of one GTP molecule, three NADP + H + molecules, and one FADH 2 molecule. The oxidation of these reducing agents in the biological oxidation chain

ion 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 provides 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 CO2 in decarboxylation reactions catalyzed by isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase.

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

• 4 ATP molecules 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 during the complete oxidation of a glucose molecule can be 40 ATP molecules. However, it should be taken into account that during the oxidation of glucose at the stage of converting glucose into glucose-6-phosphate and at the stage of converting fructose-6-phosphate into fructose-1,6-diphosphate, two ATP molecules were consumed. Therefore, the "net" energy output during the oxidation of a glucose molecule is 38 ATP molecules.

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

Under aerobic conditions, out of 688 kcal of a gram-molecule of glucose in 38 ATP molecules, 380 kcal were obtained. Thus, the efficiency of glucose utilization under aerobic conditions is about 19 times higher than in anaerobic glycolysis.

It should be pointed out 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 Phneor (the Pasteur effect). This means that the resulting NADH + H + molecule in oxidation reactions has a choice between the reactions of the respiratory system, which transfer hydrogen to oxygen, and the LDH enzyme, which transfers 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 disturbing the functioning of the cycle itself. Various factors are involved in the regulation of the activity of the tricarboxylic acid cycle. Among them, first of all, we should mention the intake of acetyl-CoA molecules, the activity of the pyruvate dehydrogenase complex, the activity of the components of the respiratory chain and the oxidative phosphorylation associated with it, as well as the level of oxaloacetic acid.

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 when electrons are transferred 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 when pyruvic acid passes into lactic acid.

In addition to the formation of ATP, the tricarboxylic acid cycle has another important significance: the cycle provides intermediary structures for various biosynthesis of the body. For example, most porphyrin atoms originate from succinyl-CoA, many amino acids are derivatives of α-keto-glutaric and oxalo-acetic acids, and fumaric acid occurs during the synthesis of urea. This manifests the integrality of the tricarboxylic acid cycle in the metabolism of carbohydrates, fats, and proteins.

As shown by the reactions of glycolysis, the ability of most cells to generate energy lies in their mitochondria. The number of mitochondria in various tissues is related to the physiological functions of tissues and reflects their ability to participate in aerobic conditions. For example, red blood cells do not have mitochondria and therefore lack the ability to generate energy using oxygen as the final electron acceptor. However, in the cardiac muscle functioning under aerobic conditions, half of the cell cytoplasm volume is represented by mitochondria. The liver also depends on aerobic conditions for its various functions, and mammalian hepatocytes contain up to 2,000 mitochondria per cell.

Mitochondria include two membranes - outer and inner. The outer membrane is simpler, consisting of 50% fat and 50% protein, 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 mediators, 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 in the citrate-synthase reaction of the tricarboxylic acid cycle and enters the cytosol in this form.

The acetyl-SCoA formed in the PVC-dehydrogenase reaction then enters into tricarboxylic acid cycle(CTC, citric acid cycle, Krebs cycle). In addition to pyruvate, keto acids coming from catabolism are involved in the cycle. amino acids or any other substances.

Tricarboxylic acid cycle

The cycle runs in mitochondrial matrix and represents oxidation molecules acetyl-SCoA in eight consecutive reactions.

In the first reaction, they bind acetyl and oxaloacetate(oxaloacetic acid) to form citrate(citric acid), then citric acid isomerizes to isocitrate and two dehydrogenation reactions with concomitant release of CO 2 and reduction of NAD.

In the fifth reaction, GTP is formed, this is the reaction substrate phosphorylation. Next, FAD-dependent dehydrogenation occurs sequentially succinate(succinic acid), hydration fumaric acid up malate(malic acid), then NAD-dependent dehydrogenation with the formation of oxaloacetate.

As a result, after eight reactions of the cycle again oxaloacetate is formed .

The last three reactions make up the so-called biochemical motif (FAD-dependent dehydrogenation, hydration and NAD-dependent dehydrogenation, it is used to introduce a keto group into the succinate structure. This motif is also present in fatty acid β-oxidation reactions. In reverse order (reduction, de hydration and recovery) this motif is observed in fatty acid synthesis reactions.

DTC functions

1. Energy

• generation hydrogen atoms for the operation of the respiratory chain, namely three NADH molecules and one FADH2 molecule,
• single molecule synthesis GTP(equivalent to ATP).

2. Anabolic. In the CTC are formed

• heme precursor succinyl-SCoA,
• keto acids that can be converted into amino acids - α-ketoglutarate for glutamic acid, oxaloacetate for aspartic,
• lemon acid, used for the synthesis of fatty acids,
• oxaloacetate, used for glucose synthesis.

Anabolic reactions of the TCA

Regulation of the tricarboxylic acid cycle

Allosteric regulation

Enzymes catalyzing the 1st, 3rd and 4th reactions of TCA are sensitive to allosteric regulation metabolites:

Regulation of oxaloacetate availability

chief and basic the regulator of the TCA is oxaloacetate, or rather its availability. The presence of oxaloacetate involves acetyl-SCoA in the TCA cycle and starts the process.

Usually the cell has balance between the formation of acetyl-SCoA (from glucose, fatty acids or amino acids) and the amount of oxaloacetate. The source of oxaloacetate is pyruvate, (formed from glucose or alanine), derived from aspartic acid as a result of transamination or the AMP-IMF cycle, and also from fruit acids the cycle itself (succinic, α-ketoglutaric, malic, citric), which can be formed during the catabolism of amino acids or come from other processes.

Synthesis of oxaloacetate from pyruvate

Regulation of enzyme activity pyruvate carboxylase carried out with the participation acetyl-SCoA. It is allosteric activator enzyme, and without it, pyruvate carboxylase is practically inactive. When acetyl-SCoA accumulates, the enzyme starts to work and oxaloacetate is formed, but, of course, only in the presence of pyruvate.

Also most amino acids during their catabolism, they are able to turn into metabolites of TCA, which then go to oxaloacetate, which also maintains the activity of the cycle.

Replenishment of the pool of TCA metabolites from amino acids

Cycle replenishment reactions with new metabolites (oxaloacetate, citrate, α-ketoglutarate, etc.) are called anaplerotic.

The role of oxaloacetate in metabolism

An example of a significant role oxaloacetate serves to activate the synthesis of ketone bodies and ketoacidosis blood plasma at inadequate the amount of oxaloacetate in the liver. This condition is observed during decompensation of insulin-dependent diabetes mellitus (type 1 diabetes) and during starvation. With these disorders, the process of gluconeogenesis is activated in the liver, i.e. the formation of glucose from oxaloacetate and other metabolites, which entails a decrease in the amount of oxaloacetate. Simultaneous activation of fatty acid oxidation and accumulation of acetyl-SCoA triggers a backup pathway for the utilization of the acetyl group - synthesis of ketone bodies. In this case, the body develops acidification of the blood ( ketoacidosis) with a characteristic clinical picture: weakness, headache, drowsiness, decreased muscle tone, body temperature and blood pressure.

Change in the rate of TCA reactions and the reasons for the accumulation of ketone bodies under certain conditions

The described method of regulation with the participation of oxaloacetate is an illustration of the beautiful formulation " Fats burn in the flame of carbohydrates". It implies that the "burning flame" of glucose leads to the appearance of pyruvate, and pyruvate is converted not only into acetyl-SCoA, but also into oxaloacetate. The presence of oxaloacetate guarantees the inclusion of an acetyl group formed from fatty acids in the form of acetyl-SCoA, in the first reaction of the TCA.

In the case of a large-scale "burning" of fatty acids, which is observed in the muscles during physical work and in the liver fasting, the rate of entry of acetyl-SCoA in the TCA reaction will directly depend on the amount of oxaloacetate (or oxidized glucose).

If the amount of oxaloacetate in hepatocyte not enough (no glucose or it is not oxidized to pyruvate), then the acetyl group will go to the synthesis of ketone bodies. This happens when prolonged fasting and type 1 diabetes.

I talked about what it is in general, why the Krebs cycle is needed and what place it occupies in metabolism. Now let's get down to the actual reactions of this cycle.

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

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

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

First reaction

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

Second reaction

Now we take citric acid and turn it into 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 so that you remember better. If necessary, you can add the missing step here if you remember everything else.

In fact, the two functional groups were simply swapped.

Third reaction

So, we got isocitric acid. Now it needs to be decarboxylated (that is, pinch off COOH) and dehydrate (that is, pinch off H). The resulting substance is a-ketoglutarate.

This reaction is remarkable in that the NADH 2 complex is formed here. This means that the NAD transporter picks up hydrogen to start the respiratory chain.

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

fourth reaction

Again, how the clock works nicotineAmideAdenineDinucleotide, that is ABOVE. This glorious carrier appears here, as in the last step, to capture the hydrogen and carry it into the respiratory chain.

By the way, the resulting substance - succinyl-CoA, should not scare you. Succinate is another name for succinic acid, well known to you since 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 last step, we said that succinyl-CoA is an ester of succinic acid. And now we'll get ourselves succinic acid, i.e. 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 PO 3 phosphorus group to GDP or ATP in order to obtain a complete GTP, or, respectively, ATP. The substrate differs in that this same phosphorus group is detached from any substance containing it. Well, simply put, it is transferred from SUBSTRATE to HDF or ADP. That is why it is called “substrate phosphorylation”.

Once again: at the moment of 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 - GDP or ADP "is completed" to a molecule with three phosphoric acid residues to get guanosine TRIphosphate or adenosine TRIphosphate. This process occurs during the conversion of succinyl-CoA to succinate (that is, to succinic acid).

On the diagram you can see the letters F (n). It means "inorganic phosphate". Inorganic phosphate passes from the substrate to GDP, so that the reaction products contain good, high-grade GTP. Now let's look at the reaction itself:

sixth reaction

next transformation. This time, the succinic acid that we received in the previous step will turn into fumarate note the new double bond.

The diagram clearly shows how the reaction is involved FAD: This tireless proton and electron carrier picks up hydrogen and drags it directly into the respiratory chain.

Seventh reaction

We are already at the finish line. The penultimate stage of the Krebs cycle is the conversion of fumarate to L-malate. L-malate is another name L-malic acid, familiar from the course of bioorganic chemistry.

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 NADH 2, which will carry electrons to the respiratory chain. Don't forget also reactions 3,4 and 6, where electron and proton carriers for the respiratory chain are also formed.

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. In green, I highlighted the reaction in which substrate phosphorylation occurs, and GTP is obtained.

How to remember all this?

Actually, it's not that difficult. Having fully read my two articles, 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, choosing an association for each that suits your memory.

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

You can also use mnemonic memos such as: A Whole Pineapple And A Slice Of Soufflé Today Is Actually My Lunch, which corresponds to the series - citrate, cis-aconitate, isocitrate, alpha-ketoglutarate, succinyl-CoA, succinate, fumarate, malate, oxaloacetate. There are many more like it.

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

By the way, in some universities, for tests and exams in biochemistry, teachers do not require knowledge of the reactions themselves. You only need to know what the Krebs cycle is, where it occurs, what are its features and significance, and, of course, the chain of transformations itself. Only a chain can be named without formulas, using only the names of substances. This approach makes no sense, in my opinion.

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

(TsTK, citrate cycle, Krebs cycle)

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

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

Four out of nine MtO substrates are involved in the reactions of this cycle.

A series of dehydrogenase reactions occur. Of these, the 3rd, 4th and 8th occur with the participation of NAD-dependent dehydrogenases, and each of these reactions allows you to get 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 for 1 cycle of the TCA cycle.

The meaning of the TCA is that the residues of acetic acid are broken down with the formation of a large amount of ATP. In addition, CO 2 and H 2 O are formed from acetate residues as end products of metabolism.

CO 2 is formed during the CTC twice:

1. at the third stage (oxidation of isocitrate)

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

If one more molecule of CO 2 is added, which is formed before the beginning of the CTC - during the conversion of PVC into Acetyl-CoA, then we can talk about three CO 2 molecules formed during the breakdown of PVC. In total, these molecules, formed during the breakdown of PVC, account for up to 90% of carbon dioxide, which is excreted from the body.

FINAL CTC EQUATION

BIOLOGICAL SIGNIFICANCE OF CTC

THE MAIN ROLE OF THE CTC IS THE FORMATION OF A LARGE AMOUNT OF ATP.

1. CTK 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 CO 2 and H 2 O.

2. CTC is a universal terminal stage of catabolism of substances of all classes.

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

From citrate → fatty acid synthesis

From alpha-ketoglutarate and PAA → amino acid synthesis

From pike → carbohydrate synthesis

From succinyl-CoA → synthesis of heme hemoglobin

AUTONOMOUS SELF-REGULATING CTC

There are two key enzymes in TCA:

1) citrate synthase (1st reaction)

2) isocitrate dehydrogenase (3rd reaction)

Both enzymes are allosterically inhibited by excess ATP and NADH 2 . Isocitrate dehydrogenase is strongly activated by ADP. If there is no ADP, then this enzyme is inactive. Under conditions of energy rest, the ATP concentration increases, and the rate of TCA 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 the concentration gradient by facilitated diffusion. In the cytoplasm, citrate is converted to Acetyl-CoA, which is involved in the synthesis of fatty acids.

4. Tricarboxylic acid cycle

The second component of the overall catabolism pathway is the CTC. This cycle was discovered in 1937 by Krebs and Johnson. In 1948, Kennedy and Lehninger proved that the TCA enzymes are localized in the mitochondrial matrix.

4.1. Chemistry of the tricarboxylic acid cycle. Free acetic acid cannot be oxidized by dehydrogenation. Therefore, it is in its active form (acetyl-CoA) previously associated with oxaloacetate (PAA, oxaloacetic acid), resulting in the formation of citrate.

1. Acetyl-CoA combines with oxaloacetate in an aldol condensation reaction catalyzed by citrate synthase. Citril-CoA is formed. Citryl-CoA is hydrolyzed with the participation of water to citrate and HS-CoA.

2. Aconitate hydratase (a conitase) catalyzes the conversion of citrate to isocitrate via a cis-aconitic acid step. According to the mechanism of action, aconitase is both a hydratase and an isomerase.

3. isocitrate dehydrogenase catalyzes the dehydrogenation of isocitric acid to oxalosuccinate (oxalosuccinic acid), which is then decarboxylated to 2-oxoglutarate (α-ketoglutarate). The coenzyme is NAD+ (in mitochondria) and NADP+ (in cytosol and mitochondria).

4. 2-Oxoglutarate dehydrogenase complex (α-ketoglutarate dehydrogenase complex) catalyzes the oxidative decarboxylation of 2-oxoglutarate to succinyl-CoA. multi-enzymatic 2-oxoglutarate dehydrogenase the complex is similar to the pyruvate dehydrogenase complex and the process proceeds similarly to the oxidative decarboxylation of pyruvate.

5. Succinylthiokinase catalyzes the breakdown of succinyl-CoA into succinic acid and coenzyme A. The energy for the breakdown of succinyl-CoA accumulates in the form of guanosine triphosphate (GTP). In a coupled rephosphorylation reaction, ADP is phosphorylated to ATP, and the released GDP molecules can be re-phosphorylated ( substrate phosphorylation). In plants, the enzyme is specific for ADP and ATP.

6. Succinate dehydrogenase catalyzes the conversion of succinate to fumaric acid. The enzyme is stereospecific, is an integral protein, as it is embedded in the inner membrane of mitochondria and contains FAD and iron-sulfur proteins as prosthetic groups. FADH 2 is not separated from the enzyme, and two electrons are further transferred to coenzyme Q of the electron transport chain of the inner mitochondrial membrane.

7.Fumarate hydratase (fumarase) catalyzes the conversion of fumaric acid to malic acid (malate) with the participation of water. The enzyme is stereospecific, forming only L-malate.

8.Malate dehydrogenase catalyzes the oxidation of malic acid to oxaloacetate. Coenzyme malate dehydrogenase - NAD +. Further, oxaloacetate condenses again with acetyl-CoA and the cycle repeats.

4.2. Biological significance and regulation of the tricarboxylic acid cycle. The tricarboxylic acid cycle is a component of the overall catabolism pathway in which the fuel molecules of carbohydrates, fatty acids, and amino acids are oxidized. Most of the fuel molecules enter the TCA in the form of acetyl-CoA (Fig. 1). All TCA reactions proceed in a coordinated manner in the same direction. The total value of D G 0 ¢ = -40 kJ / mol.

Among doctors, there has long been a catchphrase "Fats burn in the flame of carbohydrates." It should be understood as the oxidation of acetyl-CoA, the main source of which is the β-oxidation of fatty acids, after condensation with oxaloacetate, formed mainly from carbohydrates (during the carboxylation of pyruvate). With carbohydrate metabolism disorders or starvation, an oxaloacetate deficiency is created, leading to a decrease in the oxidation of acetyl-CoA in the TCA.

Fig.1. The role of TCA in cellular respiration. Stage 1 (CTC) extraction of 8 electrons from the acetyl-CoA molecule; stage 2 (electron transport chain) reduction of two oxygen molecules and formation of a proton gradient (~36 H +); Stage 3 (ATP synthase) using the energy of the proton gradient to form ATP (~9 ATP) (Berg J .M ., Tymoczko J .L ., Stryer L . Biochemistry . N-Y : W .H .Freeman and Company , 2002 ).

The main metabolic role of TCA can be represented as two processes: 1) a series of redox reactions, as a result of which the acetyl group is oxidized to two CO2 molecules; 2) quadruple dehydrogenation, leading to the formation of 3 NADH + H + molecules and 1 FADH 2 molecule. Oxygen is required for the functioning of the CTC indirectly as an electron acceptor at the end of electron transport chains and for the regeneration of NAD+ and FAD.

The synthesis and hydrolysis of ATP is of primary importance for the regulation of TCA.

1. Isocitrate dehydrogenase is allosterically activated by ADP by increasing the affinity of the enzyme for the substrate. NADH inhibits this enzyme by replacing NAD + . ATP also inhibits isocitrate dehydrogenase. It is important that the conversion of metabolites into TCA requires NAD + and FAD at several stages, the amount of which is sufficient only under conditions of low energy charge.

2. The activity of the 2-oxoglutarate dehydrogenase (α-ketoglutarate dehydrogenase) complex is regulated similarly to the regulation of the pyruvate dehydrogenase complex . This complex is inhibited by succinyl-CoA and NADH (end products of transformations catalyzed by the 2-oxoglutarate dehydrogenase complex). In addition, the 2-oxogluttarate dehydrogenase complex is inhibited by the high energy charge of the cell. So, the rate of transformations in the TCA decreases with a sufficient supply of ATP to the cell (Fig. 11.2). In a number of bacteria, citrate synthase is allosterically inhibited by ATP by increasing the KM for acetyl-CoA.

The scheme of regulation of the general pathway of catabolism is shown in Figure 2.

Rice. 2. Regulation of the general path of catabolism. The main molecules that regulate the functioning of the TCA are ATP and NADH. The main points of regulation are isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase complex.

4.3. Energetic role of the general pathway of catabolism

In the general catabolism pathway, 3 molecules of CO 2 are formed from 1 molecule of pyruvic acid in the following reactions: during the oxidative decarboxylation of pyruvic acid, during the decarboxylation of isocitric acid, and during the decarboxylation of 2-oxoglutaric acid. In total, during the oxidation of 1 molecule of pyruvic acid, five pairs of hydrogen atoms are taken away, of which one pair is from succinate and enters FAD with the formation of FADH 2, and four pairs - for 4 molecules of NAD + with the formation of 4 molecules of NADH + H + during oxidative decarboxylation of pyruvic acid. , 2-oxoglutaric acids, dehydrogenation of isocitrate and malate. Ultimately, hydrogen atoms are transferred to oxygen with the formation of 5 H 2 O molecules, and the released energy is accumulated in oxidative phosphorylation reactions in the form of ATP molecules.

Grand total:

1. Oxidative decarboxylation of pyruvate ~ 2.5 ATP.

2. In the TCA and associated respiratory chains ~ 9 ATP.

3. In the reaction of substrate phosphorylation of CTK ~ 1 ATP.

In TCA and related reactions of oxidative phosphorylation, approximately 10 ATP are formed during the oxidation of the acetyl group of one acetyl-CoA molecule

In total, in the general path of catabolism, as a result of the transformations of 1 molecule of pyruvic acid, approximately 12.5 ATP molecules are released.