What stage does the tricarboxylic acid cycle take place? Tricarboxylic acid cycle (Krebs cycle). Replenishment of the TCA cycle metabolite pool from amino acids

4. Tricarboxylic acid cycle

The second component of the general catabolic pathway is the TCA cycle. This cycle was discovered in 1937 by Krebs and Johnson. In 1948, Kennedy and Lehninger proved that TCA cycle 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 in its active form (acetyl-CoA) is preliminarily bound to oxaloacetate (OA, oxaloacetic acid), resulting in the formation of citrate.

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

2. Aconitate hydratase (A conitasis) catalyzes the conversion of citrate to isocitrate through a cis-aconitic acid step. Aconitase's mechanism of action 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. Multienzyme 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 cleavage of succinyl-CoA into succinic acid and coenzyme A. The energy from the cleavage of succinyl-CoA is stored in the form of guanosine triphosphate (GTP). In the coupled rephosphorylation reaction, ADP is phosphorylated into ATP, and the released GDP molecules can be phosphorylated again ( 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, since it is embedded in the inner membrane of mitochondria and contains FAD and iron-sulfur proteins as prosthetic groups. FADN 2 is not separated from the enzyme, and two electrons are then 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, producing only L-malate.

8.Malate dehydrogenase catalyzes the oxidation of malic acid to oxaloacetate. Malate dehydrogenase coenzyme - NAD +. Next, 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 general catabolic pathway in which the oxidation of fuel molecules of carbohydrates, fatty acids and amino acids occurs. Most fuel molecules enter the TCA cycle in the form of acetyl-CoA (Fig. 1). All reactions of the TCA cycle proceed consistently in one direction. The total value of D G 0 ¢ = -40 kJ/mol.

There has long been a catchphrase among doctors: “Fats burn in the flames of carbohydrates.” It must 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). In cases of carbohydrate metabolism disorders or starvation, a deficiency of oxaloacetate is created, leading to a decrease in the oxidation of acetyl-CoA in the TCA cycle.

Fig.1. The role of the TCA cycle in cellular respiration. Stage 1 (TCA cycle) 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) uses 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 the TCA cycle can be represented in the form of two processes: 1) a series of redox reactions, as a result of which the acetyl group is oxidized to two CO 2 molecules; 2) fourfold dehydrogenation, leading to the formation of 3 molecules of NADH + H + and 1 molecule of FADH 2 . Oxygen is required for the functioning of the TCA cycle 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 the TCA cycle.

1. Isocitrate dehydrogenase is allosterically activated by ADP by increasing the enzyme's affinity for the substrate. NADH inhibits this enzyme by replacing NAD+. ATP also inhibits isocitrate dehydrogenase. It is important that the transformation of metabolites into the TCA cycle 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 (the end products of the transformations catalyzed by the 2-oxoglutarate dehydrogenase complex). In addition, the 2-oxoglutarate dehydrogenase complex is inhibited by the high energy charge of the cell. So, the rate of transformation into the TCA cycle 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 regulation scheme of the general catabolic pathway is presented in Figure 2.

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

4.3. Energy role of the common catabolic pathway

In the general path of catabolism, 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 removed, of which one pair is from succinate and goes to FAD with the formation of FADH 2, and four pairs are taken into 4 molecules of NAD + with the formation of 4 molecules of NADH + H + during the oxidative decarboxylation of pyruvic acid , 2-oxoglutaric acids, dehydrogenation of isocitrate and malate. Ultimately, hydrogen atoms are transferred to oxygen to form 5 H2O 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. There are ~9 ATP in the TCA cycle and associated respiratory chains.

3. In the reaction of substrate phosphorylation of the TCA cycle, ~ 1 ATP.

In the TCA cycle and associated 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 molecules of ATP are released.

I talked about what it actually 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 activity 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.

First reaction

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 HADH 2 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 will 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 PO 3 to GDP 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’s why it’s 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” into 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. 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 NADH 2, 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 discovered in 1937 by G. Krebs. In this regard, it was called the “Krebs cycle”. This process is the central pathway of metabolism. It occurs in the cells of organisms at different stages of evolutionary development (microorganisms, plants, animals).

The initial substrate of the tricarboxylic acid cycle is acetyl coenzyme A. This metabolite is the active form of acetic acid. Acetic acid acts as a common intermediate breakdown product of almost all organic substances contained in the cells of living organisms. This is because organic molecules are carbon compounds that can naturally break down into two-carbon acetic acid units.

Free acetic acid has a relatively weak reactivity. Its transformations occur under rather harsh conditions, which are unrealistic in a living cell. Therefore, acetic acid is activated in cells by combining it with coenzyme A. As a result, a metabolically active form of acetic acid is formed - acetyl-coenzyme A.

Coenzyme A is a low molecular weight compound that consists of phosphoadenosine, a pantothenic acid residue (vitamin B3) and thioethanolamine. The acetic acid residue is added to the sulfhydryl group of thioethanolamine. In this case, a thioether is formed - acetyl-coenzyme A, which is the initial substrate of the Krebs cycle.

Acetyl coenzyme A

A diagram of the transformation of intermediate products in the Krebs cycle is shown in Fig. 67. The process begins with the condensation of acetyl coenzyme A with oxaloacetate (oxaloacetic acid, OCA), resulting in the formation of citric acid (citrate). The reaction is catalyzed by the enzyme citrate synthase.

Figure 67 – Scheme of the transformation of intermediate products in the cycle

tricarboxylic acids

Further, under the action of the enzyme aconitase, citric acid is converted into isocitric acid. Isocitric acid undergoes oxidation and decarboxylation processes. In this reaction, catalyzed by the enzyme NAD-dependent isocitrate dehydrogenase, the products are carbon dioxide, reduced NAD, and a-ketoglutaric acid, which is then involved in the process of oxidative decarboxylation (Fig. 68).

Figure 68 – Formation of a-ketoglutaric acid in the Krebs cycle

The process of oxidative decarboxylation of a-ketoglutarate is catalyzed by the enzymes of the a-ketoglutarate dehydrogenase multienzyme complex. This complex consists of three different enzymes. It requires coenzymes to function. Coenzymes of the a-keto-glutarate dehydrogenase complex include the following water-soluble vitamins:

· vitamin B 1 (thiamine) – thiamine pyrophosphate;

· vitamin B 2 (riboflavin) – FAD;

· vitamin B 3 (pantothenic acid) – coenzyme A;

· vitamin B 5 (nicotinamide) – NAD;

· vitamin-like substance – lipoic acid.

Schematically, the process of oxidative decarboxylation of a-keto-glutaric acid can be represented as the following balance reaction equation:

The product of this process is a thioester of the succinic acid residue (succinate) with coenzyme A - succinyl-coenzyme A. The thioester bond of succinyl-coenzyme A is macroergic.

The next reaction of the Krebs cycle is the process of substrate phosphorylation. In it, the thioester bond of succinyl-coenzyme A is hydrolyzed under the action of the enzyme succinyl-CoA synthetase with the formation of succinic acid (succinate) and free coenzyme A. This process is accompanied by the release of energy, which is immediately used for phosphorylation of HDP, which results in the formation of a high-energy molecule GTP phosphate. Substrate phosphorylation in the Krebs cycle:

where Fn is orthophosphoric acid.

GTP formed during oxidative phosphorylation can be used as an energy source in various energy-dependent reactions (in the process of protein biosynthesis, activation of fatty acids, etc.). In addition, GTP can be used to generate ATP in the nucleoside diphosphate kinase reaction

The product of the succinyl-CoA synthetase reaction, succinate, is further oxidized with the participation of the enzyme succinate dehydrogenase. This enzyme is a flavin dehydrogenase, which contains the FAD molecule as a coenzyme (prosthetic group). As a result of the reaction, succinic acid is oxidized to fumaric acid. At the same time, FAD is restored.

where E is the FAD prosthetic group associated with the polypeptide chain of the enzyme.

Fumaric acid formed in the succinate dehydrogenase reaction, under the action of the fumarase enzyme (Fig. 69), attaches a water molecule and is converted into malic acid, which is then oxidized in the malate dehydrogenase reaction into oxaloacetic acid (oxaloacetate). The latter can be used again in the citrate synthase reaction for the synthesis of citric acid (Fig. 67). Due to this, transformations in the Krebs cycle are cyclic in nature.

Figure 69 – Metabolism of malic acid in the Krebs cycle

The balance equation of the Krebs cycle can be presented as:

It shows that in the cycle there is complete oxidation of the acetyl radical of the residue from acetyl-coenzyme A to two molecules of CO 2. This process is accompanied by the formation of three molecules of reduced NAD, one molecule of reduced FAD and one molecule of high-energy phosphate - GTP.

The Krebs cycle occurs in the mitochondrial matrix. This is due to the fact that this is where most of its enzymes are located. And only a single enzyme, succinate dehydrogenase, is built into the inner mitochondrial membrane. The individual enzymes of the tricarboxylic acid cycle are combined into a functional multienzyme complex (metabolon) associated with the inner surface of the inner mitochondrial membrane. By combining enzymes into a metabolon, the efficiency of functioning of this metabolic pathway is significantly increased and additional opportunities for its fine regulation appear.

Features of the regulation of the tricarboxylic acid cycle are largely determined by its significance. This process performs the following functions:

1) energy. The Krebs cycle is the most powerful source of substrates (reduced coenzymes - NAD and FAD) for tissue respiration. In addition, energy is stored in it in the form of high-energy phosphate - GTP;

2) plastic. Intermediate products of the Krebs cycle are precursors for the synthesis of various classes of organic substances - amino acids, monosaccharides, fatty acids, etc.

Thus, the Krebs cycle performs a dual function: on the one hand, it is a general pathway of catabolism, playing a central role in the energy supply of the cell, and on the other, it provides biosynthetic processes with substrates. Such metabolic processes are called amphibolic. The Krebs cycle is a typical amphibolic cycle.

The regulation of metabolic processes in the cell is closely related to the existence of “key” enzymes. The key enzymes in the process are those that determine its speed. Typically, one of the “key” enzymes in a process is the enzyme that catalyzes its initial reaction.

The “key” enzymes are characterized by the following features. These enzymes

· catalyze irreversible reactions;

· have the least activity compared to other enzymes involved in the process;

· are allosteric enzymes.

The key enzymes of the Krebs cycle are citrate synthase and isocitrate dehydrogenase. Like key enzymes in other metabolic pathways, their activity is regulated by negative feedback: it decreases as the concentration of Krebs cycle intermediates in mitochondria increases. Thus, citric acid and succinyl-coenzyme A act as citrate synthase inhibitors, and reduced NAD acts as isocitrate dehydrogenase.

ADP is an activator of isocitrate dehydrogenase. Under conditions of increasing cell need for ATP as an energy source, when the content of breakdown products (ADP) increases in it, prerequisites arise for increasing the rate of redox transformations in the Krebs cycle and, consequently, increasing the level of its energy supply.

TRICARBOXYLIC ACIDS CYCLE

TRICARBOXYLIC ACIDS CYCLE - the citric acid cycle or the Krebs cycle is a widely represented pathway in the organisms of animals, plants and microbes for the oxidative transformations of di- and tricarboxylic acids formed as intermediate products during the breakdown and synthesis of proteins, fats and carbohydrates. Discovered by H. Krebs and W. Johnson (1937). This cycle is the basis of metabolism and performs two important functions - supplying the body with energy and integrating all the main metabolic flows, both catabolic (biodegradation) and anabolic (biosynthesis).

The Krebs cycle consists of 8 stages (intermediate products are highlighted in two stages in the diagram), during which the following occurs:

1) complete oxidation of the acetyl residue to two CO2 molecules,

2) three molecules of reduced nicotinamide adenine dinucleotide (NADH) and one reduced flavin adenine dinucleotide (FADH2) are formed, which is the main source of energy produced in the cycle and

3) one molecule of guanosine triphosphate (GTP) is formed as a result of the so-called substrate oxidation.

In general, the path is energetically favorable (DG0" = –14.8 kcal.)

The Krebs cycle, localized in mitochondria, begins with citric acid (citrate) and ends with the formation of oxaloacetic acid (oxaloacetate - OA). The substrates of the cycle include tricarboxylic acids - citric, cis-aconitic, isocitric, oxalosuccinate (oxalosuccinate) and dicarboxylic acids - 2-ketoglutaric (KG), succinic, fumaric, malic (malate) and oxaloacetic. Substrates of the Krebs cycle also include acetic acid, which in its active form (i.e. in the form of acetyl coenzyme A, acetyl-SCoA) participates in condensation with oxaloacetic acid, leading to the formation of citric acid. It is the acetyl residue included in the structure of citric acid that is oxidized; carbon atoms are oxidized to CO2, hydrogen atoms are partially accepted by coenzymes of dehydrogenases, and partially pass into solution, that is, into the environment in protonated form.

Pyruvic acid (pyruvate), which is formed during glycolysis and occupies one of the central places in intersecting metabolic pathways, is usually indicated as the starting compound for the formation of acetyl-CoA. Under the influence of an enzyme with a complex structure - pyruvate dehydrogenase (CP1.2.4.1 - PDHase), pyruvate is oxidized to form CO2 (first decarboxylation), acetyl-CoA and reduced by NAD (see diagram). However, the oxidation of pyruvate is far from the only way to form acetyl-CoA, which is also a characteristic product of the oxidation of fatty acids (thiolase enzyme or fatty acid synthetase) and other reactions of the decomposition of carbohydrates and amino acids. All enzymes involved in the reactions of the Krebs cycle are localized in mitochondria, most of them are soluble, and succinate dehydrogenase (KF1.3.99.1) is tightly associated with membrane structures.

The formation of citric acid, with the synthesis of which the cycle proper begins, with the help of citrate synthase (EC4.1.3.7 - the condensing enzyme in the diagram), is an endergonic reaction (with energy absorption), and its implementation is possible due to the use of the energy-rich bond of the acetyl residue with KoA [CH3CO~SKoA]. This is the main stage of regulation of the entire cycle. This is followed by the isomerization of citric acid into isocitric acid through the intermediate stage of the formation of cis-aconitic acid (the enzyme aconitase KF4.2.1.3, has absolute stereospecificity - sensitivity to the location of hydrogen). The product of further transformation of isocitric acid under the influence of the corresponding dehydrogenase (isocitrate dehydrogenase KF1.1.1.41) is apparently oxalosuccinic acid, the decarboxylation of which (the second CO2 molecule) leads to CG. This stage is also strictly regulated. In a number of characteristics (high molecular weight, complex multicomponent structure, stepwise reactions, partially the same coenzymes, etc.) KH dehydrogenase (EC1.2.4.2) resembles PDHase. The reaction products are CO2 (third decarboxylation), H+ and succinyl-CoA. At this stage, succinyl-CoA synthetase, otherwise called succinate thiokinase (EC6.2.1.4), is activated, catalyzing the reversible reaction of the formation of free succinate: Succinyl-CoA + Pneorg + GDP = Succinate + KoA + GTP. During this reaction, so-called substrate phosphorylation occurs, i.e. formation of energy-rich guanosine triphosphate (GTP) at the expense of guanosine diphosphate (GDP) and mineral phosphate (Pneorg) using the energy of succinyl-CoA. After the formation of succinate, succinate dehydrogenase (KF1.3.99.1), a flavoprotein, comes into action, leading to fumaric acid. FAD is linked to the protein portion of the enzyme and is the metabolically active form of riboflavin (vitamin B2). This enzyme is also characterized by absolute stereospecificity in hydrogen elimination. Fumarase (EC4.2.1.2) ensures the balance between fumaric acid and malic acid (also stereospecific), and malic acid dehydrogenase (malate dehydrogenase EC1.1.1.37, which requires the coenzyme NAD +, is also stereospecific) leads to the completion of the Krebs cycle, that is, to formation of oxaloacetic acid. After this, the condensation reaction of oxaloacetic acid with acetyl-CoA is repeated, leading to the formation of citric acid, and the cycle resumes.

Succinate dehydrogenase is part of the more complex succinate dehydrogenase complex (complex II) of the respiratory chain, supplying reducing equivalents (NAD-H2) formed during the reaction to the respiratory chain.

Using the example of PDHase, you can get acquainted with the principle of cascade regulation of metabolic activity due to phosphorylation-dephosphorylation of the corresponding enzyme by special kinase and phosphatase PDHase. Both of them are connected to PDGase.

TRICARBOXYLIC ACIDS CYCLE

It is assumed that the catalysis of individual enzymatic reactions is carried out as part of a supramolecular “supercomplex”, the so-called “metabolon”. The advantages of such an organization of enzymes are that there is no diffusion of cofactors (coenzymes and metal ions) and substrates, and this contributes to more efficient operation of the cycle.

The energy efficiency of the processes considered is low, however, 3 moles of NADH and 1 mole of FADH2 formed during the oxidation of pyruvate and subsequent reactions of the Krebs cycle are important products of oxidative transformations. Their further oxidation is carried out by enzymes of the respiratory chain also in mitochondria and is associated with phosphorylation, i.e. the formation of ATP due to esterification (formation of organophosphorus esters) of mineral phosphate. Glycolysis, the enzymatic action of PDHase and the Krebs cycle - a total of 19 reactions - determine the complete oxidation of one molecule of glucose to 6 molecules of CO2 with the formation of 38 molecules of ATP - this "energy currency" of the cell. The process of oxidation of NADH and FADH2 by enzymes of the respiratory chain is energetically very efficient, occurs using atmospheric oxygen, leads to the formation of water and serves as the main source of energy resources of the cell (more than 90%). However, enzymes of the Krebs cycle are not involved in its direct implementation. Each human cell has from 100 to 1000 mitochondria, which provide vital energy.

The basis of the integrating function of the Krebs cycle in metabolism is that carbohydrates, fats and amino acids from proteins can ultimately be converted into intermediates (intermediates) of this cycle or synthesized from them. The removal of intermediates from the cycle during anabolism must be combined with the continuation of the catabolic activity of the cycle for the constant formation of ATP necessary for biosynthesis. Thus, the loop must perform two functions simultaneously. At the same time, the concentration of intermediates (especially OA) may decrease, which can lead to a dangerous decrease in energy production. To prevent this, there are “safety valves” called anaplerotic reactions (from the Greek “to fill”). The most important reaction is the synthesis of OA from pyruvate, carried out by pyruvate carboxylase (EC6.4.1.1), also localized in mitochondria. As a result, a large amount of OA accumulates, which ensures the synthesis of citrate and other intermediates, which allows the Krebs cycle to function normally and, at the same time, ensure the removal of intermediates into the cytoplasm for subsequent biosynthesis. Thus, at the level of the Krebs cycle, an effectively coordinated integration of the processes of anabolism and catabolism occurs under the influence of numerous and subtle regulatory mechanisms, including hormonal ones.

Under anaerobic conditions, instead of the Krebs cycle, its oxidative branch functions to KG (reactions 1, 2, 3) and its reductive branch functions from OA to succinate (reactions 8®7®6). In this case, much energy is not stored and the cycle supplies only intermediates for cellular synthesis.

When the body transitions from rest to activity, the need arises to mobilize energy and metabolic processes. This, in particular, is achieved in animals by shunting the slowest reactions (1–3) and predominant oxidation of succinate. In this case, KG, the initial substrate of the shortened Krebs cycle, is formed in the rapid transamination reaction (amine group transfer)

Glutamate + OA = CG + aspartate

Another modification of the Krebs cycle (the so-called 4-aminobutyrate shunt) is the conversion of KG to succinate through glutamate, 4-aminobutyrate and succinic semialdehyde (3-formylpropionic acid). This modification is important in brain tissue, where about 10% of glucose is broken down through this pathway.

The close coupling of the Krebs cycle with the respiratory chain, especially in animal mitochondria, as well as the inhibition of most enzymes of the cycle under the influence of ATP, determine a decrease in the activity of the cycle at a high phosphoryl potential of the cell, i.e. at a high ATP/ADP concentration ratio. In most plants, bacteria and many fungi, the tight coupling is overcome by the development of uncoupled alternative oxidation pathways, which allow simultaneous respiration and cycle activity to be maintained at a high level even at a high phosphoryl potential.

Igor Rapanovich

TRICARBOXYLIC ACIDS CYCLE

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Literature

Strayer L. Biochemistry. Per. from English M., Mir, 1985

Bohinski R. Modern views in biochemistry. Translated from English, M., Mir, 1987

Knorre D.G., Myzina S.D. Biological chemistry. M., Higher School, 2003

Kolman J., Rem K.-G. Visual biochemistry. M., Mir, 2004

Brief historical information

Our favorite cycle is the TCA cycle, or the tricarboxylic acid cycle - life on Earth and under the Earth and in the Earth... Stop, in general this is the most amazing mechanism - it is universal, it is a way of oxidizing the breakdown products of carbohydrates, fats, proteins in the cells of living organisms, as a result We get energy for the activities of our body.

This process was discovered by Hans Krebs himself, for which he received the Nobel Prize!

He was born in August 25 - 1900 in the German city of Hildesheim. He received a medical education from the University of Hamburg and continued biochemical research under the leadership of Otto Warburg in Berlin.

In 1930, together with his student, he discovered the process of neutralizing ammonia in the body, which was present in many representatives of the living world, including humans. This cycle is the urea cycle, which is also known as the Krebs cycle #1.

When Hitler came to power, Hans emigrated to Great Britain, where he continues to study science at the Universities of Cambridge and Sheffield. Developing the research of the Hungarian biochemist Albert Szent-Györgyi, he received an insight and made the most famous Krebs cycle No. 2, or in other words, the “Szent-Györgyö – Krebs cycle” - 1937.

The research results are sent to the journal Nature, which refuses to publish the article. Then the text flies to the magazine "Enzymologia" in Holland. Krebs received the Nobel Prize in 1953 in physiology or medicine.

The discovery was surprising: in 1935 Szent-Györgyi found that succinic, oxaloacetic, fumaric and malic acids (all 4 acids are natural chemical components of animal cells) enhance the oxidation process in the pectoral muscle of the pigeon. Which was shredded.

It is in it that metabolic processes occur at the highest speed.

F. Knoop and K. Martius in 1937 found that citric acid is converted into isocitric acid through an intermediate product, cis - aconitic acid. In addition, isocitric acid could be converted into a-ketoglutaric acid, and that into succinic acid.

Krebs noticed the effect of acids on the absorption of O2 by the pectoral muscle of a pigeon and identified an activating effect on the oxidation of PVC and the formation of Acetyl-Coenzyme A. In addition, the processes in the muscle were inhibited by malonic acid, which is similar to succinic acid and could competitively inhibit enzymes whose substrate is succinic acid .

When Krebs added malonic acid to the reaction medium, the accumulation of a-ketoglutaric, citric and succinic acids began. Thus, it is clear that the combined action of a-ketoglutaric and citric acids leads to the formation of succinic acid.

Hans examined more than 20 other substances, but they did not affect oxidation. Comparing the data obtained, Krebs received a cycle. At the very beginning, the researcher could not say for sure whether the process began with citric or isocitric acid, so he called it the “tricarboxylic acid cycle.”

Now we know that the first is citric acid, so the correct name is the citrate cycle or the citric acid cycle.

In eukaryotes, TCA cycle reactions occur in mitochondria, while all enzymes for catalysis, except 1, are contained in a free state in the mitochondrial matrix; the exception is succinate dehydrogenase, which is localized on the inner membrane of the mitochondrion and is embedded in the lipid bilayer. In prokaryotes, the reactions of the cycle occur in the cytoplasm.

Let's meet the participants of the cycle:

2) PIKE – Oxaloacetate - Oxaloacetic acid:
seems to consist of two parts: oxalic and acetic acid.

3-4) Citric and Isocitric acids:

5) a-Ketoglutaric acid:

6) Succinyl-Coenzyme A:

7) Succinic acid:

8) Fumaric acid:

9) Malic acid:

How do reactions occur? In general, we are all accustomed to the appearance of the ring, which is shown below in the picture. Below everything is described step by step:

1. Condensation of Acetyl Coenzyme A and Oxaloacetic acid ➙ citric acid.

The transformation of Acetyl Coenzyme A begins with condensation with Oxaloacetic acid, resulting in the formation of citric acid.

The reaction does not require the consumption of ATP, since the energy for this process is provided as a result of hydrolysis of the thioether bond with Acetyl Coenzyme A, which is high-energy:

2. Citric acid passes through cis-aconitic acid into isocitric acid.

Isomerization of citric acid into isocitric acid occurs. The conversion enzyme - aconitase - first dehydrates citric acid to form cis-aconitic acid, then connects water to the double bond of the metabolite, forming isocitric acid:

3. Isocitric acid is dehydrogenated to form α-ketoglutaric acid and CO2.

Isocitric acid is oxidized by a specific dehydrogenase, the coenzyme of which is NAD.

Simultaneously with oxidation, decarboxylation of isocitric acid occurs. As a result of transformations, α-ketoglutaric acid is formed.

4. Alpha-ketoglutaric acid is dehydrogenated by ➙ succinyl-coenzyme A and CO2.

The next stage is the oxidative decarboxylation of α-ketoglutaric acid.

Catalyzed by the α-ketoglutarate dehydrogenase complex, which is similar in mechanism, structure and action to the pyruvate dehydrogenase complex. As a result, succinyl-CoA is formed.

5. Succinyl coenzyme A ➙ succinic acid.

Succinyl-CoA is hydrolyzed to free succinic acid, the energy released is stored by the formation of guanosine triphosphate. This stage is the only one in the cycle at which energy is directly released.

6. Succinic acid is dehydrogenated ➙ fumaric acid.

The dehydrogenation of succinic acid is accelerated by succinate dehydrogenase, its coenzyme is FAD.

7. Fumaric acid is hydrated ➙ malic acid.

Fumaric acid, which is formed by dehydrogenation of succinic acid, is hydrated and malic acid is formed.

8. Malic acid is dehydrogenated ➙ Oxalic-acetic acid - the cycle closes.

The final process is dehydrogenation of malic acid, catalyzed by malate dehydrogenase;

The result of the stage is the metabolite with which the tricarboxylic acid cycle begins - Oxalic-Acetic acid.

In reaction 1 of the next cycle, another quantity of Acetyl Coenzyme A will enter.

How to remember this cycle? Just!

2) Another long poem:

PIKE ate acetate, it turns out citrate,
Through cisaconitate it will become isocitrate.
Having given up hydrogen to NAD, it loses CO2,
Alpha-ketoglutarate is extremely happy about this.
Oxidation is coming - NAD has stolen hydrogen,
TDP, coenzyme A takes CO2.
And the energy barely appeared in succinyl,
Immediately ATP was born and what remained was succinate.
Now he got to the FAD - he needs hydrogen,
The fumarate drank from the water and turned into malate.
Then NAD came to malate, acquired hydrogen,
The PIKE showed up again and quietly hid.

3) The original poem - in short:

PIKE ACETYL LIMONIL,
But the horse was afraid of narcissus,
He is above him ISOLIMON
ALPHA - KETOGLUTARASED.
SUCCINALIZED WITH COENZYME,
AMBER FUMAROVO,
Stored up some APPLES for the winter,
Turned into a PIKE again.