BIOCHEMISTRY: A TEXTBOOK FOR HIGHER EDUCATION - E. S. Severin - 2004

SECTION 6. ENERGY METABOLISM

III. The Final Stage of Catabolism — The Main Source of Hydrogen Donors for the Electron Transport Chain

CARBOHYDRATES, Fatty acids, and Most Amino Acids are ultimately oxidized via The Citric Acid Cycle to CO₂ and H₂O. Before these substances enter The final stage of Catabolism, their carbon skeletons are converted into a two-carbon fragment in the form of acetyl-CoA (Fig. 6-19). It is in this form that the majority of fuel molecules enter The Citric Acid cycle.

Class="center">Fig. 6-19. Catabolism of major nutrients. 1 — 3 — Digestion; 4 — 8 — specific Catabolic pathways; 9 — 10 — final (common) pathway of catabolism; 11 — Electron Transport Chain; 12 — Oxidative Phosphorylation.

Acetyl-CoA is formed in specific catabolic reactions of Fatty Acids and Certain amino acids (see sections 8 and 9). However, the primary source of acetyl-CoA is pyruvic acid, which is produced during the catabolism of glucose and certain amino acids (see sections 7 and 9).

The conversion of Pyruvate to acetyl-CoA is catalyzed by a multienzyme system structurally organized as the pyruvate dehydrogenase complex (PDC). The acetyl moiety of acetyl-CoA is subsequently oxidized in the citric acid cycle to CO₂ and H₂O. These oxidation reactions involve NAD- and FAD-dependent dehydrogenases, which supply electrons and protons to The electron transport chain, where they are ultimately transferred to O₂.

A. Oxidative Decarboxylation of Pyruvate

The oxidative decarboxylation of pyruvate takes place in the mitochondrial matrix. Pyruvate is transported across The inner mitochondrial membrane into the matrix by a specific carrier protein via an H⁺-symport mechanism (Fig. 6-20).

Fig. 6-20. Transport of pyruvate across the mitochondrial membrane.

The conversion of pyruvate into acetyl-CoA can be represented by the following overall equation:

СН₃-СО-СООН + NAD+ + HSКоА —> СН₃-СО ~ SКоА + NADH + Н⁺ + СO₂.

During this reaction, oxidative decarboxylation of pyruvate takes place, resulting in the removal of the carboxyl group as CO₂ and the incorporation of the acetyl group into acetyl-CoA. One hydrogen atom is incorporated into NADH, while the other is released into the medium as H⁺. The reaction is irreversible, as ΔG°′ = -33.5 kJ/mol.

1. Structure OF THE Pyruvate Dehydrogenase Complex

The oxidative decarboxylation of pyruvate is catalyzed by the highly complex pyruvate dehydrogenase complex. The PDC comprises three Enzymes: pyruvate decarboxylase (E₁), dihydrolipoyl transacetylase (E₂), and dihydrolipoyl dehydrogenase (E₃), as well as five Coenzymes: Thiamine diphosphate (TDP), Lipoic Acid, FAD, NAD⁺, and CoA. In addition, the complex contains Regulatory Subunits: protein kinase and phosphoprotein phosphatase (Table 6-5).

Table 6-5. Mammalian Pyruvate Dehydrogenase Complex (PDC)

All of these Enzymes and Coenzymes are assembled into a multienzyme system containing varying amounts of each enzyme component and having a molecular weight greater than 6 x 10⁶.

Dihydrolipoyl transacetylase (E₂) is located at the center of the complex, forming its core. Molecules of pyruvate decarboxylase (E₁) and dihydrolipoyl dehydrogenase (E₃) are attached to the dihydrolipoyl transacetylase core.

Pyruvate decarboxylase contains TDP tightly bound to its protein moiety, whereas dihydrolipoyl dehydrogenase contains bound FAD.

The lipoyllisine residues of the core enzyme (E₂) function as flexible swinging arms ("tethers") that transfer hydrogen atoms and acetyl groups from one enzyme component of the complex to another.

2. Mechanism of Pyruvate Oxidative Decarboxylation

The conversion of pyruvate to acetyl-CoA proceeds in five stages (Fig. 6-21).

Fig. 6-21. Sequence of Reactions Catalyzed by the PDC. I — E₁ catalyzes pyruvate decarboxylation and transfer of the C₂-fragment to TPP; II — E₂ catalyzes oxidation of the hydroxyethyl group and transfer of the C₂-fragment to lipoic acid (LA); III — acetylated dihydrolipoyl transacetylase reacts with CoA to form the reduced form of lipoic acid and acetyl-CoA; IV — the oxidized form of transacetylase is regenerated with the participation of E₃; V — the oxidized form of E₃ is regenerated with the participation of NAD⁺.

Stage I. At this stage, pyruvate binds to TPP within E₁ and undergoes decarboxylation.

Pyruvate + E₁-TPP —> Hydroxyethyl-TPP + СО₂

This reaction yields a TPP derivative containing a hydroxyethyl group attached to the thiazole ring (Fig. 6-22).

Fig. 6-22. Thiamine diphosphate (TPP) and hydroxyethyl-TPP. The functional part of TPP is the thiazole ring, to which the pyruvate decarboxylation product—the hydroxyethyl group—attaches.

Stage II. Dihydrolipoyl transacetylase (E₂) catalyzes The transfer of a hydrogen atom and an acetyl group from TPP to the oxidized form of lipoylysine residues, forming the acetyl thioester of lipoic acid (Fig. 6-21).

Stage III. In stage III, CoA reacts with the acetyl derivative of E₂, resulting in the Formation of Acetyl-CoA and a fully reduced lipoyl residue, which serves as the prosthetic group of E₂ (Fig. 6-23).

Fig. 6-23. Lipoic acid as part of dihydrolipoyl transacetylase (E₂). Lipoic acid or the lipoyl group can exist in oxidized (disulfide LA-SS) and reduced (LA-(SН)₂) forms. Within dihydrolipoyl transacetylase, lipoic acid is linked to the protein via a Lysine residue through an amide bond. Lipoic acid acts as a vitamin or growth factor in certain microorganisms, whereas higher animals are capable of synthesizing it.

Stage IV. In stage IV, dihydrolipoyl dehydrogenase (E₃) catalyzes the transfer of hydrogen atoms from the reduced lipoyl groups to FAD, the prosthetic group of the E₃ enzyme.

Stage V. In stage V, the reduced FADH₂ transfers hydrogen to NAD⁺ to form NADH.

The pyruvate dehydrogenase complex is characterized by a large negative redox potential, which, along with coenzyme reduction (NADH), ensures The formation of a high-energy thioester bond in acetyl-CoA.

The structural association of Three types of enzymes enables the coordination of individual steps in this complex enzymatic reaction. All Intermediates of the oxidative decarboxylation of pyruvate remain tightly bound to the complex, which increases the overall reaction rate and minimizes Side Reactions.

Like all Proteins involved in TCA cycle reactions, the pyruvate dehydrogenase complex is encoded by nuclear DNA. The transport of PDC subunits into the Cell/35.html">Mitochondria is a complex process driven by ATP energy or the transmembrane Electrochemical Potential, aided by heat Shock proteins, or chaperones (see Section 1), which prevent their premature folding prior to entering the mitochondrial matrix or the inner mitochondrial membrane.

3. Link between the oxidative decarboxylation of pyruvate and the ETC

The oxidative decarboxylation of pyruvate is accompanied by the formation of NADH, which supplies electrons to the Respiratory Chain and drives the synthesis of 3 moles of ATP per mole of pyruvate via oxidative phosphorylation.

Since the ADP/ATP and NADH/NAD⁺ ratios in The Cell are relatively constant, accelerated ATP utilization leads to an increased ADP concentration and speeds up The oxidation of NADH in the respiratory chain. An elevated NAD⁺ concentration, in turn, stimulates the oxidative decarboxylation of pyruvate. Conversely, elevated ATP and NADH concentrations decrease The rate of this process. Thus, shifts in the ADP/ATP and NADH/NAD⁺ ratios serve as vital signals reflecting the energy demands of the cell and regulating the rate of pyruvate oxidative decarboxylation. The catalytic activity of the pyruvate dehydrogenase complex decreases when Cells have an adequate supply of "fuel" in the form of fatty acids and acetyl-CoA.

B. Citric acid cycle

The citric acid cycle (citrate cycle, Krebs cycle, Tricarboxylic Acid Cycle, TCA cycle) is the final stage of catabolism, in which the carbon of the acetyl-CoA acetyl moiety is oxidized to 2 molecules of СО₂. Hydrogen atoms released during redox reactions are delivered to the ETC via NAD- and FAD-dependent dehydrogenases, resulting in Water synthesis and the Oxidative phosphorylation of ADP. The carbon-carbon bonds in acetyl-CoA are resistant to oxidation. Under physiological conditions, the oxidation of the acetyl group occurs in a series of steps forming a cyclic pathway of 8 reactions (Fig. 6-24).

1. Sequence of reactions in the citrate cycle

Formation of citrate

In the citrate synthesis reaction, the methyl carbon atom of acetyl-CoA condenses with the carbonyl group of oxaloacetate (Fig. 6-24); simultaneously, the thioester bond is cleaved and coenzyme A is released (∆G°′ = -37.6 kJ/mol).

The reaction equilibrium in the cell strongly favors the forward direction, as indicated by the negative Standard Free energy change. The reaction is accompanied by the release of a large amount of energy as heat. It is catalyzed by citrate synthase, an enzyme localized in the mitochondrial matrix.

Conversion of Citrate to Isocitrate

The second reaction of the citric acid cycle is the reversible conversion of citrate to isocitrate (Fig. 6-24). The enzyme catalyzing this reaction is named aconitase after the intermediate product, cis-aconitic acid, which is presumed to be formed during the reaction. However, this compound is not detected in a free state because it does not dissociate from the Active Site of the enzyme until the reaction is complete.

Oxidative Decarboxylation of Isocitrate

This reaction is catalyzed by isocitrate dehydrogenase. There are two forms of isocitrate dehydrogenase: one contains NAD⁺ as a coenzyme, and the second contains NADP⁺. The NAD-dependent enzyme is localized in the mitochondria and participates in the TCA cycle (whereas the NADP-dependent enzyme, present in both mitochondria and Cytoplasm, plays a different metabolic role). The action of this enzyme on isocitrate yields α-ketoglutarate (see Fig. 6-24).

The reaction catalyzed by the NAD-dependent isocitrate dehydrogenase is the slowest step of the citric acid cycle. ADP acts as an allosteric activator of the enzyme.

Oxidative Decarboxylation of α-Ketoglutarate

In this reaction, α-ketoglutarate undergoes oxidative decarboxylation, yielding succinyl-CoA, CO₂, and NADH + H⁺ as end products. The outcome of this reaction is the formation of succinyl-CoA (see Fig. 6-24).

Fig. 6-24. General scheme of the citric acid cycle. Numbers 1–8 denote the Reactions of the cycle. The cycle begins with the Condensation of an acetyl group with oxaloacetate, forming the six-carbon compound citrate. One molecule of oxaloacetate is consumed to form citrate in each turn of the cycle, and oxaloacetate is regenerated upon completion of the cycle. Thus, a single molecule of oxaloacetate can be reused multiple times for the oxidation of acetyl groups.

The reaction is catalyzed by the α-ketoglutarate dehydrogenase complex, which is structurally and functionally similar to the pyruvate dehydrogenase complex (PDC). Like the PDC, it consists of three enzymes: α-ketoglutarate decarboxylase, dihydrolipoyl transsuccinylase, and dihydrolipoyl dehydrogenase. In addition, this enzyme complex includes five coenzymes: thiamine diphosphate, coenzyme A, lipoic acid, NAD⁺, and FAD. A key difference between this enzyme system and the PDC is that it lacks the complex regulatory mechanism characteristic of the PDC. Specifically, this complex lacks regulatory subunits. The equilibrium of the α-ketoglutarate oxidative decarboxylation reaction is strongly shifted toward the formation of succinyl-CoA and can be considered unidirectional.

Conversion of Succinyl-CoA to Succinate

Succinyl-CoA is a high-energy compound. The change in standard Free energy of Hydrolysis for this thioester is ∆G°′ = -35.7 kJ/mol. In the mitochondria, the Cleavage of the thioester bond of succinyl-CoA is coupled with the phosphorylation of guanosine diphosphate (GDP) to guanosine triphosphate (GTP).

Succinyl-CoA -> Succinate (∆G° = -10.36 kJ/mol).

This coupled reaction (see Fig. 6-24) is catalyzed by succinate thiokinase. An intermediate step of the reaction is the phosphorylation of the enzyme molecule at one of the Histidine residues in the active site. Subsequently, the phosphate group is transferred to GDP, forming GTP.

The terminal phosphate group of GTP can be transferred to ADP to form ATP; this reversible reaction is catalyzed by nucleoside diphosphate kinase.

GTP + ADP <-> GDP + ATP.

The formation of a high-energy phosphoanhydride bond driven by substrate energy (succinyl-CoA) is an example of substrate-level phosphorylation.

Dehydrogenation of Succinate

The succinate formed in the previous step is converted into fumarate by the action of succinate dehydrogenase (see Fig. 6-24). This enzyme is a flavoprotein whose molecule contains a tightly bound FAD coenzyme.

Succinate dehydrogenase is firmly bound to the inner mitochondrial membrane. It consists of two subunits, one of which is bound to FAD. In addition, both subunits contain iron-sulfur centers: one contains Fe₂S₂ and the other Fe₄S₄. Within the iron-sulfur centers, iron atoms change their valence state as they participate in electron transport.

Formation of Malate from Fumarate

The formation of malate proceeds via the enzyme fumarate hydratase (see Fig. 6-24). This enzyme is more commonly known as fumarase.

Fumarase is an oligomeric protein consisting of four identical polypeptide chains. It is located in the mitochondrial matrix. Fumarase is classified among enzymes with absolute substrate Specificity: it catalyzes the Hydration exclusively of the trans-isomer of fumarate.

Dehydrogenation of Malate

In the final stage of the citric acid cycle, malate is dehydrogenated to yield oxaloacetate (see Fig. 6-24). The reaction is catalyzed by the NAD-dependent malate dehydrogenase located in the mitochondrial matrix.

The equilibrium of the malate dehydrogenase reaction lies far to the left. Nevertheless, in intact cells this reaction proceeds from left to right because the reaction product, oxaloacetate, is actively consumed in the citrate synthase reaction. The Cytosol contains an isoform of malate dehydrogenase that is also NAD-dependent but does not take part in the citric acid cycle. Both malate dehydrogenase isoforms are dimers.

2. Cytology/cytology/25.html">General characteristics and Bioenergetic Significance of the Citric Acid Cycle

The formation of oxaloacetate completes one turn of the citric acid cycle. During a single turn of the citric acid cycle, 2 molecules of CO₂ are produced through two decarboxylation reactions (the conversion of isocitrate to α-ketoglutarate, and of α-ketoglutarate to succinyl-CoA). In addition, four dehydrogenation steps yield reduced coenzymes: 3 molecules of NАDН + Н⁺ and 1 molecule of FАDН₂ as part of succinate dehydrogenase.

Finally, each turn of the cycle consumes 2 water molecules: one in the citrate formation step, and the second in the hydration of fumarate.

The reduced coenzymes (3 molecules of NАDН and 1 molecule of FАDН₂) generated in the citric acid cycle donate their electrons to the electron transport chain (ETC) onto oxygen, the terminal electron acceptor. Reduced oxygen then reacts with protons to form water.

For every molecule of NАDН oxidized during water formation in cellular Respiration, 3 molecules of ATP are synthesized, whereas each molecule of FАDН₂ yields 2 molecules of ATP (Fig. 6-25).

Fig. 6-25. Interrelation scheme of the common catabolic pathway and the ETC.

Thus, each turn of the citric acid cycle is accompanied by the synthesis of 11 ATP molecules via oxidative phosphorylation. One additional ATP molecule is produced by substrate-level phosphorylation.

Consequently, for every acetyl residue entering the citrate cycle, 12 molecules of ATP are generated.

3. Regulation of the Common Catabolic Pathway

The rate of ATP synthesis closely matches the energy demands of the cell. This is achieved through Coordinated regulation of all Stages of the terminal catabolic pathway, which includes the conversion of pyruvate to acetyl-CoA, the citric acid cycle, and the ETC. In most Tissues, where The primary function of the common catabolic pathway is energy supply, Respiratory Control plays a major regulatory role.

An increased rate of ATP utilization for various cellular activities raises the ADP concentration, which accelerates NADH oxidation in the ETC and, consequently, increases the rate of reactions catalyzed by NAD-dependent dehydrogenases. The Oxidation of Pyruvate and acetyl-CoA can only proceed if electrons and protons from NADH and FADH₂ are transferred to the ETC. Thus, the ADP/ATP and NADH/NAD⁺ ratios are the primary modulators of reaction rates in the common catabolic pathway (CCP).

As is well known, the flux through metabolic pathways designed to maintain a constant level of end products, such as ATP, is regulated at the level of reactions catalyzed by regulatory enzymes. In the terminal stage of catabolism, the most important regulatory enzymes are the pyruvate dehydrogenase complex, citrate synthase, isocitrate dehydrogenase, and the α-ketoglutarate dehydrogenase complex.

Regulation of the pyruvate dehydrogenase complex. Regulation at the level of the PDC is crucial for supplying the citric acid cycle with fuel molecules of acetyl-CoA.

The formation of acetyl-CoA from pyruvate is an irreversible, key metabolic checkpoint. Animals are incapable of converting acetyl-CoA into glucose. The activity of the pyruvate dehydrogenase complex is regulated by several mechanisms: substrate availability, product inhibition, allosteric control, and covalent modification.

Covalent Modification of the PDC is mediated by phosphorylation and dephosphorylation. The PDC comprises two regulatory subunits. One of these, PDC kinase, phosphorylates the PDC at specific Serine residues, which inactivates the enzyme. The other regulatory subunit, a phosphatase, dephosphorylates the enzyme, converting it back into its active form (Fig. 6-26).

Fig. 6-26. Regulation of the pyruvate dehydrogenase complex. The PDC is allosterically activated by ADP, NАD⁺, CoA, Ca²⁺, and pyruvate; acetyl-CoA, NАDН, and ATP activate the kinase and inhibit the PDC. The phosphatase is activated by Ca²⁺.

When ADP levels rise, the PDC remains in its unphosphorylated, active form. In certain cells, this effect is further enhanced by an increase in intracellular Ca²⁺ concentration, which activates PDC phosphatase. This activation mechanism is particularly important in Muscle and adipose tissue.

The products of the pyruvate dehydrogenase reaction (acetyl-CoA and NАDН) allosterically activate PDC kinase. The activated kinase then phosphorylates and inactivates the PDC. Thus, accumulation of NADH and acetyl-CoA suppresses the conversion of pyruvate to acetyl-CoA. This situation occurs, for example, in the Liver during starvation, when fatty acids mobilized from fat depots are delivered to The Liver and converted into acetyl-CoA. In the presence of long-chain fatty acids, the inhibition of the PDC is further amplified. Under these conditions, pyruvate is spared from oxidation and can instead be channeled into Gluconeogenesis (see Section 7).

Pyruvate allosterically activates the unphosphorylated form of the PDC, acting in concert with other substrates—NAD⁺ and CoA. PDC activation is also promoted by Insulin. One of the effects of insulin is an increase in intramitochondrial Ca²⁺ concentration, which subsequently activates the PDC (see Fig. 6-26). This mechanism is particularly prominent in adipose tissue, where acetyl-CoA is required for fatty acid synthesis (see Section 8). In myocardial cells, the PDC is activated by adrenaline, although this effect is independent of changes in cAMP levels.

Regulation of the citric acid cycle. In most cases, the rate of reactions in metabolic cycles is dictated by their initial steps. In the citric acid cycle, the key regulatory step is the formation of citrate from oxaloacetate and acetyl-CoA, catalyzed by citrate synthase. This reaction is accelerated by rising concentrations of oxaloacetate (a substrate) and inhibited by the reaction product, citrate. When the NADH/NAD⁺ ratio drops, the rate of malate oxidation to oxaloacetate increases, and the resulting higher oxaloacetate concentration accelerates the citrate synthase reaction. Conversely, the reaction rate slows down when ATP, succinyl-CoA, and long-chain fatty acid levels rise; however, the precise mechanism by which these metabolites affect citrate synthase remains incompletely understood (Fig. 6-27).

Fig. 6-27. Regulation of the common catabolic pathway. 1 — PDC is activated by pyruvate, NAD⁺, and CoA, and inhibited by NADH and acetyl-CoA; 2 — citrate synthase (the reaction is accelerated by increased oxaloacetate and slowed down by increased citrate, NADH, ATP, and succinyl-CoA); 3 — isocitrate dehydrogenase is allosterically activated by ADP and Calcium Ions, and inhibited by NADH; 4 — α-ketoglutarate dehydrogenase complex is inhibited by NADH, ATP, and succinyl-CoA, and activated by calcium ions.

Isocitrate dehydrogenase, an oligomeric enzyme, consists of 8 subunits. The binding of isocitrate to the first subunit induces a cooperative conformational change in the others, thereby increasing the substrate binding rate. The enzyme is allosterically activated by ADP and Ca²⁺, which bind to different allosteric sites on the enzyme. In the presence of ADP, the conformation of all subunits changes in such a way that isocitrate binding occurs significantly faster. Thus, at the isocitrate concentration normally found in the mitochondrial matrix, small fluctuations in ADP concentration can trigger a dramatic change in the reaction rate. An increase in isocitrate dehydrogenase activity lowers the citrate concentration, which, in turn, reduces product inhibition of citrate synthase. Elevated NADH concentrations lead to a decrease in enzyme activity.

The α-ketoglutarate dehydrogenase complex, structurally similar to the pyruvate dehydrogenase complex, lacks regulatory subunits, unlike the latter. The primary mechanism for regulating the α-ketoglutarate dehydrogenase complex is the inhibition of the reaction by NADH and succinyl-CoA.

Like isocitrate dehydrogenase, the α-ketoglutarate dehydrogenase complex is activated by Ca²⁺, whereas elevated ATP levels reduce the rates of both reactions.

There are numerous additional regulatory mechanisms within the citrate cycle that ensure the required pool of metabolites and their participation in other metabolic pathways.

Compartmentalization of the enzymes involved in pyruvate oxidative decarboxylation and the citric acid cycle plays a vital role in regulating these processes.

The inner mitochondrial membrane is impermeable to anions and cations, including the intermediates of the citrate cycle, which can only cross the membrane via specific transport proteins. Consequently, the Enzymes of the citrate cycle have a greater opportunity to interact with the products of preceding reactions than they would if these products were freely removed from the mitochondria.

Substrate availability is also enhanced through the formation of enzyme complexes. Malate dehydrogenase and citrate synthase form labile complexes in which citrate synthase can utilize the oxaloacetate produced directly by malate dehydrogenase.

In the PDC and the α-ketoglutarate dehydrogenase complex, substrates are transferred directly from one enzyme to another: only transacylase can interact with the TPP-bound intermediate, while dihydrolipoyl dehydrogenase interacts exclusively with dihydrolipoic acid.

NAD⁺, NADH, CoA, acetyl-CoA, and succinyl-CoA lack transport proteins in the mitochondrial membrane. Therefore, these compounds cannot cross the mitochondrial membrane.

The accumulation of acyl-CoA derivatives, such as acetyl-CoA or succinyl-CoA, in the mitochondrial matrix inhibits other reactions that require CoA.

The close coupling between The Tricarboxylic Acid Cycle and the ETC is maintained through the utilization of a shared pool of NAD⁺ and NADH.

B. Anabolic Functions of the tricarboxylic acid cycle

The citric acid cycle is one of the amphibolic metabolic pathways. It not only drives the oxidative breakdown of energy substrates to the end products CO₂ and H₂O, but also generates precursors for other metabolic pathways (Fig. 6-28).

Fig. 6-28. Utilization of TCA cycle metabolites in the synthesis of various compounds. Synthesis of non-Essential Amino Acids (1, 2, 3), glucose (4, 5, 6), fatty acids (7), and heme (8).

Certain intermediates of the citric acid cycle—such as α-ketoglutarate, succinate, and oxaloacetate—can be utilized for the synthesis of non-essential amino acids (see Section 9).

The depletion of cycle intermediates is replenished by reactions catalyzed by specific enzymes. Under normal conditions, the reactions that draw intermediates out of the cycle and those that replenish them are in a state of dynamic equilibrium, ensuring that the concentration of these intermediates in the mitochondria remains constant.

Reactions that maintain the pool of TCA cycle intermediates are termed anaplerotic (replenishing) reactions. The most important of these is the synthesis of oxaloacetate from pyruvate, a reaction catalyzed by the mitochondrial enzyme pyruvate carboxylase.

Pyruvate carboxylase is a complex oligomeric enzyme. The enzyme molecule contains 4 prosthetic groups represented by biotin (see Section 3), which is covalently linked via an amide bond to the ε-amino groups of lysine residues located in the active site of the enzyme (Fig. 6-29).

Fig. 6-29. The prosthetic group of pyruvate carboxylase. The carboxyl group of biotin forms an amide bond with the ε-amino group of lysine in the enzyme's active site. CO₂ is activated to form an N-carboxy derivative of biotin.

If the citric acid cycle lacks sufficient oxaloacetate or any other intermediate, pyruvate carboxylation is accelerated. This reaction utilizes ATP as an energy source.

The reaction proceeds in 2 stages. In The First stage, CO₂ is activated by attaching to one of the nitrogen atoms in the biotin molecule. This reaction is coupled with the hydrolysis of ATP.

ATP + CO₂ + E-biotin + H₂O —> ADP + H₃PO₄ + E-biotin-COO^- + 2 H⁺.

At the Second Stage, the activated carboxyl group is transferred to pyruvate.

Е-биотин-СОО^- + Пируват —> Е-биотин + Оксалоацетат.

Pyruvate carboxylase is a regulatory enzyme. When the concentration of acetyl-CoA increases, it acts as an allosteric activator of pyruvate carboxylase, accelerating the formation of oxaloacetate. Thus, an excess of acetyl-CoA promotes the activation of the citrate cycle.

The metabolites of the citrate cycle are used not only as substrates for the Synthesis of the carbon

Skeleton of various compounds, but also serve as hydrogen Donors for the formation of reduced coenzymes involved in the synthesis of fatty acids, Steroids, and other substances (see sections 8, 10, 11). Two metabolites of the citrate cycle can be dehydrogenated via NADP-dependent dehydrogenases: malate and isocitrate. For instance, malate can be transported from the mitochondria into the cell cytosol. The cytosol contains an NADP-dependent dehydrogenase (malic enzyme) that catalyzes the reaction:

Malate and isocitrate provide about half of the total NADPH pool used in reductive biosyntheses; the second half is generated via the Pentose Phosphate Pathway of glucose METABOLISM (see section 7).

G. Hypoenergetic States

All living cells constantly require ATP to carry out various vital activities.

Brain cells consume large amounts of ATP for neurotransmitter synthesis, nerve cell regeneration, Maintenance of the necessary Na⁺ and K⁺ gradients, and Nerve Impulse Conduction; the Kidneys use ATP during the reabsorption of various substances in Urine Formation; the liver synthesizes Glycogen, fats, proteins, and many Other Compounds; the myocardium constantly performs the mechanical work required for Blood Circulation; skeletal Muscles consume negligible amounts of ATP at rest, but during physical exertion, these demands increase dozens of times (Table 6-6).

Table 6-6. Rates of O₂ and ATP consumption in various tissues

At the same time, practically no ATP reserves exist within cells. For example, if ATP Synthesis in the myocardium stops, its reserves are depleted within a few seconds.

As we already know, for continuous ATP synthesis, cells require an influx of metabolites as respiratory substrates and oxygen as the terminal electron acceptor in oxidation reactions coupled with ATP synthesis.

Impairments at any stage of metabolism that lead to the cessation of ATP synthesis are fatal to the cell.

Conditions characterized by decreased ATP synthesis are collectively termed "hypoenergetic states." Causes of hypoenergetic states may include starvation, deficiencies of Vitamins B₁, PP, or B₂, and Hypoxia.

Hypoxia may arise from: a deficiency of oxygen in inhaled air; pulmonary diseases and impaired pulmonary ventilation; Circulatory Disorders caused by heart disease, vascular spasm and thrombosis, or blood loss. Hypoxia may also be caused by hereditary or acquired structural abnormalities of Hemoglobin (see sections 1, 4). A frequent cause of hypoenergetic states can be impaired oxygen utilization processes within cells.

The causes of these impairments may include:

✵ the action of inhibitors and uncouplers in the electron transport chain (ETC);

✵ iron-deficiency anemias;

✵ a decrease in the levels of hemoglobin and other iron-containing proteins (Cytochromes, Fe-S proteins), resulting in disrupted Electron Transport and ATP synthesis;

✵ hereditary defects in enzymes of the ETC and the citrate cycle.

Approximately 13 out of 100 proteins involved in oxidative phosphorylation are encoded by Mitochondrial DNA: 7 subunits of complex I, a subunit of complex III, 3 subunits of complex IV, and 2 subunits of complex V, as well as the necessary components for their Translation. The remaining mitochondrial proteins are synthesized in The Nucleus.

Nuclear DNA encodes more than 70 protein subunits involved in oxidative phosphorylation. Impairments of oxidative phosphorylation are mainly associated with Mutations in mitochondrial DNA, which occur approximately 10 times more frequently than in nuclear DNA. Tissues with a high demand for ATP (the Central Nervous system, skeletal

muscles, myocardium, kidneys, and liver) are the most sensitive to impairments in oxidative phosphorylation.

Mitochondrial DNA defects are maternally inherited, as mitochondria from sperm cells do not enter the fertilized egg. Mitochondrial DNA mutations are a common cause because mitochondria lack an efficient DNA Repair system comparable to that of the nucleus (see Section 4). Even in healthy individuals, somatic mutations impair oxidative phosphorylation capacity with age. In such cases, the ATP synthesis capacity falls below the tissue-specific threshold of normal cells.