BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 12. CARBOHYDRATE METABOLISM

12.1. Catabolic Pathways of Carbohydrates

12.1.3. Glycolysis

The main PATHWAYS OF GLUCOSE breakdown include Glycolysis, the Pentose Phosphate Pathway, and the phosphoketolase pathway.

Glycolysis is a sequence of enzymatic reactions resulting in The breakdown of glucose and other substrates to Pyruvate or lactate, accompanied by The Biosynthesis of ATP. This catabolic process was discovered in the first half of the last century through the pioneering work of G. Embden, O. Meyerhof, and J. Parnas, which is why it is alternatively known as the Embden-Meyerhof-Parnas pathway. Glycolysis is considered the central pathway of Carbohydrate Catabolism found in the Cells of animals, plants, and most microorganisms. Depending on environmental conditions, cells can degrade CARBOHYDRATES to pyruvate (aerobic glycolysis) or to lactate, propionate, butyrate, ethanol, glycerol, and other products (anaerobic glycolysis).

The anaerobic breakdown of sugars to generate metabolic energy, carried out by anaerobic organisms, is known as Fermentation. Depending on the final product of the process, this includes lactic acid, propionic acid, butyric acid, alcoholic, and other Types of fermentation. Anaerobic glycolysis also occurs in certain Tissues of aerobic organisms, notably in erythrocytes, the retina, the renal medulla, intensely contracting skeletal Muscles, and other animal cells that are permanently or temporarily subjected to hypoxic conditions.

Under aerobic conditions in the vast majority of cells, glycolysis serves as the initial stage of the Complete oxidation of carbohydrates into ultimate metabolites—CO₂ and H₂O molecules. The process of glycolytic breakdown of glucose and other substrates, which takes place in the Cytoplasm, can be divided into two stages:

✵ activation of substrate molecules (preparatory stage);

✵ energy-harvesting stage.

In the Reactions of the First stage, substrate molecules are activated via phosphorylation and converted into a single common intermediate metabolite: glyceraldehyde-3-phosphate. In the second stage, glyceraldehyde-3-phosphate molecules are oxidized to pyruvate, which is accompanied by the trapping of a portion of the oxidation energy in ATP and NADH molecules (Fig. 12.6).

During the ENZYMATIC REACTIONS OF The first stage of glycolysis, glucose molecules are sequentially phosphorylated at the expense of ATP. First, under the action of hexokinase at the sixth carbon atom, and subsequently—following The conversion of glucose-6-phosphate to fructose-6-phosphate by phosphoglucose isomerase—fructose is phosphorylated at the first carbon atom by Phosphofructokinase to yield fructose-1,6-bisphosphate:

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Fig. 12.6. The Glycolytic Pathway of glucose breakdown and other glycolytic substrates:

1 - hexokinase; 2 - phosphoglucose isomerase; 3 - phosphofructokinase; 4 - aldolase; 5 - Triosephosphate isomerase;

6 - glyceraldehyde-3-phosphate dehydrogenase; 7 - phosphoglycerate kinase; 8 - phosphoglyceromutase; 9 - enolase;

10 - pyruvate kinase; 11 - Lactate dehydrogenase.

The numbers in parentheses to the right of the glycolytic metabolite names indicate the number of Molecules Participating in the reaction

Both phosphorylation reactions are physiologically irreversible. The preparatory stage of glycolysis concludes with the Cleavage of fructose-1,6-bisphosphate by aldolase into glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Although under the dynamic equilibrium of this reversible reaction dihydroxyacetone phosphate accounts for 95%, it is rapidly converted into glyceraldehyde-3-phosphate by triosephosphate isomerase, since only glyceraldehyde-3-phosphate molecules undergo further transformations in the subsequent steps of glycolysis:

Molecules of other hexoses—such as mannose, fructose, and galactose—also enter glycolysis at this stage through phosphorylation and subsequent conversion into glyceraldehyde-3-phosphate.

In the cells of skeletal muscles, Kidneys, intestines, and other peripheral tissues, fructose and mannose are phosphorylated by hexokinase at carbon-6 to form fructose-6-phosphate and mannose-6-phosphate, the latter of which is also converted into fructose-6-phosphate by phosphomannose isomerase:

D-Galactose enters glycolysis via a longer pathway than other hexoses. First, galactokinase phosphorylates it to galactose-1-phosphate, which then reacts with uridine diphosphate glucose (UDPG) in an exchange reaction catalyzed by galactose-1-phosphate uridylyltransferase to form glucose-1-phosphate and uridine diphosphate galactose (UDPGal). In the next reaction, the galactose residue in UDPGal is epimerized by UDP-glucose 4-epimerase into the glucosyl residue of UDPG, which can then interact with another molecule of galactose-1-phosphate or be cleaved by UDP-glucose pyrophosphorylase into glucose-1-phosphate and UTP. The glucose-1-phosphate molecules generated in these reactions are converted into glucose-6-phosphate through the action of phosphoglucomutase:

In addition to hexoses, glycerol can also serve as a substrate for glycolysis. Glycerol is one of the products of the Enzymatic Hydrolysis of endogenous and exogenous triacylglycerols. The conversion of glycerol into dihydroxyacetone phosphate, an intermediate metabolite of glycolysis, occurs mainly in the Liver through the sequential phosphorylation and oxidation Reactions Catalyzed by two Enzymes: glycerol kinase and glycerol phosphate dehydrogenase:

In the liver of vertebrates, fructose is phosphorylated at carbon-1 by fructokinase. The resulting fructose-1-phosphate is subsequently cleaved by aldolase into dihydroxyacetone phosphate and glyceraldehyde. Glyceraldehyde is then phosphorylated at its primary alcohol group by glyceraldehyde phosphokinase to yield glyceraldehyde-3-phosphate:

Thus, during the preparatory stage of glycolysis, substrate molecules are transformed into a single common metabolite, glyceraldehyde-3-phosphate. These transformations require the consumption of two ATP molecules for the Activation of a hexose molecule, or one ATP phosphate group for the phosphorylation of a glycerol molecule.

During the Second Stage of glycolysis, the aldehyde group of glyceraldehyde-3-phosphate is oxidized by an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase. The energy of oxidation is initially trapped in the high-energy phosphoester bond at carbon-1 of 1,3-bisphosphoglycerate; this high-energy phosphate group is then transferred by phosphoglycerate kinase to an ADP molecule, yielding the first molecule of ATP and 3-phosphoglycerate:

Both of these coupled, reversible reactions of glycolysis are referred to as substrate-level phosphorylation, a process in which ADP is phosphorylated using The energy released during The oxidation of a specific substrate (glyceraldehyde-3-phosphate).

In the subsequent reversible reactions, the phosphate group of 3-phosphoglycerate migrates to carbon-2 via the action of phosphoglyceromutase, forming 2-phosphoglycerate. The subsequent dehydration of 2-phosphoglycerate, catalyzed by enolase, leads to an intramolecular redistribution of energy, resulting in The formation of the high-energy phosphoester bond in phosphoenolpyruvate:

The transfer of this high-energy phosphate group from phosphoenolpyruvate to ADP by pyruvate kinase results in the formation of a second ATP molecule and the enol form of pyruvate, which spontaneously tautomerizes into the keto form that predominates at physiological pH:

The significant standard free-energy loss of the substrate molecule in the pyruvate kinase reaction (ΔG°' = -31.4 kJ/mol) drives the substrate-level phosphorylation of ADP and ensures the irreversibility of this reaction. The overall equation for the conversion of glucose to pyruvate

after cancelling terms on both sides, is as follows:

Thus, during the glycolytic conversion of one glucose molecule into two pyruvate molecules, two molecules of ATP and two molecules of NADH are produced. Under aerobic conditions, the NADH generated in the glyceraldehyde-3-phosphate dehydrogenase reaction transfers reducing equivalents to the Mitochondrial Electron Transport chain, whereas in an anaerobic environment (or under Hypoxia), it participates in the reduction of pyruvate.

The subsequent fate of pyruvate—one of the key central metabolites—depends primarily on the environmental conditions of the cells and their energy status.

In certain animal cells (such as vigorously contracting Skeletal Muscle cells) under conditions of severe hypoxia, as well as in lactic acid Bacteria, pyruvate is reduced to lactate by lactate dehydrogenase:

The NAD⁺ molecules regenerated in this reaction are funneled back into the glyceraldehyde-3-phosphate dehydrogenase reaction. After being reduced to NADH in that step, they are reoxidized via the lactate dehydrogenase reaction (Fig. 12.6).

The operation of this glycolytic oxidoreductase cycle enables glycolysis to proceed under anaerobic conditions, where Organic compounds serve as both Donors and acceptors of hydride ions (H^-). Consequently, the overall equation for anaerobic glycolysis shows no net redox changes:

Thus, the anaerobic breakdown of a glucose molecule into two lactate molecules is accompanied by the biosynthesis (via Substrate-Level Phosphorylation Reactions) of four ATP molecules. Accounting for the two ATP molecules consumed in the phosphorylation of glucose and fructose-6-phosphate, the net yield is only two ATP molecules.

Under standard conditions in the endergonic reaction

Standard Free energy change ΔG⁰' = 61.2 kJ/mol; in the exergonic conversion of glucose to lactic acid

ΔΘ⁰' is -196.9 kJ/mol. Consequently, the efficiency of glycolysis under standard conditions is [(61.2/196.9)· 100%] ~ 31%. However, in cells under physiological concentrations of glucose, lactate, ATP, ADP, and inorganic phosphate, the efficiency of free energy conservation—released during glycolysis into the phosphoester bonds of ATP molecules—is twice as high (>60%).

Under normal physiological conditions, lactate production is also observed in the Cells of the gastrointestinal tract, the Brain, and in erythrocytes. Due to the absence of Cell/35.html">Mitochondria, the METABOLISM/26.html">Energy Metabolism of the latter is virtually sustained by glycolysis even under aerobic conditions.

In cells of various tissues, lactate dehydrogenase (LDH), an oligomeric protein with a Molecular Weight of 134 kDa, is represented by five isozymic forms—tetramers formed by various combinations of two polypeptide subunits, H and M. For instance, in myocardial cells, the enzyme is represented by the LDH1 isoform, composed of four H domains (H4), which exhibits a higher affinity for lactate, promoting its complete oxidation in The cardiac muscle mitochondria. In contrast, the LDH5 isozyme, localized in skeletal muscle cells, is a tetramer (M4) containing only M polypeptide chains. This isozyme has a higher affinity for pyruvate and rapidly reduces it to lactate, which is transported via the Blood to the liver, where it is re-oxidized into pyruvate by the liver LDH3 isozyme containing one H and three M subunits (HM3). Thus, the dominant direction of the lactate dehydrogenase reaction depends on the Intracellular Localization of different enzyme isoforms with varying substrate specificities. During tissue damage, the enzyme content in the blood increases, which makes it possible to detect the presence and extent of organ damage, particularly of the myocardium, by assessing the isozyme spectrum of lactate dehydrogenase.

Fermentation. In the cells of certain microorganisms (particularly Yeast), pyruvate produced during anaerobic glycolysis is reduced to ethanol in the absence of the enzyme lactate dehydrogenase. In these cells, instead of the lactate dehydrogenase reaction, two coupled enzymatic reactions take place, in which pyruvate is first irreversibly converted into acetaldehyde by pyruvate decarboxylase, and the latter is reduced to ethanol by Alcohol dehydrogenase:

The donor of reducing equivalents in the second reaction is NADH, which was generated in the glyceraldehyde-3-phosphate dehydrogenase reaction of glycolysis (Fig. 12.6).

The products of Alcoholic Fermentation, ethanol and CO2, are the End products of sugar catabolism in these microorganisms and are therefore excreted into the environment. The overall equation for alcoholic fermentation is as follows:

Thus, although the enzymatic breakdown of glucose and other substrates under anaerobic conditions into lactate or ethanol involves redox reactions, there is no change in the oxidation state of carbon in the overall reactions of lactic acid and alcoholic fermentation. In other words, the conversion of glucose molecules into lactate or ethanol and CO2 molecules—accompanied by the release and conservation of a portion of the Free energy of the glucose molecule in the phosphate groups of ATP molecules—occurs without the oxidation of carbon atoms in the molecules of the end Products of Anaerobic glycolysis. Despite the low energy yield (~ 7% of the total free energy of a glucose molecule), anaerobic glycolysis is considered a uniquely efficient process among known catabolic transformations based almost exclusively on the oxidation of an organic substrate, as it allows cells to synthesize ATP under anaerobic conditions.

Regulation of glycolysis. The rate of glycolysis is determined primarily by the cellular energy potential as well as the availability of substrates. The entry of glucose, other hexoses, and Glycogen into glycolysis (Glycogenolysis) is regulated mainly by The activity of hexokinase and Glycogen phosphorylase, which catalyze the initial steps of glycolysis and glycogenolysis, respectively. As is well known, the activity of these enzymes is controlled by allosteric modulation and covalent modification mechanisms: skeletal muscle hexokinase is allosterically inhibited by its own product, glucose-6-phosphate, and by ATP, whereas the activity of glycogen phosphorylase can be controlled both by allosteric modulators (ATP and AMP) and via phosphorylation-dephosphorylation of Serine residues in the active sites of the enzyme.

As a rule, irreversible reactions of Metabolic pathways are catalyzed by regulatory enzymes. In glycolysis, these include three kinase reactions. In addition to hexokinase, phosphofructokinase and pyruvate kinase also participate in regulating the rate of glycolysis, with ATP, acetyl-CoA, Fatty acids, and fructose-1,6-diphosphate serving as allosteric modulators for the latter.

Phosphofructokinase, which is considered the key regulatory enzyme of glycolysis in skeletal muscles, differs from other Kinases of this pathway in its complex Structure and activity regulation. The enzyme has multiple allosteric sites located on its four subunits. Its allosteric inhibitors are ATP, citrate, and fatty acids. Therefore, in resting muscle with a high energy potential (a high ATP/AMP ratio) and a high concentration of citrate and fatty acids, the enzyme activity is minimal. The inhibitory effect of ATP, citrate, and fatty acids is relieved by an increase in the concentration of ATP hydrolysis products—ADP and, especially, AMP—which occurs during active skeletal Muscle contraction.

In other Organs and tissues, notably the liver, brain, and kidneys, the main allosteric regulator of glycolysis is fructose-2,6-diphosphate, which can simultaneously activate phosphofructokinase and inhibit the activity of fructose-1,6-diphosphatase, a regulatory enzyme of Gluconeogenesis.

The concentration of fructose-2,6-diphosphate in cells is controlled by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, which catalyzes the synthesis and dephosphorylation reactions of fructose-2,6-diphosphate:

The activating effect of fructose-2,6-diphosphate on phosphofructokinase is countered by the dephosphorylation of fructose-2,6-diphosphate. The Importance of the regulatory role of fructose-2,6-diphosphate in glycolysis is confirmed by the fact that the intracellular content of this metabolite is regulated at the level of induction of alternative enzyme isoforms with kinase or phosphatase activity.

Thus, the rate of glycolysis is controlled by its regulatory kinase enzymes, which catalyze the rate-limiting steps of the process. The activity of hexokinase, phosphofructokinase, and pyruvate kinase is modulated allosterically by activators (AMP, ADP, fructose-1,6- and -2,6-diphosphates) and inhibitors (glucose-6-phosphate, ATP, citrate, fatty acids, acetyl-CoA, etc.).

The predominance of a particular type of modulator depends mainly on the cellular energy potential (ATP/AMP ratio) as well as the availability of intermediate metabolites (citrate, acetyl-CoA, glucose-6-phosphate, etc.) for use in biosynthetic reactions.

It should be noted that under tissue oxygenation conditions, a profound inhibition of anaerobic glycolysis is observed, known as the Pasteur Effect. This phenomenon is associated with the activation of cellular Respiration, i.e., the process of complete Oxidation of Pyruvate in The Tricarboxylic Acid Cycle.

In mammalian erythrocytes, alongside the glycolytic conversion of glucose, there is the diphosphoglycerate shunt (Fig. 12.7), which produces 2,3-diphosphoglycerate, a compound that plays a vital role in regulating Oxygen transport by Hemoglobin. This compound can be synthesized via two pathways (Rapoport-Luebering shunt): the isomerization of 1,3-diphosphoglycerate mediated by diphosphoglycerate mutase, and the phosphorylation of 3-phosphoglycerate mediated by phosphoglycerate kinase. The second pathway also involves a reversible reaction catalyzed by phosphoglycerate phosphatase.

Fig. 12.7. Formation of 2,3-diphosphoglycerate during glucose conversion:

1 - diphosphoglycerate mutase; 2 - phosphoglycerate kinase; 3 — phosphoglycerate phosphatase

The presence of 2,3-diphosphoglycerate in erythrocytes decreases the affinity of hemoglobin for oxygen. This facilitates the dissociation of oxygen from oxyhemoglobin and its uptake by tissues.

Oxidative Decarboxylation of pyruvate. The primary energy-yielding transformation of pyruvate under aerobic conditions is its oxidative decarboxylation to form acetyl-CoA, the starting compound of the tricarboxylic acid (TCA) cycle:

In Eukaryotic cells, this process takes place in the mitochondria, where pyruvate enters from the cytoplasm via a specific transporter.

The simultaneous decarboxylation and dehydrogenation of pyruvate is catalyzed by the multienzyme pyruvate dehydrogenase complex, which is localized in the mitochondrial matrix of plants and animals, or attached to The Cell wall in microorganisms. The core of this complex, consisting of three enzymes and five Coenzymes, is dihydrolipoamide acetyltransferase, with which pyruvate dehydrogenase and dihydrolipoamide dehydrogenase molecules are associated.

In the first step (1) of the process, pyruvate dehydrogenase [E₁] catalyzes the removal of the carboxyl group from a pyruvate molecule and the transfer of the hydroxyethyl residue to the coenzyme thiamine pyrophosphate (TPP) to form hydroxyethyl-TPP (Fig. 12.8). Subsequent dehydrogenation of hydroxyethyl-TPP and the transfer of hydrogen atoms and the acetyl group to the prosthetic groups of the central enzyme of the complex (2) also occur with the participation of pyruvate dehydrogenase.

Fig. 12.8. Stages of oxidative decarboxylation of pyruvate catalyzed by Enzymes of the pyruvate dehydrogenase complex

Lipoamide prosthetic groups of dihydrolipoamide acetyltransferase [E₂], formed by the attachment of carboxyl groups of Lipoic Acid molecules to the amino groups of specific Lysine residues in the active sites of the enzyme, act in this complex as carriers of acetyl groups and hydrogen atoms between the prosthetic groups of the other two enzymes of the complex (Fig. 12.8).

The attachment of hydrogen atoms and the acetyl group to the oxidized (disulfide) form of the lipoamide groups of dihydrolipoamide acetyltransferase is accompanied by the Formation of the reduced, acetylated form of the enzyme's lipoamide groups. Next, the acetyl derivative of dihydrolipoamide acetyltransferase interacts with free CoA-SH (3) to form an acetyl-CoA molecule and the fully reduced (dithiol) form of the prosthetic groups. In the next step (4), the dithiol lipoamide groups of the enzyme [_E2] are oxidized by dihydrolipoamide dehydrogenase [E₃] to regenerate the original disulfide form of the dihydrolipoamide acetyltransferase prosthetic groups. The hydrogen atoms cleaved by dihydrolipoamide dehydrogenase are first transferred to its FAD coenzyme, and then, at the final step (5), passed from FADH₂ to an NAD⁺ molecule. The reduced NADH directs hydrogen atoms to the Electron Transport Chain (ETC) located on The inner mitochondrial membrane. The acetyl-CoA molecule produced from pyruvate in the reactions of the pyruvate dehydrogenase complex can either be oxidized in the tricarboxylic acid cycle within the mitochondrial matrix or translocated (as citrate) into the cytoplasm to participate in the Biosynthesis of Higher fatty acids, Cholesterol, Ketone Bodies, etc.

The oxidative decarboxylation of pyruvate, which is irreversible in animal cells, is regulated by covalent modification and allosteric modulation mechanisms. When tissue concentrations of ATP, acetyl-CoA, and TCA cycle intermediates are high, pyruvate dehydrogenase kinase—one of the two regulatory enzymes of the pyruvate dehydrogenase complex—is activated. It inhibits pyruvate dehydrogenase activity by phosphorylating serine residues in its Active Site. The inhibitory effect of serine phosphorylation is reversed by its dephosphorylation via the second regulatory enzyme of the complex, phosphopyruvate dehydrogenase phosphatase, which is stimulated by an increase in Ca²⁺ ion concentration, typically signaling a decreased ATP/AMP ratio in The Cell.

The rate of pyruvate decarboxylation is also regulated by allosteric modulation. Acetyl-CoA and NADH molecules produced in mitochondria during the oxidation of pyruvate and Higher Fatty Acids act as allosteric Inhibitors of the pyruvate dehydrogenase reaction. Therefore, during the absorptive state or the mobilization of fatty acids from cellular fat depots, the conversion of pyruvate to acetyl-CoA is suppressed.