Textbook - BIOLOGICAL CHEMISTRY - Gubsky Yu.I. - 2000

Chapter III. METABOLISM OF MAJOR CLASSES OF BIOMOLECULES

CHAPTER 11. CARBOHYDRATE METABOLISM. I. AEROBIC AND ANAEROBIC GLUCOSE OXIDATION

11.3. GLYCOLYSIS: REACTIONS, ENERGETICS, REGULATION

Class="center">General characteristics of Glycolysis

Glycolysis (Embden-Meyerhof pathway) is the central pathway of Glucose Catabolism, representing a cascade of enzymatic reactions in which a six-carbon glucose molecule C₆H₁₂O₆ is split into two three-carbon molecules of pyruvic or lactic acid. Glycolysis is a catabolic pathway of glucose in which oxygen is not directly involved; however, due to the oxidation-reduction reactions within The Glycolytic Pathway, two ATP molecules are generated As a result of glucose Cleavage.

Fig. 11.1. Gustav Embden (1874-1933), German biochemist. Professor and HEAD of the Department of Biochemistry, Rector of the University of Frankfurt am Main.

Fig. 11.2. Otto Meyerhof (1884-1951), German biochemist. Worked in Germany and the USA. Nobel Prize laureate (1922).

Glycolysis is a type of Fermentation—a biochemical process that provides energy in the form of ATP for most anaerobic organisms on Earth, as well as for aerobes functioning under conditions of insufficient molecular oxygen supply. A common type of fermentation in anaerobes is The production of ethyl alcohol from glucose, a process catalyzed by Yeast Enzymes and widely used in the production of alcoholic beverages:

In the Human and Animal body, a distinction is made between:

- aerobic glycolysis, which is accompanied by The formation of two molecules of pyruvic acid (Pyruvate) from a single glucose molecule:

Aerobic glycolysis can also be viewed as an intermediate (glycolytic) stage in the aerobic oxidation of glucose to its final products—carbon dioxide and Water (see section 11.2);

- anaerobic glycolysis, which is accompanied by the formation of two molecules of lactic acid (lactate) from a single glucose molecule:

Under normal physiological conditions, most Tissues of humans and higher animals are characterized by aerobic glycolysis, i.e., The conversion of glucose into pyruvate, which is subsequently oxidized to carbon dioxide and water. Anaerobic glycolysis occurs primarily in Muscles during intense physical activity (i.e., under relative oxygen deficiency), in certain highly specialized Cells (such as erythrocytes, which lack Cell/35.html">Mitochondria), or under specific pathological conditions (malignant tumor cells).

Glycolytic reactions take place in The Cell Cytosol and are catalyzed by enzymes localized in this compartment.

Glycolysis is divided into two stages:

1. The cleavage of a glucose molecule into two phosphotriose molecules (glyceraldehyde-3-phosphate and dihydroxyacetone phosphate). This stage involves a sequence of reactions that require the expenditure of two ATP molecules for each cleaved glucose molecule.

2. The conversion of two phosphotriose molecules into two pyruvate (or lactate) molecules. This stage involves oxidation-reduction reactions ("glycolytic oxido-reduction") accompanied by the generation of four ATP molecules.

Thus, as a result of the cleavage of a single glucose molecule via aerobic or anaerobic glycolysis, the net yield of ATP is two molecules, which can be represented by the following equation:

ENZYMATIC REACTIONS OF glycolysis

The reactions of aerobic and anaerobic glycolysis are identical in the initial stage of glucose breakdown and diverge only after the formation of pyruvate. In aerobic glycolysis, pyruvate serves as the end product, whereas in anaerobic glycolysis, it acts as an intermediate that is reduced to lactate. Furthermore, aerobic and anaerobic pathways differ in the metabolic fraction of reduced NAD⁺ (NADH) generated during the glycolytic oxidoreduction step.

Enzymatic reactions of aerobic glycolysis:

1. Activation of the glucose molecule via phosphorylation to form a sugar phosphate ester, glucose-6-phosphate. The source of the phosphate group in this reaction is an ATP molecule:

This reaction is catalyzed by the enzyme hexokinase, which exhibits its highest activity in Muscle tissue. In Liver cells, glucokinase also plays a significant role in converting free glucose into glucose-6-phosphate. Hexokinase is capable of phosphorylating various hexoses, whereas glucokinase is specific to glucose; however, the latter's activity becomes significant only when Blood glucose levels exceed the physiological glycemic range (such as in alimentary hyperglycemia or Diabetes Mellitus).

2. Conversion (isomerization) of glucose-6-phosphate into fructose-6-phosphate:

The reaction is catalyzed by the enzyme phosphoglucoisomerase and is reversible, meaning it readily proceeds in either direction depending on the relative concentrations of the substrate (glucose-6-phosphate or fructose-6-phosphate, respectively). Under physiological conditions, the equilibrium is shifted to the right because fructose-6-phosphate is continuously consumed in subsequent glycolytic reactions.

3. Phosphorylation of fructose-6-phosphate to yield fructose-1,6-bisphosphate. Similar to the first reaction of glycolysis, the phosphate donor is an ATP molecule.

The enzyme catalyzing this reaction is Phosphofructokinase, which is a key regulatory enzyme of glycolysis operating via an allosteric mechanism.

4. Cleavage of fructose-1,6-bisphosphate into two phosphotriose molecules via the rupture of the covalent -C-C- bond between the 3rd and 4th carbon atoms of the six-carbon fructose-1,6-bisphosphate chain.

The reaction is catalyzed by fructose-1,6-bisphosphate aldolase (commonly known as aldolase). The aldolase reaction yields dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P):

5. Interconversion of the two phosphotrioses (DHAP and G3P), catalyzed by the enzyme Triosephosphate isomerase:

Since physiological equilibrium heavily favors the forward reaction due to the continuous utilization of glyceraldehyde-3-phosphate in subsequent glycolytic steps, the net result of these first five glucose degradation reactions is the conversion of a single glucose molecule into two molecules of G3P:

6. Oxidation of glyceraldehyde-3-phosphate to 3-phosphoglyceric acid (3-PGA). This process (glycolytic oxidoreduction) consists of two stages catalyzed by separate enzymes:

6.1. Oxidation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate (1,3-bisphosphoglyceric acid — 1,3-BPGA).

The reaction is catalyzed by glyceraldehyde-3-phosphate dehydrogenase, which utilizes NAD⁺ as a coenzyme to accept reducing equivalents (electrons) from the aldehyde group of G3P. Inorganic phosphate also participates in this reaction:

The resulting 1,3-BPGA is a mixed anhydride of phosphoric and 3-phosphoglyceric acids (an acyl phosphate) containing a high-energy phosphate bond; thus, 1,3-BPGA is a compound with a high Phosphate group transfer potential (see Chapter 8).

The second product of the glyceraldehyde-3-phosphate dehydrogenase reaction is NADH ("glycolytic NADH"), which under aerobic conditions transfers its reducing equivalents to intramitochondrial NAD⁺ and subsequently to the Mitochondrial Electron Transport chain, thereby generating three ATP molecules via Oxidative Phosphorylation.

6.2. Conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate (3-phosphoglyceric acid — 3-PGA).

This reaction is accompanied by The transfer of a high-energy phosphate group from 1,3-BPGA to ADP, yielding an ATP molecule, and is catalyzed by the enzyme phosphoglycerate kinase:

7. Conversion of 3-phosphoglycerate to 2-phosphoglycerate (2-phosphoglyceric acid — 2-PGA).

The reaction is catalyzed by the enzyme phosphoglycerate mutase:

8. Dehydration of 2-phosphoglycerate to yield phosphoenolpyruvate (PEP).

The reaction is catalyzed by the enzyme enolase:

This reaction is the second step in glycolysis that generates a high-energy bond (within the PEP molecule).

9. Formation of pyruvate from phosphoenolpyruvate.

The reaction is catalyzed by the enzyme pyruvate kinase and involves the transfer of a high-energy phosphate group from the PEP molecule to ADP:

The pyruvate kinase reaction marks the culmination of aerobic glycolysis. It results in the formation of two pyruvate molecules and two ATP molecules (per each glucose molecule entering the glycolytic pathway). Like phosphofructokinase, pyruvate kinase is a regulatory enzyme that serves as a key control point in the Regulation of glycolysis.

Enzymatic Reactions of Anaerobic Glycolysis

As indicated by the reactions above, under aerobic conditions, the end product of glycolysis in animal tissues is pyruvate; meanwhile, the NADH produced during The oxidation of glyceraldehyde-3-phosphate is reoxidized in the mitochondria at the expense of molecular oxygen. Under anaerobic conditions (such as in vigorously contracting skeletal muscles or in lactic acid Bacteria), glycolytic NADH does not pass its reducing equivalents into the mitochondrial Respiratory Chain, but instead is utilized to reduce pyruvate to L-lactate:

The reaction is catalyzed by the enzyme Lactate dehydrogenase, which exists in five distinct isoenzymatic forms (LDH₁–LDH₅) that differ in their kinetic properties (K_M, V_max, and the degree of allosteric inhibition by pyruvate).

The overall sequence of enzymatic reactions in glycolysis is illustrated in Fig. 11.3.

Fig. 11.3. Metabolic map of glycolysis.

Thus, the enzymatic reactions of anaerobic glycolysis are nearly identical to those of aerobic glycolysis, differing only at the stage following the formation of pyruvate: in aerobic glycolysis, pyruvate serves as a substrate for conversion into acetyl-coenzyme A and subsequent oxidation (see: AEROBIC GLUCOSE OXIDATION), whereas in anaerobic glycolysis, pyruvate is reduced to lactate by means of the NADH generated during the glycolytic oxidoreductase reactions. In other words, following pyruvate formation, its further metabolic fate can proceed via one of two alternative pathways, depending on the redox state within a given tissue:

- under aerobic conditions, Oxidative Decarboxylation of pyruvate yields acetyl-CoA, which is subsequently oxidized to CO₂ and H₂O in the Krebs cycle; the NADH produced during the oxidation of glyceraldehyde-3-phosphate transfers its reducing equivalents to the mitochondrial respiratory chain via specialized shuttle mechanisms;

- under anaerobic conditions (or Hypoxia), the reoxidation of glycolytic NADH is driven by lactate dehydrogenase, which reduces pyruvate to lactate; running in this direction, the lactate dehydrogenase reaction generates NAD⁺, which is reused to oxidize glyceraldehyde-3-phosphate, thereby leading to the accumulation of lactate as the end product of anaerobic glycolysis. This sequence of reactions is most characteristic of vigorously exercising skeletal muscles; besides skeletal muscles and erythrocytes, cells of certain other Organs and tissues (the Brain, gastrointestinal tract, renal medulla, retina, and Skin) partially meet their energy demands through anaerobic glycolysis, producing lactic acid.

The relationship between aerobic glucose oxidation, aerobic glycolysis, and anaerobic glycolysis is shown in Fig. 11.4.

Fig. 11.4. The relationship between aerobic glucose oxidation, aerobic glycolysis, and anaerobic glycolysis.

Shuttle mechanisms of NADH oxidation

Mitochondrial membranes are impermeable to NADH; therefore, glycolytic NADH generated in the cytosol cannot be directly oxidized by the NADH dehydrogenase of the mitochondrial Electron Transport Chain. Shuttle systems exist to oxidize cytosolic NADH by transporting reducing equivalents from this compound into the mitochondria via an indirect pathway. The Essence of these processes is that glycolytic NADH in the cytosol reduces a specific metabolite capable of crossing The inner mitochondrial membrane into the mitochondrial matrix, where it is oxidized—thereby reducing intramitochondrial NAD⁺—and then returns to the cytosol.

The transport of cytosolic (glycolytic) hydrogen into the mitochondria is carried out by the malate-aspartate and glycerophosphate shuttle systems.

The malate-aspartate shuttle system Functions by reducing oxaloacetate to malate (a cytosolic reaction) using hydrogen from the (НАДН⁺ + Н⁺) system, followed by the transport of malate into the mitochondria and its re-oxidation to oxaloacetate with the reduction of NAD⁺ (a mitochondrial reaction); the reverse transport of oxaloacetate into the cytosol occurs after its conversion into aspartate (via a reversible Transamination reaction), as shown in Fig. 11.5.

Fig. 11.5. The malate-aspartate shuttle for transporting reducing equivalents of glycolytic NADH⁺ into the mitochondria. E₁, E₂ represent the mitochondrial and cytoplasmic malate dehydrogenase Isoenzymes, respectively; T denotes mitochondrial membrane translocases.

The glycerophosphate shuttle system operates by reducing dihydroxyacetone phosphate to glycerol-3-phosphate (a cytosolic process) using glycolytic hydrogen, transporting glycerol-3-phosphate into the mitochondria, and oxidizing it within the respiratory chain back to dihydroxyacetone phosphate, which then returns to the cytosol.

Regulation of glycolysis

Glycolysis is regulated by METABOLISM/18.html">The Influence of negative and positive modulators (inhibitors and activators) on the catalytic activity of regulatory enzymes that catalyze the irreversible reactions of glycolysis:

- Hexokinase — in the muscle enzyme, the reaction product glucose-6-phosphate acts as an allosteric inhibitor;

- Phosphofructokinase — inhibitors include citrate (a Tricarboxylic Acid Cycle metabolite) and ATP; activators include the enzyme substrate fructose-6-phosphate and AMP. A high rate of oxidative processes, characterized by the accumulation of TCA cycle substrates and ATP within the cell, utilizes this mechanism to preserve the glucose pool. Phosphofructokinase catalyzes the rate-limiting step of glycolysis;

- Pyruvate kinase — the enzyme is inhibited by ATP, as well as by Citric Acid Cycle substrates such as acetyl-CoA and Fatty acids, which ensures the downregulation of glycolysis under conditions of high oxidative activity. The liver isoform of pyruvate kinase is regulated by covalent modification via cAMP-dependent phosphorylation (the dephosphorylated form is active, while the phosphorylated form is inactive). Additionally, hepatocyte pyruvate kinase is an inducible enzyme whose synthesis is stimulated by increased dietary carbohydrate intake and rising Insulin levels.

The inhibition of glycolytic reactions resulting from the suppression of phosphofructokinase and pyruvate kinase catalytic activities during active cellular Respiration forms the Molecular Basis of the Pasteur Effect.