Principles of Biochemistry Volume 2 - A. Lehninger 1985

Bioenergetics and Metabolism
Glycolysis: The Central Pathway of Glucose Catabolism
Energy is stored during the second stage of glycolysis

The Second Stage of Glycolysis (see the reaction sequence in Fig. 15-5) involves phosphorylation reactions in which the Free energy originally contained in the glucose molecule is released and captured in the form of ATP. Because each glucose molecule yields two molecules of glyceraldehyde 3-phosphate, both halves of the glucose molecule undergo the exact same reactions during this second stage. The conversion of two molecules of glyceraldehyde 3-phosphate into two molecules of Pyruvate is accompanied by The formation of four molecules of ATP from ADP. However, the net yield of ATP per split glucose molecule is only two, because two molecules of ATP are consumed During the first stage of glycolysis to phosphorylate the hexose molecule at positions 1 and 6.

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Fig. 15-4. Fate of the carbon atoms of glucose during the Formation of glyceraldehyde 3-phosphate.

A. The aldolase and triose phosphate isomerase reactions. B. Through the triose phosphate isomerase reaction, the two halves of the original glucose molecule are converted into two molecules of glyceraldehyde 3-phosphate. Each of the three carbon atoms in glyceraldehyde 3-phosphate originates from one of the two glucose carbon atoms, as shown in the figure. The numbering of carbon atoms in glyceraldehyde 3-phosphate does not correspond to that in D-glucose. This must be kept in mind when interpreting the results of experiments with D-glucose labeled at only a single carbon atom.

Fig. 15-5. The second stage of glycolysis.

i. Oxidation of glyceraldehyde 3-phosphate to 3-phosphoglyceryl phosphate

This is the first of two glycolytic reactions that lead to energy conservation in the form of ATP (Fig. 15-5). The enzyme glyceraldehyde 3-phosphate dehydrogenase catalyzes the reversible reaction

As a result of this complex reaction, the aldehyde group of D-glyceraldehyde 3-phosphate is oxidized. However, instead of yielding a carboxylic acid as one might expect, it produces a mixed anhydride of phosphoric and 3-phosphoglyceric acids: 3-phosphoglyceryl phosphate. This type of anhydride, known as an acyl phosphate, is characterized by a very high Standard Free Energy of Hydrolysis, ∆G^0' (-11.8 kcal/mol), placing it in the category of super-high-energy phosphorylated compounds (Section 14.9). For the second phosphate group of 3-phosphoglyceryl phosphate—that is, the phosphate group at position 3—the standard Free energy of hydrolysis is only about 3.2 kcal/mol. Thus, a significant portion of the free energy released during The oxidation of the aldehyde group of glyceraldehyde 3-phosphate is conserved in the high-energy phosphate group of the acyl phosphate (at C-1).

The coenzyme NAD⁺ (Fig. 15-6) acts as the hydrogen acceptor in the glyceraldehyde 3-phosphate dehydrogenase reaction. It represents the oxidized form of nicotinamide adenine dinucleotide, which contains the vitamin nicotinamide (Section 10.6). When NAD⁺ is converted to its reduced form (designated as NADH; Fig. 15-6), a hydride ion (:H^-) is transferred enzymatically from the aldehyde group of glyceraldehyde 3-phosphate to position 4 of the nicotinamide ring of NAD⁺, resulting in reduction at positions 1 and 4. Simultaneously, the second hydrogen atom of the substrate is released into the medium as an H⁺ ion. The equation describing the enzymatic reduction of NAD⁺ reflects this process:

Fig. 15-6. A. Structure of nicotinamide adenine dinucleotide in its oxidized form (NAD⁺).

B. Reduction of NAD⁺ via The transfer of a hydride ion (^:H-) from the substrate RH₂ to position 4 of the nicotinamide ring. See also Fig. 10-7.

Fig. 15-7. A. Scheme illustrating the MECHANISM OF ACTION of glyceraldehyde 3-phosphate dehydrogenase. A covalent bond forms between the substrate and the SH group in the Active Site of the enzyme, yielding a thiohemiacetal. This intermediate, which is an enzyme-substrate complex, is oxidized by NAD⁺ (also bound to the active site), producing a thioester covalent intermediate called an acyl-enzyme. The bond between the acyl group and the thiol group of the enzyme is characterized by a very high standard free energy of hydrolysis. In the final step, the thioester bond undergoes phosphorolysis, which regenerates the free enzyme and produces an acyl phosphate that retains a large fraction of The energy released during the oxidation of the aldehyde group. B. Iodoacetate is a potent inhibitor of glyceraldehyde 3-phosphate dehydrogenase because it forms a covalent bond with an essential functional SH group of the enzyme, thereby inactivating it.

The Mechanism of action of glyceraldehyde 3-phosphate dehydrogenase is quite complex (Fig. 15.7). First, the substrate interacts with the SH group of a Cysteine residue that plays a crucial role in the enzyme's active site. Next, the enzyme catalyzes the transfer of a hydride ion from the covalently bound substrate to NAD⁺, which is also tightly bound to the active site. This process generates a high-energy covalent acyl-enzyme complex. This complex then reacts with inorganic phosphate to yield free 3-phosphoglyceryl phosphate and regenerate the free enzyme. The NADH produced in this reaction is subsequently reoxidized to NAD⁺ so that it can participate in The breakdown of many more glucose molecules into pyruvate. If this reoxidation of NADH did not occur, glycolysis would quickly grind to a halt due to the depletion of the cellular pool of NAD⁺, which is present in limited amounts.

Glyceraldehyde 3-phosphate dehydrogenase has been isolated in crystalline form from Skeletal Muscle of the rabbit. Its molecular weight is 140,000. The enzyme molecule consists of four identical subunits, each being a single polypeptide chain containing approximately 330 amino acid residues. Glyceraldehyde 3-phosphate dehydrogenase is inhibited by iodoacetate (Section 9.12), which binds to an essential functional SH group of the enzyme and thereby abolishes its catalytic activity (Fig. 15-7, B). The discovery that iodoacetate inhibits glycolysis played a historically important role in The Study of enzyme systems (Section 13.13).

b. Transfer of the phosphate group from 3-phosphoglyceryl phosphate to ADP

The enzyme phosphoglycerate kinase catalyzes the transfer of the high-energy phosphate group from the carboxyl group of 3-phosphoglyceryl phosphate to ADP, yielding ATP and 3-phosphoglycerate:

This glycolytic reaction, together with the preceding one, provides energy coupling. Writing the equations for these two reactions consecutively makes it immediately clear that 3-phosphoglyceryl phosphate serves as a common intermediate here: it is produced in the first reaction, and In the second, its high-energy phosphate group is transferred to ADP to form ATP:

The overall equation for these two sequential reactions, coupled through a common intermediate (3-phosphoglyceryl phosphate), is as follows:

The net result of these two reactions, which are reversible under intracellular conditions, is that the energy released during the oxidation of the aldehyde group to a carboxyl group is conserved through the coupled formation of ATP from ADP and phosphate. This type of ATP synthesis, coupled with the enzymatic conversion of a "substrate" (i.e., one of the metabolic intermediates, such as glyceraldehyde 3-phosphate), is known as substrate-level phosphorylation. We will encounter other Examples of this type of process below.

c. Conversion of 3-Phosphoglycerate to 2-Phosphoglycerate

The enzyme phosphoglycerate mutase catalyzes the reversible intramolecular transfer of a phosphate group from one position to another within the substrate molecule.

This reaction, in which the phosphate group of glycerate is shifted from carbon 3 to carbon 2 (Fig. 15-5), requires Mg²⁺. The term mutase is commonly used to designate Enzymes that catalyze the intramolecular rearrangement of functional groups.

d. Dehydration of 2-Phosphoglycerate to Form Phosphoenolpyruvate

This is the second glycolysis reaction that yields a high-energy phosphorylated compound: the enzyme enolase catalyzes the reversible dehydration of 2-phosphoglycerate to form phosphoenolpyruvate (Fig. 15-5):

Despite the relatively small standard free-energy change for this reaction, The values of ∆G^0' for the hydrolysis of the phosphate groups of the reactant and the product differ markedly. For phosphoglycerate (a low-energy phosphorylated compound), this value is approximately — 4.2 kcal, whereas for phosphoenolpyruvate (a super-high-energy phosphorylated compound), it is — 14.8 kcal (Sec. 14.9). Although the total energy content of 2-phosphoglycerate and phosphoenolpyruvate is nearly identical, the removal of a Water molecule from 2-phosphoglycerate causes a redistribution of energy within the molecule. This internal redistribution accounts for the fact that the hydrolytic Cleavage of the phosphate group from phosphoenolpyruvate is accompanied by a much greater decrease in free energy.

Enolase (molecular weight 85,000) has been isolated in crystalline form from several sources. Its activity requires Mg²⁺ ions, with which the enzyme forms a complex prior to binding the substrate. Enolase is characteristically inhibited by fluoride (F^-) in the presence of phosphate; the true inhibitors in this case are fluorophosphate ions, which sequester Mg²⁺ ions.

e. Transfer of a Phosphate Group from Phosphoenolpyruvate to ADP

The final step of glycolysis is the transfer of the high-energy phosphate group from phosphoenolpyruvate to ADP (Fig. 15-5). Catalyzed by pyruvate kinase, this reaction provides yet another example of substrate-level phosphorylation. The reaction product, pyruvate, is initially formed in its enol form:

however, this enol form rapidly and nonenzymatically tautomerizes into the keto form, which predominates at pH 7.0:

The equilibrium of this reaction lies far to the right, which, in accordance with the law of mass action, also pulls the preceding pyruvate kinase reaction forward. The overall equation for the pyruvate kinase reaction coupled with the nonenzymatic formation of ketopyruvate is:

This overall reaction is characterized by a very large negative value of ∆G^0', which is largely driven by the spontaneous Conversion of the enol form of pyruvate into the keto form. The standard free-energy change for the hydrolysis of phosphoenolpyruvate is — 14.8 kcal/mol. Approximately half of this energy is conserved as ATP (∆G^0' = -7.3 kcal/mol), while the other half (-7.5 kcal/mol) provides the strong driving force that decisively pulls the reaction equilibrium to the right. Under cellular conditions, the pyruvate kinase reaction is essentially irreversible.

Pyruvate kinase has been crystallized (molecular weight 250,000). Its activity requires K⁺ ions, as well as either Mg²⁺ or Mn²⁺. This enzyme is among the key regulatory enzymes, and its function will be discussed in detail below.

f. Reduction of Pyruvate to Lactate

The pivotal role of pyruvate in Carbohydrate Catabolism stems from the fact that it sits at the crossroads of various Catabolic pathways. Under aerobic conditions in animal Tissues, pyruvate is the end product of glycolysis, and the NADH generated during the oxidation of glyceraldehyde 3-phosphate is reoxidized (i.e., converted back to NAD⁺) via molecular oxygen (Ch. 17). The situation is quite different under anaerobic conditions, such as in vigorously working skeletal muscle or in lactic acid Bacteria. Under these conditions, the NADH produced during glycolysis is reoxidized not by oxygen (which is absent), but by pyruvate, which is thereby reduced to lactate. The electrons transferred initially from glyceraldehyde 3-phosphate to NAD⁺ are transferred via NADH to pyruvate. The reduction of pyruvate is catalyzed by the enzyme Lactate dehydrogenase, yielding the L-isomer of lactate as the product of the lactate dehydrogenase reaction:

The equilibrium of this reaction lies far to the right, as indicated by its large negative ∆G^0' value. The oxidation of two molecules of glyceraldehyde 3-phosphate derived from each glucose molecule consumes two molecules of NAD⁺ and synthesizes two molecules of NADH. Consequently, the regeneration of two molecules of NAD⁺ through the reduction of two pyruvate molecules to lactate ensures that NAD can be used repeatedly in glycolysis.

As we already know (Sec. 9.23), lactate dehydrogenase in most tissues exists in five distinct isoforms that differ in properties such as the K_m for pyruvate, turnover number (or V_max), and the degree of allosteric inhibition by pyruvate. The cardiac muscle isozyme (designated H₄) is composed of four identical polypeptide chains of the H type. It is characterized by a low K_m for pyruvate and strong inhibition by pyruvate. Another isomeric form of this enzyme found in skeletal muscle (designated M₄) has a higher K_m for pyruvate, is not inhibited by pyruvate, and exhibits a higher catalytic activity than The Heart isozyme.

Numerous attempts have been made to provide a satisfactory explanation for the function and Physiological Role of lactate dehydrogenase isoforms across various tissues, particularly the heart, skeletal muscle, and Liver. Nevertheless, this subject remains a matter of considerable controversy and debate. The exact physiological role of these isoforms, as well as the two genes responsible for their synthesis, is still unclear. A particularly intriguing finding is that in a 64-year-old male subject, heart-type lactate dehydrogenase was completely absent due to a genetic defect; remarkably, this individual exhibited neither cardiac dysfunction nor any other metabolic abnormalities. This observation suggests that perhaps not all cellular enzymes or Proteins are strictly essential; some may be vestigial remnants that are no longer functionally utilized.

F. Overall Energy balance of Glycolysis

We can now formulate the overall energy balance of glycolysis, taking into account: 1) The Fate of the carbon Skeleton of glucose, 2) The pathway of electrons in oxidation-reduction reactions, and 3) the consumption of ADP and phosphate alongside the net yield of ATP per molecule of glucose degraded. The left-hand side of the equation below lists all the reactants utilized during glycolysis—namely ATP, P_i, ADP, NAD⁺, NADH, and H⁺ (see Figs. 15-4 and 15-5)—while the right-hand side lists all the glycolytic products (recalling that each glucose molecule yields two molecules of glyceraldehyde 3-phosphate):

Canceling out identical terms on both sides of the equation gives the overall net equation for anaerobic glycolysis in skeletal muscle under anaerobic conditions, as well as for Lactic acid Fermentation:

As a result of this process, one molecule of D-glucose is converted into two molecules of lactate (the carbon pathway). Two molecules of ADP and two molecules of inorganic phosphate are converted into two molecules of ATP (the phosphate group pathway). Four electrons (in the form of two hydride ions) are transferred by two molecules of NAD⁺ from two molecules of glyceraldehyde 3-phosphate to two molecules of pyruvate, yielding two molecules of lactate (the electron pathway). Although glycolysis includes two oxidation-reduction steps, there is no net change in the oxidation state of carbon over the course of the pathway. This can be verified by comparing the empirical formulas of glucose (C₆H₁₂O₆) and lactic acid (C₃H₆O₃). It is readily apparent that The ratio of C, H, and O atoms is identical in both compounds; consequently, the conversion of glucose to lactic acid does not entail the net oxidation of carbon. Nonetheless, anaerobic glycolysis successfully extracts a fraction of the free energy inherent in the glucose molecule—an amount sufficient to drive the net synthesis of two ATP molecules per molecule of glucose cleaved.

Under aerobic conditions, the end product of glycolytic glucose breakdown is pyruvate rather than lactate. In this case, the NADH produced by the oxidation of two molecules of glyceraldehyde 3-phosphate is reoxidized through a mechanism other than the reduction of pyruvate. The overall equation for glycolysis under these conditions is

The two NADH molecules generated during cytosolic glycolysis are reoxidized to NAD⁺ under aerobic conditions by donating their electrons to the Electron Transport Chain, which in Eukaryotic Cells is located within the Cell/35.html">Mitochondria. Here, the electrons are ultimately transferred to molecular oxygen, reducing it to H₂О: