13.1: Glycolysis

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Learning Goals 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

Thermodynamic Framework for Glycolysis

• Apply the relationship between ΔG°, reaction quotient, and actual free energy change to explain how thermodynamically unfavorable glycolytic steps are driven forward by reaction coupling, product removal, or mass action effects.
• Distinguish between the energy-investing and energy-recovering phases of glycolysis, and calculate the net ATP yield per glucose, accounting for ATP consumed and produced at specific steps.

The Ten Reactions of Glycolysis: Chemistry and Enzymology

• Describe the chemical logic of each glycolytic reaction — including the type of bond made or broken, the class of enzyme involved, and the mechanistic role of any cofactors or metal ions — for all ten steps from glucose to pyruvate.
• Explain how hexokinase uses an induced-fit conformational change to exclude water from the active site, thereby preventing ATP hydrolysis and ensuring phosphoryl transfer to glucose.
• Explain why the aldolase reaction (step 4) is thermodynamically unfavorable yet proceeds in the forward direction in vivo, and describe how Class I and Class II aldolases differ in their mechanisms of C–C bond cleavage.
• Explain the chemical logic of the glyceraldehyde-3-phosphate dehydrogenase reaction (step 6) — including how a single oxidative step produces a mixed anhydride with a phosphoryl transfer potential exceeding that of ATP — and describe how this couples oxidation to substrate-level phosphorylation in step 7.
• Explain how the sequential actions of phosphoglycerate mutase (step 8) and enolase (step 9) reposition and then eliminate a phosphate group to generate phosphoenolpyruvate, a compound with a phosphoryl transfer potential sufficient to drive ATP synthesis in step 10.

Regulation, Physiological Context, and Anaerobic Metabolism

• Identify the committed and rate-limiting step of glycolysis, explain how PFK1 is regulated by the ATP:ADP ratio and by fructose-2,6-bisphosphate, and connect this regulation to cellular energy status.
• Explain the metabolic rationale for converting pyruvate to lactate under anaerobic conditions, specifically in terms of regenerating NAD⁺ to sustain continued glycolytic flux.

Introduction

This chapter provides a historical overview of glycolysis and introduces you to the 10 enzymatic reactions in the pathway. Our main goal is to understand how the oxidation of our major food molecules, sugars in the case of glycolysis, can lead to ATP synthesis.  We detail each reaction mechanism for two reasons:

• Glycolysis is the central metabolic pathway for extracting energy from glucose, producing ATP.  It is found universally in almost all organisms.  The intermediates in the pathway can also be siphoned off to provide building blocks for biosynthesis
• It's the first opportunity to interrogate a metabolic pathway in detail, combining structural analyses, thermodynamics, organic chemistry, and kinetics. It provides a template for studying and understanding all other metabolic pathways in living cells. 

Before we begin our journey into the glycolytic pathway, reviewing the concept of reaction free energy is useful. For a reaction to be spontaneous, the change in the free energy within the system must be negative. From the equation in Figure \(\PageIndex{1}\), you can see that the change in free energy of a reaction depends on the concentration of the reactants and the products, as well as the temperature within the system.

Figure \(\PageIndex{1}\): The Change in Free Energy of a Reaction. The change in the free energy of a reaction is equal to the standard free energy change for the reaction plus the gas constant (R) multiplied by the Temperature in Kelvin (T) and the natural log (ln) of the concentrations of the products of the reaction (noted as C and D in the example) over the concentration of the reactants (noted as A and B in the example).

You will note that some reactions of glycolysis and other metabolic pathways that we will investigate are not favored. Thus, it is necessary to drive the reaction to become spontaneous by either coupling the reaction with a spontaneous reaction that can generate enough free energy to drive the nonspontaneous reaction forward or by using Le Chatelier’s principle and removing the products from the enzyme’s area as soon as they are made. This will help drive the reaction forward. In this way, a small amount of product can be formed spontaneously. Essentially, the continued removal of the product or the addition of excess reactant will drive the reaction forward. It is worth noting that the reaction temperature can also influence the change in free energy. However, in biological systems, temperature changes that significantly alter free energy are usually incompatible with the maintenance of most life forms. Thus, it will not be a large consideration in our metabolic discussions here.

When reactions are coupled together to obtain a spontaneous reaction, the overall free energy change for a chemically coupled series of reactions is equal to the sum of the free energy changes of the individual steps, as noted in Figure \(\PageIndex{2}\).

Figure \(\PageIndex{2}\): The Coupling of Two Reactions. The top reaction is nonspontaneous, with a positive free-energy change of 21 kJ/mol. This reaction can be driven forward by coupling it with a strongly spontaneous reaction that releases -34 kJ/mol. The overall net reaction has a negative change in free energy and is spontaneous.

The metabolic reactions of carbohydrates and other nutrients play an important role in generating ATP. For carbohydrates, this begins with the metabolic process known as glycolysis (or the breakdown, "lysis," of sugars, "glycol"). At the end of the 1910s, Otto Meyerhof mapped some metabolic conversions by measuring heat trends and oxygen consumption in frog muscles. When the muscle is working, lactic acid is formed from carbohydrates. Otto Meyerhof showed that lactic acid is reprocessed into carbohydrates.

Concurrently, Archibald Hill also outlined these processes in frog muscles. In opposition to the prevailing view that mechanical movement and chemical processes were parallel sequences, Hill could show, through measurements of heat generated by the mechanical processes, that these were delayed relative to the movements. The chemical sequence consists of a work phase, which is independent of oxygen supply, and a recovery phase, which requires oxygen. Together, their work has opened the door to understanding aerobic and anaerobic metabolism, beginning with glycolysis. They both shared the Nobel Prize in Physiology and Medicine in 1922 for their work in these processes Figure \(\PageIndex{3}\).

Figure \(\PageIndex{3}\): The Nobel Prize for Physiology and Medicine in 1922. Archibald V. Hill and Otto Fritz Meyerhof received this award for their work on sugar metabolism and breakdown. Photos from Nobel Prize.Org

The glycolytic pathway consists of 10 enzymatic steps that convert glucose to pyruvate. This conversion generates a small amount of energy. The pyruvate can then be converted to lactic acid (lactate) in vertebrates or to ethanol in yeast in an anaerobic (or oxygen-independent) pathway, or it can be fully oxidized to carbon dioxide in an aerobic (or oxygen-requiring) pathway that takes place within the mitochondria (which is shown in green in Figure \(\PageIndex{4}\)). Aerobic oxidation yields about 18 times as much energy as anaerobic pathways, making aerobic pathways favored over anaerobic ones.  Most animal tissues can only survive short anaerobic bursts in isolation and do not involve the entire organism. The aerobic pathway is required to sustain life. Yeast also prefers to grow using the aerobic, mitochondrial pathway. However, if oxygen is unavailable, yeast and other fungi can switch to anaerobic growth and produce ethanol as a byproduct. The production of alcoholic beverages through this fermentation process is quite popular.

Figure \(\PageIndex{4}\): Overview of Aerobic and Anaerobic Oxidation of Glucose. Figure modified from Kim, Y. et al (2011) PLoS ONE 6(12):e28293

Figure \(\PageIndex{5}\) summarizes the glycolytic pathway coupled to the oxidative phosphorylation pathway that occurs within the mitochondria.

Figure \(\PageIndex{5}\): A Summary of the Glycolytic and Oxidative Phosphorylation Pathways. The glycolytic pathway is shown in blue on the left-hand side. During aerobic metabolism, pyruvate is converted to acetyl-CoA, which then enters the Krebs cycle within the mitochondrial matrix. Future chapters will focus on mitochondrial reactions. Our focus in this chapter will be on the cytosolic reactions of the glycolytic pathway.

The major reactions of glycolysis are shown in Figure \(\PageIndex{6}\). The pathway can be divided into two major sections: (1) the energy-consuming reactions and (2) the energy-generating reactions. An adage states that ‘It takes money to make money.’ The same can be thought about the glycolytic pathway. The first section requires an energy investment to generate energy in the second half of the reaction pathway. In this pathway, glucose, a 6-carbon hexose, is converted to two 3-carbon molecules: pyruvate. Figure \(\PageIndex{6}\) shows the entire pathway using Lewis wedge/dash representations plus the anaerobic conversion of pyruvate to lactate.

Figure \(\PageIndex{6}\): Glycolytic pathway. The chemical steps of the glycolytic pathway are shown using Lewis wedge/dash representations. Enzymes required at each step are labeled in red. The energy-consuming steps encompass reactions 1-3, whereas the energy-producing steps occur in the second half of the pathway, from reactions 4-10. The conversion of pyruvate to lactate by lactate dehydrogenase represents anaerobic respiration in mammals.

Glycolysis is the key anaerobic pathway for energy production in all organisms, except lithotrophs, which use the oxidation of inorganic molecules for energy. In aerobic systems, glycolysis releases energy quickly within the body, as glycogen metabolism can quickly release glucose for utilization. Given the centrality of glycolysis to all of life, we will explore each reaction in detail below.

Reaction 1: Glucose → Glucose-6-Phosphate. ΔG^o = -4.0 kcal/mol (-16.7 kJ/mol).

The first step in glycolysis is catalyzed by hexokinases. The hexokinase family of enzymes typically has broad specificity for various hexoses and catalyzes the phosphorylation of carbon 6. Hexokinase phosphorylates glucose using ATP as the phosphate donor, producing glucose-6-phosphate, a more reactive form of glucose. Notably, this reaction prevents the phosphorylated glucose molecule from further interacting with GLUT transport proteins that shuttle glucose into and out of the cell. Thus, once phosphorylated, glucose cannot leave the cell. Do note that both sugar forms (the free sugar and the phosphorylated version) can shift back and forth between the ring-closed and ring-opened conformation. Hexokinases require glucose to be in the closed conformation for phosphorylation. Figure \(\PageIndex{7}\) shows the reaction, which is catalyzed by hexokinase (a kinase that transfers the γ-phosphate from ATP to a hexose in the closed-ring form).

Figure \(\PageIndex{7}\) : Summary reaction - hexokinase

This reaction is a nucleophilic substitution reaction on the γ-phosphate of ATP. A phosphoanhydride bond is broken as a phosphoester bond is formed, yielding glucose 6-phosphate. Hence, the reaction proceeds with a negative ΔG^o.

Vertebrates have four hexokinase isoforms, I-IV (also called A-D). The regulation of these enzymes is presented in more detail in Chapter 15.5. Hexokinases I-III bind glucose more tightly, as reflected by low K_M values. Type IV, or glucokinase, binds it less tightly and is found at high concentrations in vertebrate livers. Yeast has three isozymes: P1, PII (hexokinase B), and glucokinase. Most prokaryotic hexokinases are in the glucokinase (Type IV) class.

In addition to hexokinases, there are many other sugar kinase enzymes. Notably, all sugar kinases must prevent the spurious hydrolysis of ATP by water, which can also be considered a phosphotransfer to water rather than the preferred transfer of the γ phosphate of ATP to a sugar ROH (alcohol), an "alcoholysis" reaction. Hexokinase does this through an "induced fit" mechanism, in which glucose binding triggers a large conformational change that closes off the active site to water.

This conformational change is illustrated in Figure \(\PageIndex{8}\) below, which shows an interactive iCn3D model of the yeast hexokinase PI in the absence (2YHX) and presence (3B8A) of glucose.

Figure \(\PageIndex{8}\): Yeast hexokinase PI in the absence (2YHX) and presence (3B8A) of glucose. (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...84YGEd1ob3th7A

Within Figure \(\PageIndex{8}\), the gray structure is hexokinase without glucose (2YHX). However, it has a glucose analog bound, o-tolyoylglucosamine (spacefill, yellow highlights), which binds at the glucose-binding site but does not cause a subsequent conformational change. The cyan structure (in the actual iCn3D model) shows the enzyme with glucose shown as colored sticks. Note the large conformational change in the actual binding of glucose, a classic example of an "induced fit" mechanism.

Figure \(\PageIndex{9A}\) shows the surface of both enzymes using the same color coding as in the above figure. These images better show how the active site of hexokinase is occluded in the glucose-bound form. This prevents access to water and hydrolysis of bound ATP, rather than "alcoholysis" of ATP (transfer of phosphate to glucose). The gray structure on the left shows the glucose analog bound, which doesn't alter the protein's global conformation.

Figure \(\PageIndex{9A}\): Surface of human hexokinase I with bound o-tolyoylglucosamine (spacefill, left, 2YHX) and with bound glucose (spacefill, right, 3B8A)

Figure \(\PageIndex{9B}\) below shows a surface rendering depicting the conformation change of Sulfolobus tokodaii hexokinase in the apoform without bound glucose (cyan,2E2N) to the glucose-bound form (magenta,2EON).

Figure \(\PageIndex{9B}\): Conformation change of Sulfolobus tokodaii hexokinase in the apoform without bound glucose (cyan, 2E2N) to the glucose-bound form (magenta, 2EON).   Note that a small part of glucose is present in the center of the bound form. 

Hexokinase I is the key form in the brain. It forms a complex with porin in the mitochondrial outer membrane and ATP/ADP translocase or carrier protein in the mitochondrial inner membrane, facilitating the hexokinase reaction. Figure \(\PageIndex{10}\) below shows the mechanism for the human hexokinase I reaction.

Figure \(\PageIndex{10}\): Mechanism of human hexokinase I.

Ribeiro AJM et al. (2017), Nucleic Acids Res, 46, D618-D623. Mechanism and Catalytic Site Atlas (M-CSA): a database of enzyme reaction mechanisms and active sites. DOI:10.1093/nar/gkx1012. PMID:29106569. https://www.ebi.ac.uk/thornton-srv/m-csa/entry/696/. Creative Commons Attribution 4.0 International (CC BY 4.0) License.

Figure \(\PageIndex{11}\) below shows an interactive iCn3D model of human hexokinase I with glucose and ADP in the active site (1dgk).

Figure \(\PageIndex{11}\): Human hexokinase I with glucose and ADP in the active site (1dgk). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...aQy4gf6A2bWtp8

The key catalytic residues are shown in colored sticks and labeled. Glucose, ADP, and PO₄^2- are shown as colored spheres.

Note that hexokinase I and forms II and III have spatially distinct halves similar to those in the yeast enzymes. Forms I-III have a molecular weight of about 100K. Form IV (glucokinase) has a molecular weight of 50K, similar to the distinct halves of I-III. This suggests that I-III arose through gene duplication.

Each half also binds glucose and ADP, but the N-terminal domains of I and III are catalytically inactive. Hexokinase II, with both halves active, is the primary enzyme involved in glycolysis in most mammalian tissues. Another difference is that glucose-6-phosphate, a product, inhibits I-III. PO₄^2- relieves G6P product inhibition in form I but not the others. ADP binding at multiple sites in hexokinase I likely causes a conformational change that affects structure and activity.

Sugar kinases, in general

We will encounter many kinases that use ATP to phosphorylate sugars, so exploring their similarities and differences at the beginning of our studies on carbohydrate metabolism is useful.

Figure \(\PageIndex{12}\) below shows the comparative structures of the five carbohydrate kinase classes.

Figure \(\PageIndex{12}\): Structures of the five carbohydrate kinase classes. All images are shown in rainbow format (blue: N-terminus, red: C-terminus). Roy, S.; Vivoli Vega, M.; Harmer, N.J. Carbohydrate Kinases: A Conserved Mechanism Across Differing Folds. Catalysts 2019, 9, 29. https://doi.org/10.3390/catal9010029. Creative Commons Attribution License

Panel (a) shows the structure of human glucokinase (hexokinase class; PDB (protein data bank) ID: 4IWV).

Panel (b) shows the structure of Bacillus subtilis fructokinase dimer, the second molecule shown in raspberry (ROK kinase class; PDB ID: 1XC3 [17]).

Panel (c) shows the structure of Escherichia coli ribokinase (ribokinase class; PDB ID: 1RKD; [18]).

Panel (d) shows the structure of Aquifex aeolicus IspE (GHMP kinase class; PDB ID: 2V2Z; [19]).

Panel (e) shows the structure of human PIK3C3 (phosphatidylinositol phosphate kinase class; PDB ID: 3IHY).

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Table \(\PageIndex{1}\): Overview of the Five Carbohydrate Kinase Classes.

Carbohydrate Kinase Family	Common Substrates	Native Phosphate Donors (Minor Donors in Parentheses)	Pfam ID
Hexokinase	Glucose, mannose, fructose	ATP (ITP)	PF00349, PF03727, PF02685
ROK Kinase	Glucose, allose, fructose, N-acetylglucosamine, N-acetylmannosamine	ATP (polyphosphate)	PF00480
Ribokinase	Ribose, 2-deoxy-d-ribose, adenosine	ATP, ADP (GTP, ribonucleotide)	PF00294
GHMP Kinase	Galactose, N-acetylgalactosamine	ATP (GTP, ITP)	PF00288
Phosphatidylinositol kinase	Phosphatidylinositol, phosphatidylinositol phosphates	ATP (GTP)	PF00454, PF01504

ROK is a bacterial Repressor, Open reading frame, Kinase. Ribokinases include adenosine kinases, fructokinases, and phosphofructokinases. GHMP Kinases include Galactokinase, Homoserine kinase, Mevalonate kinase, and Phosphomevalonate kinase. Roy et al. Ibid

Many of these kinases are regulated by allosteric effectors.

Figure \(\PageIndex{13}\) shows a common mechanism for all, using glucose as an example substrate.

Figure \(\PageIndex{13}\): Generic reaction mechanism for carbohydrate kinases (hexokinase shown). Roy et al., Ibid. The generic reaction is initiated by a catalytic base abstracting a proton from the reactive hydroxyl (left). The oxygen atom then attacks the -phosphate of ATP (second left), forming a pentacoordinate transition state (second right). This is stabilized by a divalent cation and by the protein (not shown). This transition state resolves, leaving ADP and the phosphorylated carbohydrate (right).

Reaction 2: Glucose-6-Phosphate ↔ Fructose-6-Phosphate. ΔG^o = +0.4 kcal/mol (+1.7 kJ/mol)

In the second step of glycolysis, phosphoglucose isomerase (PGI) converts glucose-6-phosphate into its isomer, fructose-6-phosphate. Recall that an isomerase is an enzyme that catalyzes the conversion of a molecule into one of its isomers. In this reaction, the aldohexose glucose-6-phosphate is converted to the ketohexose fructose-6-phosphate. This conversion is essential for the eventual splitting of the sugar into two three-carbon molecules. We'll present this reaction, catalyzed by phosphoglucose isomerase (PGI), in several different types of representations (chair, wedge/dash, and Fischer projection), as shown in Figure \(\PageIndex{14}\) below.

Figure \(\PageIndex{14}\): Summary reaction, phosphoglucoisomerase

From a functional perspective, as seen more clearly in the linear Fischer structure, the C=O has been moved to the C2 position to form the ketose structure. The carbonyl O atom is now positioned to act as an electron sink, facilitating electron flow in reaction 4. This isomerization reaction would be expected to have a ΔG^o of about 0.

It has been proposed that the mechanism of PGI requires the enzyme to open the Glucose-6-Phosphate ring before the actual isomerization step, which proceeds through the formation of a cis-enediol intermediate and keto-enol tautomers before conversion back to the cyclic form.

Figure \(\PageIndex{15}\) below shows a proposed mechanism for rabbit phosphoglucose isomerase

Figure \(\PageIndex{15}\): Mechanism for phosphoglucose isomerase. Ribeiro AJM et al. Ibid

Figure \(\PageIndex{16}\) below shows an interactive iCn3D model of rabbit phosphoglucose isomerase with bound 6-phosphogluconic acid (1DQR)

Figure \(\PageIndex{16}\): Rabbit phosphoglucose isomerase with bound 6-phosphogluconic acid (1DQR). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...xo1SgF1ZpAySm9

One monomer of the dimer is shown in gray, and the other in cyan. The catalytic residues Lys518 and His388 are shown in both subunits as colored sticks, labeled. Additional residues that contribute to specificity (Ser209, Ser159, Thr214, Thr217, and Thr211) are shown in the gray subunit, with color-coded sticks and labels. 6-phosphogluconic acid, a competitive inhibitor, is shown in color spacefill.

The enzyme also has other functions besides its role in glycolysis, so it is a member of a group called "moonlighting" proteins (a term referring to proteins that perform a secondary role). Phosphoglucoisomerase acts outside the cell as a nerve growth factor and cytokine. It is also called autocrine motility factor (PGI/AMF), and its cytokine activity is associated with aggressive cancers.

Reaction 3: Fructose-6-Phosphate → Fructose-1,6-Bisphosphate. ΔG^o = -3.4 kcal/mol (-14 kJ/mol)

The third step is the phosphorylation of fructose-6-phosphate by phosphofructokinase-1 (PFK1). A second ATP molecule donates a high-energy phosphate to fructose-6-phosphate, producing fructose-1,6-bisphosphate. Bisphosphate is used when two phosphate groups are joined to a molecule at different positions. In this case, one phosphate is at the 1-carbon position, and the other is at the 6-carbon position. This differs from the term diphosphate, which is used when the phosphate groups are joined in a sequence, as in the molecule ADP. In ADP, both phosphate groups are joined in tandem from the 5-carbon position on the ribose ring structure.

In the glycolytic pathway, PFK1 is a rate-limiting enzyme. The mechanism of PFK1 regulation is discussed in greater detail in Chapter 15.5.  However, we will introduce the process here. Essentially, the enzyme is sensitive to the energy load within the cell. Recall that ATP is a recycled molecule and exists in a pool of interconverting  ATP, ADP, and AMP. A chief outcome of glycolysis is to shift the ATP pool towards higher levels to drive endergonic processes, such as muscle contraction.  PFK1 activity is sensitive to the ATP:ADP ratio within the cell. PFK1 is more active when the concentration of ADP is high, and the concentration of ATP is low, and it is conversely less active when ADP levels are low, and the concentration of ATP is high. This is a type of end-product inhibition since ATP is the end product of glucose catabolism. Note, however, that ATP is also a substrate for PFK1. ATP is required as the phosphate donor in the reaction. This is the second energy-intensive step in the glycolytic pathway. At the end of the PFK1 step, 2 ATP molecules have been broken down in the glycolytic pathway.

The PFK1 enzymatic step is also important within the glycolytic pathway as it is the committed step. Glucose-6-phosphate may be used for other purposes within the cell, and the isomerase step that converts glucose-6-phosphate to fructose-6-phosphate is readily reversible. The PFK1 enzyme catalyzes only the forward reaction to produce fructose-1,6-bisphosphate. It cannot operate in the reverse direction and recover the reactant. Thus, it is considered the committed step in the glycolytic pathway, as fructose-1,6-bisphosphate is predominantly converted to pyruvate through the remaining enzymatic steps.

The reaction catalyzed by PFK1 is shown in Figure \(\PageIndex{17}\) below.

Figure \(\PageIndex{17}\): Summary reaction - phosphofructokinase

By phosphorylating this intermediate, both products of the cleavage of this 6C molecule will be phosphorylated, keeping both more readily inside the cell. This reaction is a nucleophilic substitution reaction on the gamma phosphate of ATP. In ATP, a phosphoanhydride bond is broken, and in fructose-1,6-bisphosphate, a phosphoester bond is formed. As in reaction 1, this reaction proceeds with a negative ΔG^o.

The PFK1 enzyme exists as a tetramer, and, like another "famous" tetramer, hemoglobin, it can adopt T- and R-symmetry states. Hence, it is an allosteric enzyme and has many allosteric regulators. One important allosteric activator of eukaryotic (not prokaryotic) PFK is fructose-2,6-bisphosphate. This is formed by a separate phosphofructokinase enzyme named PFK2. This regulatory pathway will be described in greater detail in Chapter 15.5. Hence, the number 1 is added to the name of the glycolytic enzyme, PFK1, which forms fructose 1,6-bisphosphate, to indicate the position of phosphorylation. Mammals have three isoforms of PFK1: muscle (PFKM), liver (PFKL), and platelet (PFKP).

The mechanism for E. Coli phosphofructokinase is shown in Figure \(\PageIndex{18}\) below. A metal cofactor (Mg²⁺) coordinates the positioning of the ATP and stabilizes the gamma phosphate for nucleophilic attack by the fructose alcohol group at position 1. Activation of the fructose alcohol group is mediated by proton abstraction by Asp₁₂₇ of PFK1.

Figure \(\PageIndex{18}\): Proposed mechanism for E. Coli phosphofructokinase. Ribeiro AJM et al. Ibid

Figure \(\PageIndex{19}\) below shows an interactive iCn3D model of the E. Coli phosphofructokinase with bound F1,6-bisphosphate and ADP products (1PFK). (long load)

Figure \(\PageIndex{19}\): E. Coli phosphofructokinase with bound F1,6-bisphosphate and ADP products (1PFK). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...t4KKgLAGzHrzo8

Each monomer of the tetramer is shown in a different color. The gray monomers show key binding and catalytic residues described in the mechanism above. F1,6-bisphosphate is shown as spacefill, and ADP is shown as sticks.

The structure of the human platelet PFK1 tetramer has been determined in the presence of ATP and ADP. Figure \(\PageIndex{20}\) below shows an interactive iCn3D model of the human phosphofructokinase-1 dimer (for clarity) in a complex with ATP and Mg (4XYJ). (long load)

Figure \(\PageIndex{20}\): Human phosphofructokinase-1 dimer in complex with ATP and Mg (4XYJ). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...AKfonyGsNq6Cs9 (long load). Here is a link to the ADP-bound structure: https://structure.ncbi.nlm.nih.gov/i...N81MBS4PZfSQp9

The monomers are shown as colored surfaces with secondary structures underneath. Note that side chains from each monomer in the dimer contribute to binding and catalysis. The actual PDB structure is tetrameric. The structures have an E173S mutation.

Figure \(\PageIndex{21}\) shows the differences in orientation of the key side chains in the ADP structure (left) and the ATP structure (right) that are key in the conformation changes in the enzyme on substrate binding.

ADP-bound form of human PFK (4XYK)

ATP-bound form of human PFK (4XYJ)

Figure \(\PageIndex{21}\): Conformation changes in the active site of PFK on ATP hydrolysis

Reaction 4: Fructose-1,6-bisphosphate → Dihydroxyacetone phosphate (DHAP) + Glyceraldehyde 3-phosphate (G3P). ΔG^o = + 5.7 kcal/mol (+24 kJ/mol)

The newly added high-energy phosphates further destabilize fructose-1,6-bisphosphate. The fourth step in glycolysis employs the enzyme aldolase to cleave fructose-1,6-bisphosphate into two three-carbon compounds: dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. This reaction is shown in Figure \(\PageIndex{22}\) below in both wedge-dash and Fischer projections.

Figure \(\PageIndex{22}\): Summary reaction - aldolase

This is the first C-C bond cleavage within glucose on the path to complete cleavage during aerobic respiration, releasing 6 carbon dioxide molecules per glucose metabolized. This reaction is the reverse of an aldol condensation, when an enol or enolate reacts with a carbonyl C to form an adduct. Note that both products are phosphorylated. The reaction is not thermodynamically favored, but is driven forward by using the product in the next reaction in the pathway.

Table \(\PageIndex{2}\) Characteristics of the Three Classes of Aldolases (I, IA, and II) and the Organisms in Which They are Found.

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Pirovich et al, Frontiers in Molecular Biosciences, 8 (2021), https://www.frontiersin.org/article/...lb.2021.719678. AUTHOR=Pirovich David B., Da’dara Akram A., Skelly Patrick J. Creative Commons Attribution License (CC BY).

Class I Aldolase

These enzymes proceed through a Schiff base intermediate between a reactive lysine and the reactant/product. The enzyme is favored in the reverse direction, and it's perhaps easier to see the mechanism presented in that fashion. In rabbits, the muscle Class I aldolase (RAMA) uses Lys-229 to form a Schiff base with DHAP, as shown in a mechanism presented in Figure \(\PageIndex{23}\) below.

Figure \(\PageIndex{23}\): Class I aldolases - general mechanism (after Bolt et al., Arch Biochem Biophys. 2008 June 15; 474(2): 318–330)

Only the pro(S) proton of the dihydroxyacetone phosphate C3 carbon is removed and effectively exchanged with the glyceraldehyde-3-phosphate substrate. Figure \(\PageIndex{24}\) below reviews how the pro(R) and pro(S) hydrogens can be visually differentiated by replacing one with a deuterium and determining the stereochemistry of the now chiral C3.

Figure \(\PageIndex{24}\): Visualization of the Pro(R) and Pro(S) hydrogens on DHAP

Figure \(\PageIndex{25}\) shows key active site residues in the active site of a Class I aldolase from rabbit muscle. The first step in Schiff base formation with Lys229 is shown.

Figure \(\PageIndex{25}\): Catalytic residues in rabbit muscle Class I enolase.

Figure \(\PageIndex{26}\) shows an interactive iCn3D model of the dihydroxyacetone phosphate enamine intermediate in fructose-1,6-bisphosphate aldolase from rabbit muscle (2QUT)

Figure \(\PageIndex{26}\): Dihydroxyacetone phosphate enamine intermediate in fructose-1,6-bisphosphate aldolase from rabbit muscle (2QUT). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...uR5KZBsfN8QQh9

Only one monomer of the four in the homotetramer is shown. The ligand, 1,3-dihydroxyacetonephosphate, is shown in spacefill with CPK colors. It is in Schiff base linkage with Lys229. The key catalytic residues are shown and labeled.

The reaction proceeds between eneamine and iminium covalent intermediates. A K146M mutation (in the active site) decreases enzyme activity and allows the trapping of the K229 eneamine intermediate. A key tyrosine (Y363) in its deprotonated state, formed in the presence of the iminium phosphate and a local water, appears to abstract the C3 pro(S) proton to form the enamine.

Class II Aldolase

Figure \(\PageIndex{27}\):

Figure \(\PageIndex{27}\): Class II aldolases - general mechanism (after Bolt et al, ibid)

Reaction 5: DHAP ↔ G3P. ΔG^o= + 1.8 kcal/mol (+7.5 kJ/mol)

This reaction, catalyzed by triose phosphate isomerase (TPI), is shown in Figure \(\PageIndex{28}\).

Figure \(\PageIndex{28}\: Summary reaction - triose phosphate isomerase.

This is another simple isomerization reaction. Only one product, glyceraldehyde-3P, continues in glycolysis, so only one enzyme is needed to metabolize the cleavage products of this reaction further. As in other isomerization reactions, the ΔG^o is close to 0.

Figure \(\PageIndex{29} below shows a proposed mechanism for this reaction.

Figure \(\PageIndex{29}\): Proposed mechanism for triose phosphate isomerase (after Bolt et al., ibid)

Note that the reaction employs the deprotonation of a neutral imidazole side chain to form an imidazolate anion. This would not likely be favorable, given its pKa value.

Four possible conserved proton donors have more reasonable pKas in the active sites of TPIs, including K12, H95, E97, and E165. Mutations of E97 to E97Q and E97D lead to a 4000-fold reduction in k_cat (E97Q) but only a 100-fold reduction for E97D suggesting tha30}\) shows an interactive iCn3D model of the chicken triosephosphate isomerase-phosphoglycolohydroxamate complex (1TPH)

Figure \(\PageIndex{30}\): Chicken triosephosphate isomerase-phosphoglycolohydroxamate complex (1TPH). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...AFXpt9kg5aki5A

The four key conserved residues are shown in the gray monomer of the homodimer.

Reaction 6: G3P ↔1,3 BPG. ΔG^o= +1.5 kcal/mol (+6.1 kJ/mol)

This reaction, catalyzed by glyceraldehyde-3-phosphate dehydrogenase, is shown in Figure \(\PageIndex{31}\)

Figure \(\PageIndex{31}\: Summary reaction - glyceraldehyde-3-phosphate dehydrogenase

This is a big reaction! The ΔG^o is close to 0, but look at what happened. The carbonyl O in G3P has been oxidized to a mixed anhydride, which can donate a phosphate to ADP (in the next step) to form ATP. This is an oxidation reaction because the carbonyl C in G3P has two bonds to O, whereas the carbonyl C in 1,3 BPG has three bonds to O.

To carry out an oxidation reaction, you need an oxidizing agent. In comes NAD⁺, a modest but very prevalent oxidizing agent in biology. When glucose is oxidized completely by O₂ to CO₂ during combustion, much energy is released, so we can surmise that oxidation reactions, if carried out by powerful oxidizing agents like O₂, proceed with a large negative ΔG^o. For every oxidation reaction, the oxidizing agent is reduced. All reactions are potentially reversible, so the products formed can act as new oxidizing and reducing agents. As in acid/base reactions, which proceed from stronger to weaker conjugate acid, redox reactions proceed from a stronger to a weaker oxidizing agent. For reaction 6, we must use tables of redox potentials to calculate the actual ΔG^o. It turns out to be close to 0, which is great since a substrate-level phosphorylation reaction (using inorganic phosphate (Pi) instead of ATP) occurs in the same reaction. In summary, this reaction catalyzes the first and only glucose oxidation in glycolysis, which has paid (thermodynamically) for the generation of a mixed anhydride whose phosphorylating potential is higher than that of ATP.

Figure \(\PageIndex{32}\) shows a proposed mechanism for glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi

Figure \(\PageIndex{32}\): Proposed mechanism for glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi (after Reis et al. Phys. Chem. Chem. Phys., 2013, 15, 3772. https://pubmed.ncbi.nlm.nih.gov/23389436/)

The reaction proceeds in two parts. The first (top section) is the oxidation of glyceraldehyde-3-phosphate (G3P) by NAD⁺ to the state of a thioester attached to Cys166. The now-reduced NADH dissociates and is replaced by a new NAD⁺ for another catalytic cycle. In the meantime, inorganic phosphate (Pi) binds to and reacts with the thioester to form 1,3-bisphosphoglycerate (1,3-BPG).

The mechanism is not entirely clear. Two phosphate binding sites, Pi (red) and Ps (purple) interact with phosphate groups on the substrates. The oxidation step of the reaction appears to occur at the Pi site. Pi comprises the side chains of Thr197, Thr199, and the 2-hydroxyl group of the ribose in NAD⁺. The Pi site in the T. cruzi enzyme consists of Thr226 and Arg249, Gly227, and Ser247. The mechanism appears to involve a flip in the orientation of substrates and intermediates after NADH dissociates from the enzyme.

Figure \(\PageIndex{33}\) shows an interactive iCn3D model of the Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase with bound NAD and a 1,3-bisphosphoglycerate analogue (1QXS)

Figure \(\PageIndex{33}\): Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase with bound NAD and a 1,3-bisphosphoglycerate analogue (1QXS) . (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...WnsNSgmA3AJyw6

Only two monomers of the homotetramer are shown for clarity. The Pi site is magenta, and the Ps site is purple. The phosphonic acid analog is shown in spacefill, and NAD is shown in sticks. The active site His194 and Cys166 are also shown in sticks and labeled.

Reaction 7: 1,3 BPG + ADP + H⁺ → 3PG + ATP ΔG^o = -4.5 kcal/mol (-19 kJ/mol)

This reaction, catalyzed by phosphoglycerate kinase, is shown in Figure \(\PageIndex{34}\)

Figure \(\PageIndex{34}\): Summary reaction - phosphoglycerate kinase

It's finally happened! An ATP has been made for each of the two 1,3-BPG molecules derived from glucose. We've made back the ATP used in steps 1 and 3. A mixed phosphoanhydride bond is broken in 1,3 BPG as a phosphoanhydride bond is made in ATP. As the mixed phosphoanhydride has higher energy than its hydrolysis product compared to the phosphoanhydride in ATP, the reaction proceeds with a negative ΔG^o.

A mechanism for the reaction is shown in Figure \(\PageIndex{35}\).

Figure \(\PageIndex{35}\): Reaction mechanism for phosphoglycerate kinase

Figure \(\PageIndex{36}\) shows an interactive iCn3D model of human phosphoglycerate kinase in complex with ADP, 3PG, and magnesium trifluoride (2WZB).

Figure \(\PageIndex{36}\): Human phosphoglycerate kinase in complex with ADP, 3PG, and magnesium trifluoride (2WZB). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...w5yvu2VuGhmQXA

3-phosphoglycerate is shown in spacefill with CPK colors. ADP, together with the adjacent MgF₃, mimics the transition state for ATP synthesis. Hence, this structure shows the products/transition state in a closed active site, which, as we have seen before, prevents spurious hydrolysis of 1,3-BPG or ATP.

Figure \(\PageIndex{37}\) shows an interactive iCn3D model of the alignment of the open form of human phosphoglycerate kinase (2XE7) with bound 3PG and ADP with the closed form with bound 3PG, ADP, and MgF₃ (2WZB).

Figure \(\PageIndex{37}\): Alignment of the open form of human phosphoglycerate kinase (2XE7) with bound 3PG and ADP with the closed form with bound 3PG, ADP, and MgF₃ (2WZB). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...n5FfsJeYitiWr9

Use the "a" key to toggle back between the open form (magenta) and closed forms (cyan). In the closed state, which most closely resembles the ATP bond transition state, the two lobes of the enzyme clamp together.

Reaction 8: 3PG ↔ 2PG ΔG^o= +1.1 kcal/mol (4.6 kJ/mol)

This reaction, catalyzed by phosphoglycerate mutase (PGM), is shown in Figure \(\PageIndex{38}\)

Figure \(\PageIndex{38}\): Summary reaction - phosphoglycerate mutase

This isomerization reaction proceeds with little thermodynamic barrier. Its function is to locate the phosphate on C2 which on the next reaction (dehydration) will form a molecule whose phosphoryl transfer potential is greater than ATP. It seems so simple, but the enzymes that catalyze this reaction are diverse and quite complicated from a mechanistic perspective.

There are two types of PGMs, bisphosphoglycerate and monophosphoglycerate mutases, that carry out three different reactions involving the shuffling of phosphates from one position to another in 3C sugars or the cleavage of a phosphate from a sugar

• 3-phosphoglycerate ↔ 2-phosphoglycerate (reaction 8 of glycolysis) catalyzed by bisphosphoglycerate and monophosphoglycerate (the glycolytic enzyme) mutases
• 1,3-bisphosphoglycerate ↔ 2,3-bisphosphoglycerate by bisphosphoglycerate mutases
• 2,3-phosphoglycerate ↔ 3-phosphoglycerate + Pi by bisphosphoglycerate mutases

Within the monophosphoglcyerate mutases, and more specifically, the phosphoglycerate mutase (PGM) of glycolysis, there are two types

• one that depends on the cofactor 2,3-phosphoglycerate. These are called cofactor-dependent phosphoglycerate mutase (dPGM) and are found in mammals, yeast, and some bacteria. They do not require metal ions.
• one that does not depend on the cofactor 2,3-phosphoglycerate. These are called cofactor-independent phosphoglycerate mutase (iPGM) and are found in plants and some bacteria. These can only interconvert 3PGA and 2PGA. One family of enzymes in the class requires Mn^2+, while the other requires Mg²⁺ or Zn²⁺. These enzymes are often structurally similar to alkaline phosphatases.

The cofactor-independent and cofactor-dependent monophosphoglycerates (such as the phosphoglycerate mutase of glycolysis) are very different structurally and mechanistically, so we will look at both types of mechanisms. Within each type, the enzyme sequences are very conserved.

Mechanism of cofactor (2,3-BPG) dependent phosphoglycerate mutase (dPGM)

The reaction is much simpler than that of the cofactor-independent PGM. In E. coli, the reaction involves transferring the phosphate on the C3-OH to the nucleophilic nitrogen on histidine 8 (His 10 in other enzymes) in the active site to form a covalent pHis8 intermediate. The phosphate on pHis8 could then be transferred to the O on carbon C2 of the substrate.  An active His 181 in E. Coli may also act as a general acid/base and is adjacent to Glu 88 in the active site.  

Figure \(\PageIndex{39}\) shows an interactive iCn3D model of yeast phosphoglycerate mutase (cofactor dependent) bound to 3-phosphoglycerate (1QHF)

Figure \(\PageIndex{39}\): Yeast phosphoglycerate mutase (cofactor dependent) bound to 3-phosphoglycerate (1QHF). (Copyright; author via source). A dimer of the active tetramer is shown.  Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...9F4oc6mmr5DFs9

Mechanism of cofactor-independent phosphoglycerate mutase (iPGM)

Let's consider the mechanism for the Mn²⁺-requiring iPGM from Geobacillus stearothermophilus. Two Mn²⁺ ions are in the active site. The mechanism for the first half of the cofactor-independent phosphoglycerate mutase (iPGM) reaction is shown in Figure \(\PageIndex{40}\).

Figure \(\PageIndex{40}\): Part A - Mechanism for cofactor-independent phosphoglycerate mutase (iPGM) from Geobacillus stearothermophilus (after Bolt et al, ibid)

Arg 261 interacts with the substrate, stabilizing its negative charge and transition state. It also makes the target phosphorus more electrophilic. A Mn²⁺ ion activates Ser 62 to become more nucleophilic after the abstraction of a proton by Lys 336. Reaction 3 probably proceeds through an S_N2 mechanism.

Figure \(\PageIndex{41}\) shows the rest of the mechanism.

Figure \(\PageIndex{41}\): Part B - Mechanism for cofactor-independent phosphoglycerate mutase (iPGM) from Geobacillus stearothermophilus (after Bolt et al., ibid)

The iPGMs are monomers with two distinct domains (lobes), each containing a substrate-binding site, separated by an active site. They are connected by flexible sequences that bend to produce either an open or closed form of the enzymes (as we have seen before). In some enzymes, the two sites are merged at the domain interface. The active site Ser 62 becomes phosphorylated and then transfers its phosphate to a new site in the substrate, which is oriented differently in the enzyme.

Figure \(\PageIndex{42}\) shows an interactive iCn3D model of the Geobacillus stearothermophilus cofactor-independent Phosphoglycerate Mutase (iPGM) with bound 2-phosphoglycerate (product) (1O98)

Figure \(\PageIndex{42}\): Cofactor-Independent Phosphoglycerate Mutase with bound 2-phosphoglycerate (product) (1O98). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...71iYHXAEKMGsEA

Reaction 9: 2PG ↔ PEP + H₂O ΔG^o = +0.4 kcal/mol (+1.7 kJ/mol)

This reaction, catalyzed by enolase, is shown in Figure \(\PageIndex{43}\)

Figure \(\PageIndex{43}\: Summary reaction - enolase

Now you can see the rationale for reaction 8. In a simple dehydration reaction, a molecule with high phosphoryl-transfer potential is produced, which, in the next and final step of glycolysis, yields ATP.

This enzyme has an active-site Mg²⁺ required for catalysis. Mammals have three forms of the enzyme: α-enolase (ENO-1) found in most tissues, β-enolase (ENO-3) found mostly in muscle, and γ-enolase (ENO-2) found in neurons. Figure \(\PageIndex{44}\) shows a possible mechanism for yeast enolase.

Figure \(\PageIndex{44}\): A possible mechanism for yeast enolase

Figure \(\PageIndex{45}\) shows an interactive iCn3D model of Yeast enolase with bound 2-phosphoglycerate (7ENL)

Figure \(\PageIndex{45}\): Yeast enolase with bound 2-phosphoglycerate (7ENL). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...TgwYV9vF8cYtPA

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Enolase might seem like a nondescript, boring enzyme to some.  Yet it and hexokinase have an unlikely role in the growth of tumor cells. Both hexokinase and enolase 1 (ENO1) are overexpressed in these cells, so inhibiting them could slow tumor cell growth and potentially kill them. We will see in several subsequent chapters that cancer cells engage in glycolysis even under aerobic conditions (called the Warburg effect).  An inhibitor was recently found that did not bind at the active (orthosteric) site but at an allosteric site.  Binding of the drug (SU212, a podophyllotoxin derivative) at an allosteric site did not affect the reaction kinetics but did lead to enzyme degradation.  Figure \(\PageIndex{45B}\) shows an  iCn3D model of the drug docked (using Neurosnap) to the allosteric site of human enolase 1 (2PSN).  

Figure \(\PageIndex{45B}\): Human enolase 1 (2PSN) with SU212 docked at an allosteric site.. (Copyright; author via source).

Two identical subunits of the enolase 1 tetramer are shown (magenta and blue).  SU212 is shown in spheres.  The active site of the blue subunit is shown in green sticks and the Mg²⁺ binding site in red sticks. Tailor D, Garcia-Marques F, Bermudez A et al., Non-orthosteric inhibition of enolase 1 impedes growth of triple-negative breast cancer.  Cell Reports Medicine, 2025; 6

To view the complex in iCn3D, download this PNG file of the protein complex. If the file opens in a browers window and not to your download folder, click on the image and follow prompts to save it.

1. Open iCn3D
2. File, Open File, iCn3D PNG appendible

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Reaction 10: PEP + ADP → Pyr + ATP ΔG^o = -7.5 kcal/mol (-31 kJ/mol)

This reaction, catalyzed by pyruvate kinase, is shown in Figure \(\PageIndex{46}\)

Figure \(\PageIndex{46}\): Summary reaction - pyruvate kinase

In this step, one more ATP is made for each PEP consumed (hence 2 ATPs for both 3C PEPs). The phosphoryl transfer potential for PEP is higher than for ATP, which allows this reaction to proceed with a large negative ΔG^o (-7.5 kcal/mol, -31 kJ/mol).

The mechanism for rabbit pyruvate kinase is shown in Figure \(\PageIndex{47}\).

Figure \(\PageIndex{47}\): Mechanism for rabbit pyruvate kinase

Figure \(\PageIndex{48}\) shows an interactive iCn3D model of rabbit muscle pyruvate kinase complexed with Mn²⁺, K⁺, and pyruvate (1PKN)

Figure \(\PageIndex{48}\): Rabbit muscle pyruvate kinase complexed with Mn²⁺, K⁺, and pyruvate (1PKN). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...cZkm8DdEw2N3C6

We are done! Given that glycolysis is the central anaerobic energy-extracting pathway in all life, we examined each enzyme in detail.

This is the net reaction of the glycolytic pathway:

\[\ce{Glc + Pi + 2ADP + 2NAD^{+} -> 2 Pyr + 2 ATP + 2NADH + 2H^{+} + 2H2O} \nonumber \]

Uncoupling glycolytic oxidation and phosphorylation (ATP formation)

Since we are most interested in energy transduction, let's focus on just two key steps in glycolysis that directly lead to ATP synthesis. Only one oxidative step is found in this pathway: the oxidative phosphorylation of the 3C glycolytic intermediate glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate, a mixed anhydride (see link below for the mechanism). The oxidizing agent is NAD^+_, and the phosphorylating agent is NOT ATP but rather Pi. The enzyme is named glyceraldehyde-3-phosphate dehydrogenase. It contains an active site Cys, which helps explain how the enzyme can be inactivated by stoichiometric amounts of iodoacetamide. In the enzyme's active site, the general base abstracts a proton from Cys, which then attacks the carbonyl C of glyceraldehyde, forming a tetrahedral intermediate. Instead of the expected reaction (the protonation of the alkoxide in an overall nucleophilic addition reaction at the aldehyde), a hydride leaves from the former carbonyl C to NAD+ in an oxidation step. Notice this is a two-electron oxidation reaction similar to alcohol dehydrogenase. An acyl-thioester intermediate has formed, much like the acyl intermediate that formed in Ser proteases.

Next, inorganic phosphate (Pi) attacks the carbonyl C of the intermediate in a nucleophilic substitution reaction to form the mixed anhydride product, 1,3-bisphosphoglycerate. Although we have formed a mixed anhydride, we cleaved a sulfur ester, which is destabilized with respect to its hydrolysis products (since the reactant, the thioester, is not stabilized by resonance to the extent of regular esters owing to the poor donation of electrons from the larger S to the carbonyl-like C.) In the next step, catalyzed by the enzyme phosphoglycerate kinase, ADP acts as a nucleophile that attacks the mixed anhydride of the 1,3-bisphosphoglycerate to form ATP. Note that the enzyme is named for the reverse reaction. We have coupled the oxidation of an organic molecule (glyceraldehyde-3-phosphate) to the phosphorylation of ADP by forming a "high" energy mixed anhydride, 1,3-bisphosphoglycerate.

The linkage between the oxidation of glyceraldehyde-3-phosphate and the phosphorylation of ADP by 1,3-bisphosphoglycerate can be artificially uncoupled by adding arsenate, which has a similar structure to phosphate. The arsenate can form a mixed anhydride at C1 of glyceraldehyde-3-phosphate, but since the bridging O-As bond is longer and not as strong as in the mixed anhydride, it is easily hydrolyzed. This prevents the subsequent transfer of phosphate to ADP to form ATP.

Figure \(\PageIndex{49}\) summarizes oxidation and substrate-level phosphorylation in glyceraldehyde-3-phosphate dehydrogenase.

Figure \(\PageIndex{49}\): Oxidation and substrate-level phosphorylation in glyceraldehyde-3-phosphate dehydrogenase (after Voet and Voet)

Summary: Under anaerobic conditions, glucose (6Cs) is metabolized through glycolysis, which converts it into two pyruvate molecules (3Cs). Only one oxidation step occurs when glyceraldehyde 3-phosphate is oxidized to 1,3-bisphosphoglycerate. To regenerate NAD⁺ so glycolysis can continue, pyruvate is reduced to lactate, catalyzed by lactate dehydrogenase, as shown in Figure \(\PageIndex{50}\). These reactions occur in the cytoplasm of cells actively engaged in the anaerobic oxidation of glucose (muscle cells, for example, during sprints).

Figure \(\PageIndex{50}\): Conversion of pyruvate to lactate

We will explore the fate of pyruvate under anaerobic conditions more in the next chapter section.

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

This chapter introduces glycolysis — the universal ten-step anaerobic pathway that converts glucose to pyruvate — with emphasis on the chemical logic of each enzymatic step, the thermodynamic principles that govern pathway flux, and the mechanistic strategies employed by each enzyme.

The chapter opens with the thermodynamic framework necessary to understand metabolism. Because ΔG depends on both the standard free energy change and the actual concentrations of reactants and products, several glycolytic reactions that are unfavorable under standard conditions are driven forward in the cell either by coupling to highly exergonic reactions (such as ATP hydrolysis) or by the continuous removal of products via subsequent reactions. The overall pathway is divided into two phases: an energy-investing phase (steps 1–3) in which two ATP molecules are consumed to phosphorylate glucose and cleave it into two triose phosphates, and an energy-recovering phase (steps 4–10) in which four ATP molecules are generated, for a net yield of two ATP per glucose.

The first three reactions prepare glucose for cleavage. Hexokinase (step 1) phosphorylates glucose at C6 using a γ-phosphate from ATP, trapping glucose inside the cell; an induced-fit conformational change closes the active site around the substrates, preventing wasteful ATP hydrolysis by water. Phosphoglucose isomerase (step 2) converts glucose-6-phosphate to fructose-6-phosphate via a cis-enediol intermediate, repositioning the carbonyl to C2—an arrangement that facilitates the subsequent aldol cleavage. Phosphofructokinase-1 (step 3) phosphorylates C1 of fructose-6-phosphate in the pathway's committed and rate-limiting step; as an allosteric enzyme with T and R states analogous to hemoglobin, PFK1 is activated by ADP and inhibited by ATP, making it a sensitive sensor of cellular energy charge. Fructose-2,6-bisphosphate, produced by PFK2, is an additional allosteric activator in eukaryotes.

Aldolase (step 4) cleaves fructose-1,6-bisphosphate into dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) via a retroaldol mechanism. Class I aldolases (found in animals and plants) use a Schiff base intermediate with an active-site lysine, while Class II aldolases (found in bacteria and fungi) use a zinc ion to stabilize the enolate intermediate. Although thermodynamically unfavorable (ΔG° = +24 kJ/mol), the reaction is pulled forward by rapid consumption of G3P in subsequent steps. Triose phosphate isomerase (step 5) interconverts DHAP and G3P, ensuring that both three-carbon fragments enter the energy-recovery phase; the enzyme operates at near-diffusion-controlled rates.

The central oxidative event of glycolysis occurs at step 6, catalyzed by glyceraldehyde-3-phosphate dehydrogenase. A covalent thioester intermediate forms between the substrate and an active-site cysteine; hydride transfer to NAD⁺ oxidizes the substrate, and subsequent attack by inorganic phosphate yields 1,3-bisphosphoglycerate, a mixed anhydride with a phosphoryl transfer potential exceeding that of ATP. This coupling of oxidation to phosphorylation is the key energetic innovation of the pathway. Arsenate poisoning disrupts this coupling by substituting for phosphate, forming an unstable arsenate ester that spontaneously hydrolyzes, thereby preventing ATP synthesis. Phosphoglycerate kinase (step 7) then transfers the acyl phosphate of 1,3-BPG to ADP, regenerating the two ATP molecules invested in the first phase.

Steps 8 and 9 prepare the second substrate-level phosphorylation. Phosphoglycerate mutase relocates the phosphate from C3 to C2 through either a cofactor-dependent (histidine phosphoenzyme) or cofactor-independent (serine phosphoenzyme with metal ions) mechanism, depending on the organism. Enolase then catalyzes dehydration of 2-phosphoglycerate to produce phosphoenolpyruvate (PEP), a high-energy enol phosphate. Finally, pyruvate kinase (step 10) transfers the phosphate from PEP to ADP with a large negative ΔG°, generating the second net pair of ATPs and releasing pyruvate.

Under anaerobic conditions, pyruvate is reduced to lactate by lactate dehydrogenase, regenerating NAD⁺ from NADH and allowing glycolysis to continue. The net reaction of glycolysis — glucose + 2 Pi + 2 ADP + 2 NAD⁺ → 2 pyruvate + 2 ATP + 2 NADH + 2 H⁺ + 2 H₂O — reflects a modest but physiologically indispensable energy yield that sustains life under conditions where mitochondrial oxidative phosphorylation is unavailable or insufficient.