Tag Archives: carbohydrates


Glycolysis: contents in brief

What is glycolysis?

Glycolysis, from Greek word glykys, meaning “sweet”, and lysis, meaning “dissolution or breakdown”, can be defined as the sequence of enzymatic reactions that, in the cytosol, also in the absence of oxygen, leads to the conversion of one molecule of glucose, a six carbon sugar, to two molecules of pyruvate, a three carbon compound, with the concomitant production of two molecules of ATP, the universal energy currency in biological systems.

Fig. 1 – The Glycolytic Pathway

Glycolysis, which evolved before a substantial amount of oxygen had accumulated in the atmosphere, is the metabolic pathway with the largest flux of carbon in most living cells, and is present in almost all organisms.
This pathway, not requiring oxygen, played a crucial role in metabolic processes during the first 2 billion years of evolution of life, and probably represents the most ancient biological mechanism for extracting energy from organic molecules when oxygen availability is low. Moreover, it is a source of precursors for aerobic catabolism and for various biosynthetic processes.
Note: Glycolysis is also known as the Embden-Meyerhof pathway, named after Gustav Embden and Otto Meyerhof, the two researchers who elucidated the entire pathway in the muscle.

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Glycolysis: the first metabolic pathway to be elucidated

The development of biochemistry has gone hand in hand with the elucidation of glucose metabolism, especially glycolysis, the first metabolic pathway to have been elucidated.
Though the elucidation of this metabolic pathway was worked out in the ‘40 of the last century, the key discovery about glucose metabolism was made in 1897, quite by accident, following a problem arose a year earlier, when a German chemist, M. Hahn, in attempting to obtain and preserve cell-free protein extracts of yeast, encountered difficulties in its conservation. A colleague, Hans Buchner, remembering a method for preserving jams, suggested to add sucrose to the extract.

Fig. 2 -Eduard Buchner

Eduard Buchner, Hans’s brother, put the idea of Hans into practice, and observed that the solution produced bubbles. This prompted Eduard to conclude that a fermentation was occurring, a quite surprising discovery. Indeed fermentation, according to Pasteur’s assertion in 1860, was inextricably tied to living cells, whereas it was now demonstrated that it could also occur outside them. Briefly, these two researchers refuted the vitalist dogma and had a pivotal role in starting modern biochemistry.
Eduard Bucher was awarded the Nobel Prize for Medicine in 1907 for this research, and was the first of several researchers who won the award for their discoveries concerning the glycolytic pathway.
It was later demonstrated, working with muscle extracts, that many of the reactions of lactic fermentation  were the same of those of alcoholic fermentation , thus revealing the underlying unity in biochemistry.
As previously mentioned, glycolysis was then fully elucidated in the first half of the last century largely due to the work of researchers such as Gerty and Carl Cori, Carl Neuberg, Arthur Harden, William Young, Jacob Parnas, Otto Warburg, Hans von Euler-Chelpin, Gustav Embden and Otto Meyerhof. In particular, Warburg and von Euler-Chelpin elucidated the whole pathway in yeast, and Embden and Meyerhof in muscle in the 30’s.

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Why is glycolys so important?

Glycolysis is essential to most living cells both from the energy point of view and as a source of precursors for many other metabolic pathways. And the rate of carbon flow through glycolysis, namely, the amount of glucose converted to pyruvate per unit time, is regulated to meet these two basic needs for the cell.
From the energetic point of view, although glycolysis is a relatively inefficient pathway, it can occur in the absence of oxygen, the condition in which life evolved on Earth and that many contemporary cells, both eukaryotic and prokaryotic, experience. Here are some examples.

  • In most animals, muscles exhibit an activity-dependent anaerobiosis, namely, they can work anaerobically for short periods. For example, when animals, but also athletes, perform high intense exercises, their need for ATP exceeds body’s ability to supply oxygen to the muscle. In such situation, muscles function, albeit for a short period of time, anaerobically.
  • Another example is the cornea of the eye, a poorly vascularized tissue.
  • Many microorganisms live in environments where oxygen is low or absent, such as deep water, soil, but also skin pores. And a variety of microorganisms called obligate anaerobes cannot survive in the presence of oxygen, a highly reactive molecule. Examples are Clostridium perfringens, Clostridium tetani, and Clostridium botulinum, that cause gangrene, tetanus and botulism, respectively.

It should also be underlined that glycolysis also plays a key role in those cells and tissues in which glucose is the sole source of energy, such as:

  • red blood cells, lacking mitochondria,
  • sperm cells;
  • the brain, which can also use ketone bodies for fuel in times of low glucose;
  • the adrenal medulla.

A similar situation is also found in the plant world where many aquatic plants and some plant tissues specialized in starch accumulation, such as potato tubers, use glucose as the main source of energy.

Note: There are organisms that are facultative anaerobes, namely organisms that can survive in the presence and in the absence of oxygen, acting aerobically or anaerobically, respectively. Examples are animals belonging to the genus Mytilus, which display an habitat-dependent anaerobiosis, a condition similar to the activity-dependent anaerobiosis seen in muscle.

Finally, it should not be forgotten that under aerobic conditions, in cells with mitochondria, glycolysis constitutes the upper part of the metabolic pathway leading to the complete oxidation of glucose to carbon dioxide (CO2) and water for energy purposes.

Fig. 3 – Glycolysis: Source of Building Blocks for Biosynthesis

Some glycolytic intermediates, for example glucose 6-phosphate (G-6-P), fructose 6-phosphate (F-6-P) or dihydroxyacetone phosphate (DHAP), may be used as building blocks in several metabolic pathways, such as those leading to the synthesis of glycogen, fatty acids,  triglycerides, nucleotides, of some amino acids, or 2,3-bisphosphoglycerate (2,3-BPG).

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The steps of glycolysis

The 10 steps that make up glycolysis can be divided into two phases.
The first, called the preparatory phase, consists of 5 steps and starts with the conversion of glucose to fructose 1,6-bisphosphate (F-1,6-BP) through three enzymatic reactions, namely, a phosphorylation at C-1, an isomerization, and a second phosphorylation, this time at C-6, with consumption of 2 ATP. Fructose 1,6-bisphosphate is then cleaved into two phosphorylated three-carbon compounds, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate.  Finally, the isomerization of DHAP to a second molecule of glyceraldehyde-3- phosphate occurs. In the preparatory phase therefore a glucose is split into two molecules of glyceraldehyde 3-phosphate, and two ATP are consumed.
In the second phase, called the payoff phase, consisting of the remaining 5 steps of the pathway, the two molecules of glyceraldehyde 3-phosphate are converted into two molecules of pyruvate, with the concomitant production of 4 ATP. So, in this phase, part of the energy present in the chemical bonds of glucose is extracted and conserved in the form of ATP. Furthermore, reducing equivalents are extracted and conserved in the form of the reduced coenzyme NADH. The metabolic fate of NADH will depend on the cell type and aerobic or anaerobic conditions.

Note: Glucose metabolized in the glycolytic pathway derives both from glucose that enters the cell through specific membrane transporters, that in turn derives from the bloodstream, and glucose 6-phosphate produced by glycogen degradation.

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Reaction 1: glucose phosphorylation to glucose 6-phosphate

In the first step of the glycolytic pathway glucose is phosphorylated to glucose 6-phosphate at the expense of one ATP.

Glucose + ATP → Glucose 6-phosphate + ADP + H+

In most cells this reaction is catalyzed by hexokinase (EC, enzyme present in the cells of all organisms, and in humans with four isozyme).
Hexokinase and pyruvate kinase, the other kinase of the glycolysis, like many other kinases, require the presence of magnesium ion, Mg2+, or of another bivalent metal ion such as manganese, Mn2+, for their activity. Mg2+ binds to the ATP to form the complex MgATP2-, and in fact the true substrate of the enzyme is not ATP but this complex. It should be emphasized that the nucleophilic attack by a hydroxyl group (-OH) of glucose at the terminal phosphorus atom of the ATP is facilitated by the action of Mg2+ that interacts with the negative charges of the phosphoryl groups of the nucleoside triphosphate.
The formation of the phosphoester bond between a phosphoryl group and the hydroxyl group at C-6 of glucose is thermodynamically unfavorable and requires energy to proceed, energy that is provided by the ATP. Indeed, while the phosphorylation of glucose at C-6 by inorganic phosphate has a ΔG°’ of 13.8 kJ/mol (3.3 kcal/mol), namely, it is an endoergonic reaction, the hydrolysis of ATP to ADP and Pi has ΔG°’ of -30.5 kJ/mol (-7.3 kcal/mol), namely, it is an esoergonic reaction. The net reaction has a ΔG°’ equal to (-30.5 + 13.8) = -16.7 kJ/mol (-7.3 + 3.3 = -4.0 kcal/mol). Under cellular conditions the reaction is even more favorable, with a ΔG equal to -33.5 kJ/mol (-8.0 kcal/mol).
Therefore, this is an essentially irreversible reaction.

Note: In biochemistry, phosphorylations are fundamental reactions catalyzed by enzymes called kinases, a subclass of transferases. Kinases catalyze the transfer of the terminal phosphoryl group, or γ-phosphoryl group, of a nucleoside triphosphate to an acceptor nucleophile to form a phosphoester bond. Specifically, hexokinase catalyzes the transfer of the γ-phosphoryl group of ATP to a variety of hexoses, that is, sugars with six carbons, such as fructose and mannose), in addition to glucose.

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The importance of glucose phosphorylation

The phosphorylation of the glucose has some functions.

  • Glucose 6-phosphate, due to its negative charge and because there are no transporters for phosphorylated sugars in the plasma membrane, cannot diffuse out of the cell. Thus, after the initial phosphorylation, no further energy is needed to keep the phosphorylated molecule within the cell, despite the large difference between its intra- and extracellular concentrations.
    Similar considerations are valid for each of the eight phosphorylated intermediates between glucose 6-phosphate and pyruvate.
  • The rapid phosphorylation of glucose maintains a low intracellular concentration of the hexose, thus favoring its facilitated diffusion into the cell.
  • Phosphorylation causes an increase in the energy content of the molecule, that is, it starts to destabilize it, thus facilitating its further metabolism.

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Other possible fates of glucose 6-phosphate

Glucose 6-phosphate is a key metabolite of glucose metabolism. In fact, in addition to be metabolized in the glycolytic pathway, in anabolic conditions it can have other fates (see Fig. 3). Here are some examples.

  • It can be used in the synthesis of:

glycogen, a polysaccharide stored mainly in the liver and muscle;
complex polysaccharides present in the extracellular matrix;
glucosamine and other sugars used for protein glycosylation.

  • It can be metabolized by the pentose phosphate pathway, that provides cells with:

NADPH, needed for reductive biosynthesis, such as fatty acid, cholesterol, steroid hormone, and deoxyribonucleotide biosynthesis, and for preventing oxidative damage in cells such as erythrocytes;
ribose 5-phosphate, used in nucleotide synthesis but also in NADH, FADH2 and coenzyme A synthesis.

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Reaction 2: isomerization of glucose 6-phosphate to fructose 6-phosphate

In the second step of the glycolytic pathway, the isomerization of glucose 6-phosphate, an aldose, to fructose 6-phosphate, a ketose, occurs. This reaction is catalyzed by phosphoglucose isomerase, also known as phosphohexose isomerase or glucose phosphate isomerase (EC

Glucose 6-phosphate ⇄ Fructose 6-phosphate

Like hexokinase, phosphoglucose isomerase requires the presence of Mg2+.
The ΔG°’ of the reaction is 1.7 kJ/mol (0.4 kcal/mol), while the ΔG is -2.5 kJ/mol (-0.6 kcal/mol). These small values indicate that the reaction is close to equilibrium and is easily reversible.
The reaction essentially consists in the shift of the carbonyl group at C-1 of the open-chain form of glucose 6-phosphate to C-2 of the open-chain form of fructose 6-phosphate.

Fig. 4 – Phosphoglucose Isomerase Reaction

The enzymatic reaction can be divided at least into three steps. Since in aqueous solution both hexoses are primarily present in the cyclic form, the enzyme must first open the ring of G-6P, catalyze the isomerization, and, finally, the formation of the five-membered ring of F-6-P.
This isomerization is a critical step for glycolytic pathway, as it prepares the molecule for the subsequent two steps.

  • The phosphorylation that occurs in the third step requires the presence of an alcohol group at C-1, and not of a carbonyl group.
  • In the fourth step, the covalent bond between C-3 and C-4 is cleaved, and this reaction is facilitated by the presence of the carbonyl group at C-2.

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Reaction 3: phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate

In the third step of the glycolytic pathway, a second phosphorylation reaction occurs. Phosphofructokinase 1 or PFK-1 (EC catalyzes the phosphorylation of fructose 6-phosphate at C-1 to form fructose 1,6-bisphosphate, at the expense of one ATP.

Fructose 6-phosphate + ATP → Fructose 1,6-bisphosphate + ADP + H+

PFK-1 is so named to distinguish it from phosphofructokinase 2 or PFK-2 (EC, the enzyme that catalyzes the phosphorylation of fructose 6-phosphate to fructose 2,6-bisphosphate.
Like the reaction catalyzed by hexokinase/glucokinase, this phosphorylation, too, is an essentially irreversible step, irreversibility, once again, achieved by coupling, by phosphofructokinase 1, with the hydrolysis of ATP. In fact, phosphorylation of fructose 6-phosphate by inorganic phosphate is endergonic, with a ΔG°’ of 16.3 kJ/mol (3.9 kcal/mol), whereas, when the reaction is coupled to the hydrolysis of ATP, the overall equation becomes exergonic, with a ΔG°’ of -14.2 kJ/mol (-3.4 kcal/mol) and a ΔG of -22.2 kJ/mol (-5.3 kcal/mol).
While hexokinase allows to trap glucose inside the cell, phosphofructokinase 1 prevents glucose to be used for glycogen synthesis or the production of other sugars, but is instead metabolized in the glycolytic pathway. In fact, unlike glucose 6-phosphate, fructose 1,6-bisphosphate cannot be used directly in other metabolic pathways than glycolysis/gluconeogenesis, that is, phosphofructokinase 1 catalyzes the first “committed” step of the glycolytic pathway. Such reactions are usually catalyzed by enzymes regulated allosterically, that prevent the accumulation of both intermediates and final products. PFK-1 is no exception, being subject to allosteric regulation by positive and negative effectors that signal the energy level and the hormonal status of the organism.
Some protists and bacteria, and perhaps all plants, have a phosphofructokinase that uses pyrophosphate (PPi) as a donor of the phosphoryl group in the synthesis of F-1,6-BP. This reaction has a ΔG°’ of -2.9 kJ/mol (-12.1 kcal/mol).

Fructose 6-phosphate + PPi → Fructose 1,6-bisphosphate + Pi

Note: The prefix bis– in bisphosphate, as fructose 1,6-bisphosphate, indicates that there are two phosphoryl groups are bonded to different atoms.
The prefix di– in diphosphate, as in adenosine diphosphate, indicates that there are two phosphoryl groups connected by an anhydride bond to form a pyrophosphoryl group, namely, they are directly bonded to one another.
Similar rules also apply to the nomenclature of molecules that have three phosphoryl groups standing apart, such as inositol 1,4,5-trisphosphate, or connected by anhydride bonds, such as ATP or guanosine triphosphate or GTP.

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Reaction 4: cleavage of fructose 1,6-bisphosphate into two three-carbon fragments

In the fourth step of the glycolytic pathway, fructose 1,6-bisphosphate aldolase, often called simply aldolase (EC, catalyzes the reversible cleavage of fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate, an aldose, and dihydroxyacetone phosphate, a ketose. The enzyme cleaves the bond between C-3 and C-4.

Fructose 1,6-bisphosphate ⇄ Dihydroxyacetone phosphate + Glyceraldehyde 3-phosphate

All glycolytic intermediates downstream to this reaction are three-carbon molecules, instead of six-carbon molecules as the previous ones.
The ΔG°’ of the reaction in the direction of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate production is of 23.8 kJ/mol (5.7 kcal/mol), and the Km is approximately 10-4 M, values that would indicate that the reaction does not proceed as written from left to right. However, under normal cellular conditions, due to the lower concentrations of the reactants, the ΔG is -1.3 kJ/mol (-0.3 kcal/mol), a very small value, thus the reaction is easily reversible, that is, essentially to equilibrium.

Note: The name “aldolase” derives from the nature of the reverse reaction, from right to left as written, that is, an aldol condensation.

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Reaction 5: interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate

Of the two products of the previous reaction, glyceraldehyde 3-phosphate goes directly into the second phase of the glycolytic pathway. Conversely, DHAP is not on the direct pathway of glycolysis and must be converted, isomerized, to glyceraldehyde 3-phosphate to continue through the pathway. This isomerization is catalyzed by triose phosphate isomerase (EC

Dihydroxyacetone phosphate ⇄ Glyceraldehyde 3-phosphate

Triose phosphate isomerase, in converting dihydroxyacetone phosphate into glyceraldehyde 3-phosphate, catalyzes the transfer of a hydrogen atom from C-1 to C-2, that is, catalyzes an intramolecular oxidation-reduction. And in essence, after the enzyme reaction, the carbons C-1, C-2 and C-3 of the starting glucose to become equivalent,  chemically indistinguishable, from the carbons C-6, C-5 and C-4, respectively.
Therefore, the net result of the the last two steps of glycolysis is the production of two molecules of glyceraldehyde 3-phosphate.
The ΔG°’ of the reaction is of 7.5 kJ/mol (1.8 kcal/mol), while the ΔG is 2.5 kJ/mol (0.6 kcal/mol). Although at equilibrium dihydroxyacetone phosphate represent about 96% of the trioso phosphates, the reaction proceeds readily towards the formation of glyceraldehyde 3-phosphate because of the subsequent step of the glycolytic pathway that removes the glyceraldehyde 3-phosphate produced.
One of the distinguishing features of triose phosphate isomerase is the great catalytic efficiency. The enzyme is in fact considered kinetically perfect. Why?  The enzyme enhances the isomerization rate by a factor of 1010 compared with that obtained with a catalyst such as acetate ion. Indeed, the Kcat/KM ratio for the isomerization of glyceraldehyde 3-phosphate is equal to 2×108 M-1s-1, value close to the diffusion-controlled limit. Thus, the rate-limiting step in the reaction catalyzed by triose phosphate isomerase is diffusion-controlled encounter of enzyme and substrate.
From the energetic point of view, the last two steps of glycolysis are unfavorable, with ΔG°’ of 31.3 kJ/mol  (7.5 kcal/mol), whereas the net ΔG°’ of the first five reactions is of 2.1 kJ/mol (0.5 kcal/mol), with a Keq of about 0.43. And it is the free energy derived from the hydrolysis two ATP that, under standard-state conditions, makes the value of the overall equilibrium constant close to one. If instead we consider ΔG, it is quite negative, -56.8 kJ/mol (-13.6 kcal/mol).

Notice that dihydroxyacetone phosphate may also be reduced to glycerol 3-phosphate (see Fig. 3) in the reaction catalyzed by cytosolic glycerol 3-phosphate dehydrogenase (EC

Dihydroxyacetone phosphate + NADH + H+ ⇄ Glycerol 3-phosphate + NAD+

The enzyme acts as a bridge between glucose and lipid metabolism because the glycerol 3-phosphate produced is used in the synthesis of lipids such as triacylglycerols.
This reaction is an important sources of glycerol 3-phosphate in adipose tissue and small intestine.

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Reaction 6: oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate

In the sixth step of the glycolytic pathway, the first step of the second phase, the payoff phase, glyceraldehyde 3-phosphate dehydrogenase (EC catalyses the oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate (1,3-BPG), with the concomitant reduction of NAD+ to NADH.

Glyceraldehyde 3-phosphate + NAD+ + Pi ⇄ 1,3-Bisphosphoglycerate + NADH + H+

This is the first of the two glycolytic reactions in which the chemical energy needed for the subsequent synthesis of ATP is harvested and made available; the other reaction is catalyzed by enolase (EC Why?
This reaction is the sum of two processes.

  • In the first reaction, the oxidation of the aldehyde group to a carboxyl group occurs, step in which NAD+ is used as oxidizing agent. The reaction is quite exergonic, with a ∆G’° of -43 kJ/mol (-10.3 kcal/mol).
  • In the second reaction, the formation of the bond between the carboxylic group at C-1 of 1,3-bisphosphoglycerate and orthophosphate occurs, to form an anhydride called acyl phosphate. The reaction is quite endergonic, with a ∆G’° of 49.3 kJ/mol (11.8 kcal/mol).

These two chemical processes must not take place in succession but must be coupled in order to allow the formation of the acyl phosphate because the oxidation of the aldehyde group is used to drive the formation of the anhydride, with an overall ΔG°’ of 6.3 kJ/mol (1.5 kcal/mol), and a ΔG of 2.5 kJ/mol (0.6 kcal/mol), both slightly endoergonic.
Therefore, the free energy that might be released as heat is instead conserved by the formation of the acyl phosphate.

Note: The reversible reduction of the nicotinamide ring of NAD+ or NADP+ is due to the loss of two hydrogen atoms by another molecule, in this case the aldehyde group of glyceraldehyde 3-phosphate, that undergoes oxidation, and to the subsequent transfer of a hydride ion, the equivalent of two electrons and a proton, to the nicotinamide ring. The other proton removed from the substrate is released to the aqueous solution. Below, the half reactions for both coenzymes.

NAD+ + 2 e + 2 H+ → NADH + H+

NADP+ + 2 e + 2 H+ → NADH + H+

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Reaction 7: phosphoglycerate kinase and the first ATP forming reaction

In the seventh step of the glycolytic pathway, phosphoglycerate kinase (EC catalyzes the transfer of the high-energy phosphoryl group from the acyl phosphate of 1,3-BPG to ADP to form ATP and 3-phosphoglycerate (3-PG).

1,3-Bisphosphoglycerate + ADP + H+ ⇄ 3-Phosphoglycerate + ATP

The ΔG°’ of the reaction is of -18.5 kJ/mol (-4.4 kcal/mol), namely, it is an exergonic reaction. The ΔG is 1.3 kJ/mol (0.3 kcal/mol).
The high phosphoryl-transfer potential of the acyl phosphate is used to phosphorylate ADP. The production of ATP in this manner  is called substrate-level phosphorylation. In other words, part of the energy released during the oxidation of the aldehyde group in the sixth step is now conserved by the synthesis of ATP from the ADP and Pi.
The reaction catalyzed by phosphoglycerate kinase is the first reaction of glycolysis in which part of the chemical energy present in glucose molecule is conserved as ATP. And, because the reactions catalyzed by aldolase and triose phosphate isomerase, step 4 and 5, respectively, lead to the formation of two molecules of glyceraldehyde 3-phosphate per molecule of glucose, in this step two ATP are produced and the ATP debt created by  the preparatory phase, steps 1 and 3, respectively, is “paid off”.
It should be noted that the enzyme is named for the reverse reaction, from right to left as written, that is, the phosphorylation of 3-phosphoglycerate to form 1,3-bisphosphoglycerate at the expense of one ATP.
Indeed, this enzyme, like all other enzymes, is able to catalyze the reaction in both directions. And the direction leading to the synthesis of 1,3-bisphosphoglycerate occurs during the photosynthetic CO2 fixation and gluconeogenesis.

The sixth and seventh reactions of glycolysis, are, as a whole, an energy-coupling process in which the common intermediate is 1,3-bisphosphoglycerate. While the reaction leading to the synthesis of 1,3-BPG is endergonic, with a ΔG°’ of 6.3 kJ/mol (1.5 kcal/mol), the second reaction is strongly exergonic, with a ΔG°’ of -18.5 kJ/mol (-4,4 kcal/mol). The overall ΔG°’ is -12.2 kJ/mol (-2.9 kcal/mol), namely, the reaction catalyzed by phosphoglycerate kinase is sufficiently exergonic to pull even the previous one, too, making the overall reaction exergonic.

Glyceraldehyde 3-phosphate + ADP + Pi + NAD+ ⇄ 3-Phosphoglycerate + ATP + NADH + H+

In reality, phosphoglycerate kinase reaction is sufficiently exergonic to pull also the reactions catalyzed by aldolase and triose phosphate isomerase.

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What is substrate-level phosphorylation?

Substrate-level phosphorylation is defined as the production of ATP by the transfer of a phosphoryl group from a substrate to ADP, a process involving chemical intermediates and soluble enzymes.
There is also a second type of phosphorylation for the synthesis of ATP called oxidative phosphorylation, a process involving not chemical intermediates and soluble enzymes but transmembrane proton gradients and membrane-bound enzymes.

Because the standard free energy of hydrolysis of the phosphoryl group of 3-phosphoglycerate is equal to 12.5 kJ/mol (-3 kcal/mol), it is not sufficient to produce ATP by phosphoryl group transfer. In the two subsequent reactions of glycolysis, 3-phosphoglycerate is converted to phosphoenolpyruvate (PEP), a molecule with a phosphoryl group transfer potential sufficiently elevated to allow the synthesis of ATP.

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Reaction 8: from 3-phosphoglycerate to 2-phosphoglycerate

In the eighth step of the glycolytic pathway, 3-phosphoglycerate is converted into 2-phosphoglycerate (2-PG), in a reversible reaction catalyzed by phosphoglycerate mutase (EC The reaction requires Mg2+, and has a very small ΔG, equal to about 0.8 kJ/mol (0.2 kcal/mol) and a ΔG°’ of 4.4 kJ/mol (1.1 kcal/mol).
Phosphoglycerate mutase is a mutase, enzymes that catalyze intramolecular group transfers, in this case the transfer of a phosphoryl group from C-3 to C-2 of the 3-phosphoglycerate. Mutases, in turn, are a subclass of isomerases.
The mechanism by which this reaction takes place depends on the type of organism studied. For example, in yeast or in rabbit muscle the reaction occurs in two steps and involves the formation of phosphoenzyme intermediates. In the first step, a phosphoryl group bound to a histidine residue in the active site of the enzyme is transferred to the hydroxyl group at C-2 of 3-PG to form 2,3-bisphosphoglycerate. In the next step, the enzyme acts as a phosphatase converting 2,3-BPG into 2-phosphoglycerate; however, the phosphoryl group at C-3 is not released but linked to the histidine residue of the active site to regenerate the intermediate enzyme-His-phosphate. Schematically:

Enzyme-His-phosphate + 3-Phosphoglycerate ⇄ Enzyme-His + 2,3-Phosphoglycerate

Enzyme-His + 2,3-Bisphosphoglycerate ⇄ Enzyme-His-phosphate + 2-Phosphoglycerate

Notice that the phosphoryl group of 2-phosphoglycerate is not the same as that of the substrate 3-phosphoglycerate.
Approximately once in every 100 catalytic cycles, 2,3-BPG dissociates from the active site of the enzyme, leaving it unphosphorylated, that is, in the inactive form. The inactive enzyme may be reactivated by binding 2,3-bisphosphoglycerate, which must, therefore, be present in the cytosol to ensure the maximal activity of the enzyme. And 2,3-BPG is present in small, but sufficient amounts in most cells, except for red blood cells, where it acts as an allosteric inhibitor, too, reducing  the affinity of hemoglobin for oxygen, and has a concentration of 4-5 mM.

Note: 3-Phosphoglycerate can also be used for the biosynthesis of serine, from which glycine and cysteine derive (see Fig. 3). The biosynthesis of serine begins with the reaction catalyzed by phosphoglycerate dehydrogenase (EC The enzyme catalyzes the oxidation of 3-phosphoglycerate to 3-phosphohydroxypyruvate and the concomitant reduction of NAD+ to NADH. This reaction is also the rate-limiting step of this biosynthetic pathway, because serine inhibits the activity of the enzyme.

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Synthesis of 2,3-bisphosphoglycerate and the Rapoport-Luebering pathway

1,3-Bisphosphoglycerate can be also converted into 2,3-bisphosphoglycerate (see Fig. 3).
In red blood cells this reaction is catalyzed by the bisphosphoglycerate mutase, one of the three isoforms of phosphoglycerate mutase found in mammals. The enzyme requires the presence of 3-phosphoglycerate as it catalyzes the intermolecular transfer of a phosphoryl group from C-1 of 1,3-bisphosphoglycerate to the C-2 of 3-phosphoglycerate. Therefore, 3-phosphoglycerate becomes 2,3-BPG, while 1,3-BPG is converted into 3-phosphoglycerate. The mutase enzyme activity has EC number

Fig. 5 – Synthesis of 2,3-Bisphosphoglycerate

2,3-Bisphosphoglycerate can then be hydrolyzed to 3-phosphoglycerate in the reaction catalyzed by the phosphatase activity of bisphosphoglycerate mutase, that removes the phosphoryl group at C-2. This activity has EC number The enzyme is also able to catalyze the interconversion of 2-phosphoglycerate and 3-phosphoglycerate, therefore, it is a trifunctional enzyme. 3-Phosphoglycerate can then re-enter the glycolytic pathway. This detour from glycolysis, also called Rapoport-Luebering pathway, that leads to the synthesis of 3-phosphoglycerate without any ATP production.
The other two isoforms of phosphoglycerate mutase, phosphoglycerate mutase 1 or type M, present in the muscle, and phosphoglycerate mutase 2 or type B, present in all other tissues, are able to catalyze, in addition to the interconversion of the 2-phosphoglycerate and 3-phosphoglycerate, the two steps of Rapoport-Luebering pathway, although with less efficacy than the glycolytic reaction. Therefore they are trifunctional enzymes.

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Reaction 9: formation of phosphoenolpyruvate

In the ninth step of the glycolytic pathway, 2-phosphoglycerate is dehydrated to form phosphoenolpyruvate, an enol, in a reversible reaction catalyzed by enolase.

2-Phosphoglycerate ⇄ Phosphoenolpyruvate + H2O

The reaction requires Mg2+ that stabilizes the enolic intermediate that is formed during the process.
The ΔG°’ of the reaction is 7.5 kJ/mol (1.8 kcal/mol), while ΔG -3.3 kJ/mol (-0.8 kcal/mol).
Like 1,3-BPG, phosphoenolpyruvate has a phosphoryl group transfer potential high enough to allow ATP formation. Why does this phosphoryl group have a high free energy of hydrolysis?
Although phosphoenolpyruvate and 2-phosphoglycerate contain nearly the same amount of metabolic energy with respect to decomposition to CO2, H20 and Pi, 2-PG dehydration leads to a redistribution of energy such that the standard free energy of hydrolysis of the phosphoryl groups vary as described below:

  • -17.6 kJ/mol (-4.2 kcal/mol) for 2-phosphoglycerate, a phosphoric ester;
  • -61.9 kJ/mol (-14.8 kcal/mol) for phosphoenolpyruvate, an enol phosphate.

What happens is that the phosphoryl group traps PEP in its unstable enol form. When, in the last step of glycolysis, phosphoenolpyruvate donates the phosphoryl group to ADP, ATP and the enol form of pyruvate are formed. The enol form of pyruvate is unstable and tautomerizes rapidly and nonenzymatically to the more stable keto form, that predominates at pH 7. So, the high phosphoryl-transfer potential of PEP is due to the subsequent enol-keto tautomerization of pyruvate.

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Reaction 10: the transfer of the phosphoryl group from the phosphoenolpyruvate to the ADP

In the final step of the glycolytic pathway, pyruvate kinase (EC catalyzes the transfer of the phosphoryl group from phosphoenolpyruvate to ADP to form pyruvate and ATP. This is the second substrate-level phosphorylation of glycolysis.

Phosphoenolpyruvate + ADP + H+ → Pyruvate + ATP

The enzyme is a tetramer and, like PFK-1, is a highly regulated. Indeed, it has binding sites for numerous allosteric effectors. Moreover, in vertebrates, there are at least three isozymes of pyruvate kinase, of which the M type predominates in muscle and brain, while the L type in liver. These isozymes have many properties in common, whereas differ in the response to hormones such as glucagon, epinephrine and insulin.
The enzyme activity is stimulated by potassium ion (K+)  and some other monovalent cations.
The reaction is essentially irreversible, with a ΔG°’ of -31.4 kJ/mol (-7.5 kcal/mol), and a ΔG of -16.7 kJ/mol (-4.0 kcal/mol), largely due, as anticipated in the previous paragraph, to the tautomerization of the pyruvate from the enol form to the more stable keto form.

Fig. 6 – Spontaneous Tautomerization of Pyruvate

And, of the -61.9 kJ/mol (14.8 kcal/mol) released from the hydrolysis of the phosphoryl group of PEP, nearly half is conserved in the formation of the phosphoanhydride bond between ADP and Pi, whose ΔG°’ is of -30.5 kJ/mol (-7.3 kcal/mol). The remaining energy, -31.4 kJ/mol (-7.5 kcal/mol), is the driving force that makes the reaction proceed towards ATP production.
While the reaction catalyzed by phosphoglycerate kinase, in the seventh step of the glycolytic pathway, pays off the ATP debt of the preparatory phase, the reaction catalyzed by pyruvate kinase allows a net gain of two ATP.

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The fate of NADH and pyruvate produced in glycolysis

Glycolysis produces  2 NADH, 2 ATP, and 2 pyruvate molecules per molecule of glucose.
NADH must be reoxidized to NAD+ to allow glycolysis to proceed. NAD+, a coenzyme that is produced from the vitamin B3, also known as niacin, is present in limited amounts in the cytosol, ≤ 10-5M, a value well below than that of glucose metabolized in a few minutes, and must be continuously regenerated. Therefore, the final step of the glycolytic pathway is the regeneration of NAD+ from NADH through aerobic or anaerobic pathways, each of which involves pyruvate. Such pathways allow, therefore, maintenance of the redox balance of the cell.

Fig. 7 – Possible Catabolic Fates of the Pyruvate Produced in Glycolysis

Pyruvate is a versatile metabolite that can enter several metabolic pathways, both anabolic and catabolic, depending on the type of cell, the energy state of the cell and the availability of oxygen. With the exception of some variations encountered in bacteria, exploited, for example, in food industry for the production of various foods such as many cheeses, there are essentially three pathways in which pyruvate may enter:

This allows glycolysis to proceed in both anaerobic and aerobic conditions.
It is therefore possible to state that the catabolic fate of the carbon skeleton of glucose is influenced by the cell type, the energetic state of the cell, and the availability of oxygen.

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Lactic acid fermentation

In animals, with few exceptions, and in many microorganisms when oxygen availability is insufficient to meet the energy requirements of the cell, or if the cell is without mitochondria, the pyruvate produced by glycolysis is reduced to lactate in the cytosol, in a reaction catalyzed by lactate dehydrogenase (EC

Pyruvate + NADH + H+ ⇄ Lactate + NAD+

In the reaction, pyruvate, by accepting electrons from NADH, is reduced to lactate, while NAD+ is regenerated. And the overall equilibrium of the reaction strongly favors the formation of lactate, as shown by the value of ΔG°’ of -25.1 kJ/mol (-6 kcal/mol).
The conversion of glucose to lactate is called lactic acid fermentation. The overall equation of the process is:

Glucose + 2 Pi + 2 ADP + 2H+ → 2 Lactate + 2 ATP + 2 H2O

Notice that fermentation, discovered by Louis Pasteur who defined it “la vie sans l’air”, is a metabolic pathway that:

  • extracts energy from glucose and stores it as ATP;
  • does not consume oxygen;
  • does not change the concentration of NAD+ or NADH.

With regard to coenzymes, neither NAD+ nor NADH appears in the overall equation, although both are crucial in the process, that is, no net oxidation-reduction occurs. In other words, in the conversion of glucose, C6H12O6, to lactate, C3H6O3, the ratio of hydrogen to carbon atoms of the reactants and products does not change.
From an energy point of view, it should however be emphasized that fermentation extracts only a small amount of the chemical energy of glucose.

In humans, much of the lactate produced enters the Cori cycle for glucose production via gluconeogenesis. We can also state that lactate production shifts part of the metabolic load from the extrahepatic tissues, such as skeletal muscle during intense bouts of exercise, like a 200-meter, when the rate of glycolysis can almost instantly increase 2,000-fold, to the liver.
In contrast to skeletal muscle that releases lactate into the venous blood, the heart muscle is able to take up and use it for fuel, due to its completely aerobic metabolism and to the properties of the heart isozyme of lactate dehydrogenase, referred to as H4. Therefore, portion of the lactate released by skeletal muscle engaged in intense exercise is used by the heart muscle for fuel.

Note: Lactate produced by microorganisms during lactic acid fermentation is responsible for both the scent and taste of sauerkraut, namely, fermented cabbage, as well as for the taste of soured milk.

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Alcoholic fermentation

In microorganisms such as brewer’s and baker’s yeast, in certain plant tissues, and in some invertebrates and protists, pyruvate, under hypoxic or anaerobic conditions, may be reduced in two steps to ethyl alcohol or ethanol, with release of CO2.
The first step involves the non-oxidative decarboxylation of pyruvate to form acetaldehyde, an essentially irreversible reaction. The reaction is catalyzed by pyruvate decarboxylase (EC, an enzyme that requires Mg2+ and thiamine pyrophosphate, a coenzyme derived from vitamin thiamine or vitamin B1. The enzyme is absent in vertebrates and in other organisms that perform lactic acid fermentation.
In the second step, acetaldehyde is reduced to ethanol in a reaction catalyzed by alcohol dehydrogenase (EC, an enzyme that contains a bound zinc atom in its active site. In the reaction, NADH supplies the reducing equivalents and is oxidized to NAD+. At neutral pH, the equilibrium of the reaction lies strongly toward ethyl alcohol formation.
The conversion of glucose to ethanol and CO2 is called alcoholic fermentation. The overall reaction is:

Glucose + 2 Pi + 2 ADP + 2 H+ → 2 Ethanol + 2 CO2 + 2 ATP + 2 H2O

And, as for lactic fermentation, even in alcoholic fermentation no net oxidation-reduction occurs.

Alcoholic fermentation is the basis of the production of beer and wine. Notice that the CO2 produced by brewer’s yeast is responsible for the characteristics “bubbles” in beer, champagne and sparkling wine, while that produced by baker’s yeast causes dough to rise.

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Fate of pyruvate and NADH under aerobic conditions

In cells with mitochondria and under aerobic conditions, the most common situation in multicellular and many unicellular organisms, the oxidation of NADH and pyruvate catabolism follow distinct pathways.
In the mitochondrial matrix, pyruvate is first converted to acetyl-CoA in a reaction catalyzed by the pyruvate dehydrogenase complex. In the reaction, a oxidative decarboxylation, pyruvate loses a carbon atom as CO2, and the remaining two carbon unit is bound to Coenzyme A to form acetyl-coenzyme A or acetyl-CoA.

Pyruvate + NAD+ + CoA → acetyl-CoA + CO2 + NADH + H+

The acetyl group of acetyl-CoA is then completely oxidized to CO2 in the citric acid cycle, with production of NADH and FADH2. Pyruvate dehydrogenase therefore represents a bridge between glycolysis, which occurs in the cytosol, and the citric acid cycle, which occurs in the mitochondrial matrix.
In turn, electrons derived from oxidations that occur during glycolysis are transported into mitochondria via the reduction of cytosolic intermediates. In this way, in the cytosol NADH is oxidized to NAD+, while the reduced intermediate, once in the mitochondrial matrix, is reoxidized through the transfer of its reducing equivalents to Complex I of the mitochondrial electron transport chain. Here the electrons flow to oxygen to form H2O, a transfer that supplies the energy needed for the synthesis of ATP through the process of oxidative phosphorylation. Of course, also the electrons carried by NADH formed by pyruvate dehydrogenase and citric acid cycle and by FADH2 formed by citric acid cycle meet a similar fate.

Note: FADH2 transfers its reducing equivalents not to Complex I but to Complex II.

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Anabolic fates of pyruvate

Under anabolic conditions, the carbon skeleton of pyruvate may have fates other than complete oxidation to CO2 or conversion to lactate. In fact, after its conversion to acetyl-CoA, it may be used, for example, for the synthesis of fatty acids, or of the amino acid alanine (see Fig. 3).

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Glycolysis and ATP production

In the glycolytic pathway the glucose molecule is degraded to two molecules of pyruvate.
In the first phase, the preparatory phase, two ATP are consumed per molecule of glucose in the reactions catalyzed by hexokinase and PFK-1. In the second phase, the payoff phase, 4 ATP are produced through substrate-level phosphorylation in the reactions catalyzed by phosphoglycerate kinase and pyruvate kinase. So there is a net gain of two ATP per molecule of glucose used. In addition, in the reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase, two molecules of NADH are produced for each glucose molecule.

Fig. 8 – Free-Energy Changes of Glycolytic Reactions

The overall ΔG°’ of glycolysis is -85 kJ/mol (-20.3 kcal/mol), value resulting from the difference between the ΔG°’ of the conversion of glucose into two pyruvate molecules, -146 kJ/mol (-34,9 kcal/mol), and the ΔG°’ of the formation of ATP from ADP and Pi, 2 x 30.5 kJ/mol = 61 kJ / mol  (2  x 7.3 kcal/mol = 14.6 kcal/mol). Here are the two reactions.

Glucose + 2 NAD+ → 2 Pyruvate + 2 NADH + 2 H+

2 ADP + 2 Pi → 2 ATP + 2 H2O

The sum of the two reactions gives the overall equation of glycolysis.

Glucose + 2 NAD+ + 2 ADP + 2 Pi → 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H20

Thus, under standard conditions, the amount of released energy stored within ATP is (61/146) x 100 = 41.8%.
Notice that the overall equation of glycolysis can also be derived by considering all the reagents, ATP, NAD+, ADP, and Pi and all the products.

Glucose + 2 ATP + 2 NAD+ + 4 ADP + 2 Pi → 2 Pyruvate + 2 ADP + 2 NADH + 2 H+ + 4 ATP + 2 H20

Cancelling the common terms on both sides of the equation, we obtain the overall equation shown above.

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Glycolysis and ATP production under anaerobic conditions

Under anaerobic conditions, regardless of what is the metabolic fate of pyruvate, conversion to lactate, ethanol or other molecules, there is no additional production of ATP downstream of glycolysis.
Therefore under these conditions, glycolysis extracts only a small fraction of the chemical energy of the glucose molecule, energy equal to 2840 kJ/mol (679 kcal/mol) released as a result of its conversion to CO2 and H2O. Indeed, only 146 kJ/mol are released in the conversion of a glucose molecule to two pyruvate molecules, equal to 5%, [(146/2,840) x 100], of the available chemical energy. Therefore,  pyruvate still contains most of the chemical energy of the hexose.
Similarly, the 4 electrons carried by NADH produced in step 6 of glycolysis cannot be used for ATP production.
In lactic acid fermentation, the ΔG°’ associated with the conversion of a glucose molecule to two molecules of lactate is -183.6 kJ/mol  (-43.9 kcal/mol), and 33.2% of such free energy, [(61/183.6) x 100] is stored within ATP, whereas it is 41.8% in the conversion of a glucose molecule to two molecules of pyruvate.
It should be noted that under actual conditions the amount of free energy required for the synthesis of ATP from ADP and Pi is much higher than that required under standard conditions, namely, approximately 50%  of the energy released is stored within ATP.

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Glycolysis and ATP production under aerobic conditions

Under aerobic conditions, in cells with mitochondria, the amount of chemical energy that can be extracted from glucose and stored within ATP is much greater than under anaerobic conditions.
If we consider the two NADH produced during glycolysis, the flow of their 4 reducing equivalents along the mitochondrial electron transport chain allows the production of 2-3 ATP per electron pair through oxidative phosphorylation. Therefore, 6 to 8 ATP are produced when one molecule of glucose is converted into two molecules of pyruvate, 2 from glycolysis and 4-6 from oxidative phosphorylation.

Note: The amount of ATP produced from the reducing equivalents of NADH depends upon the mechanism  by which they are shuttled into mitochondria.

On the other hand, if we analyze the coordinated and consecutive action of glycolysis, pyruvate dehydrogenase, citric acid cycle,  mitochondrial electron transport chain and oxidative phosphorylation, much more energy can be extracted from glucose and stored within ATP. In this case, according to what reported by Lehninger, 30 to 32 ATP are produced for each glucose molecule, although recent estimates suggest a net production equal to 29.85 ATP/glucose, or 29.38 ATP/glucose if also ATP formed from GTP, in turn produced by the citric acid cycle, is exported. Considering both estimates, the production of ATP is about 15 times greater than under anaerobic condition.

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Feeder pathways for glycolysis

Other carbohydrates besides glucose, both simple and complex, can be catabolized via glycolysis, after enzymatic conversion to one of the glycolytic intermediates.  Among the most important are:

Fig. 9 – Feeder Pathways for Glycolysis

Dietary starch and disaccharides must be hydrolyzed in the intestine to the respective monosaccharides before being absorbed. Once in the venous circulation, monosaccharides reach the liver through the portal vein; this organ is the main site where they are metabolized.

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Glycogen and starch

Regarding the phosphorolytic breakdown of starch and endogenous glycogen refer to the corresponding articles.

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Under physiological conditions, the liver removes much of the ingested fructose from the bloodstream before it can reach extrahepatic tissues.
The hepatic pathway for the conversion of the monosaccharide to intermediates of glycolysis consists of several steps.
In the first step fructose is phosphorylated to fructose 1-phosphate at the expense of one ATP. This reaction is catalyzed by fructokinase (EC, and requires the presence of Mg2+.

Fructose + ATP → Fructose 1-phosphate + ADP + H+

As for glucose, fructose phosphorylation traps the molecule inside the cell.
In the second step fructose 1-phosphate aldolase catalyzes the hydrolysis, an aldol cleavage, of fructose 1-phosphate to dihydroxyacetone phosphate and glyceraldehyde.

Fructose 1-phosphate → Dihydroxyacetone Phosphate + Glyceraldehyde

Dihydroxyacetone phosphate is an intermediate of the glycolytic pathway and, after conversion to glyceraldehyde 3-phosphate, may flow through the pathway. Conversely, glyceraldehyde is not an intermediate of the glycolysis, and is phosphorylated to glyceraldehyde 3-phosphate at the expense of one ATP. The reaction is catalyzed by triose kinase (EC, and requires the presence of Mg2+.

Glyceraldehyde + ATP → Glyceraldehyde 3-phosphate + ADP + H+

In hepatocytes, therefore, a molecule of fructose is converted to two molecules of glyceraldehyde 3-phosphate, at the expense of two ATP, as for glucose.

Fructose + 2 ATP → 2 Glyceraldehyde 3-phosphate +2 ADP +2  H+

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Fructose and hexokinase

In extrahepatic sites, such as skeletal muscle, kidney or adipose tissue, fructokinase is not present, and fructose enters the glycolytic pathway as fructose 6-phosphate. In fact, as previously seen, hexokinase can catalyzes the phosphorylation of fructose at C-6.

Fructose + ATP → Fructose 6-phosphate + ADP + H+

However, the affinity of the enzyme for fructose is about 20 times lower than for glucose, so in the hepatocyte, where glucose is much more abundant than fructose, or in the skeletal muscle under anaerobic conditions, that is, when glucose is the preferred fuel, little amounts of fructose 6-phosphate are formed.
Conversely, in adipose tissue, fructose is more abundant than glucose, so that its phosphorylation by hexokinase is not competitively inhibited to a significant extent by glucose. In this tissue, therefore, fructose 6-phosphate synthesis is the entry point into glycolysis for the monosaccharide.
With regard to the metabolic effects of fructose, it is important to underline that in the liver the monosaccharide, being phosphorylated at C-1, enters glycolysis at triose phosphate level, thus downstream to the reaction catalyzed by PFK-1, an enzyme that plays a key role in the regulation of the flow of carbon through this metabolic pathway. Conversely, when fructose is phosphorylated at C-6, it enters the glycolytic pathway upstream of PFK-1.

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Fructose is the entry point into glycolysis for sorbitol, a sugar present in many fruits and vegetables, and used as a sweetener and stabilizer, too. In the liver, sorbitol dehydrogenase (EC catalyzes the oxidation of sorbitol to fructose.

Sorbitol + NAD+ → Fructose + NADH + H+

The reaction requires the presence of zinc ion, and occurs in the cytosol.

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Galactose, for the most part derived from intestinal digestion of the lactose, once in the liver is converted, via the Leloir pathway, to glucose 1-phosphate.
For a more in-depth discussion of the Leloir pathway, see the article on galactose.
The metabolic fate of glucose 1-phosphate depends on the energy status of the cell.
Under conditions promoting glucose storage, glucose 1-phosphate can be channeled to glycogen synthesis. Conversely, under conditions that favor the use of glucose as fuel, glucose 1-phosphate is isomerized to glucose 6-phosphate in the reversible reaction catalyzed by phosphoglucomutase (EC

Glucose 1-phosphate ⇄ Glucose 6-phosphate

In turn, glucose 6-phosphate can be channeled to glycolysis and be used for energy production, or dephosphorylated to glucose in the reaction catalyzed by glucose 6-phosphatase, and then released into the bloodstream.

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Mannose is present in various dietary polysaccharides, glycolipids and glycoproteins. In the intestine, it is released from these molecules, absorbed, and, once reached the liver, is phosphorylated at C-6 to form mannose 6-phosphate, in the reaction catalyzed by hexokinase.

Mannose + ATP → Mannose 6-phosphate + ADP + H+

Mannose 6-phosphate is then isomerized to fructose 6-phosphate in the reaction catalyzed by mannose 6-phosphate isomerase (EC

Mannose 6-phosphate ⇄ Fructose 6-phosphate

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Regulation of glycolysis

The flow of carbon through the glycolytic pathway is regulated in response to metabolic conditions, both inside and outside the cell, essentially to meet two needs: the production of ATP and the supply of precursors for biosynthetic reactions.
And in the liver, to avoid wasting energy, glycolysis and gluconeogenesis are reciprocally regulated so that when one pathway is active, the other slows down. As explained in the article on gluconeogenesis, during evolution this was achieved by selecting different enzymes to catalyze the essentially irreversible reactions of the two pathways, whose activity are regulated separately. Indeed, if these reactions proceeded simultaneously at high speed, they would create a futile cycle or substrate cycle. A such fine regulation could not be achieved if a single enzyme operates in both directions.
The control of the glycolytic pathway involves essentially the reactions catalyzed by hexokinase, PFK-1, and pyruvate kinase, whose activity is regulated through:

  • allosteric modifications, that occur on a time scale of  milliseconds and are instantly reversible;
  • covalent modifications, that is, phosphorylations and dephosphorylation, that occur on a time scale of seconds;
  • changes in enzyme concentrations, resulting from changes in the rate of their synthesis and/or degradation, that occur on a time scale of hours.

Note: The main regulatory enzymes of gluconeogenesis are pyruvate carboxylase (EC and fructose 1,6-bisphosphatase (EC

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In humans, hexokinase has four tissue specific isozymes, designated as hexokinase I, II, III, and IV, encoded by as many genes.
Hexokinase I is the predominant isozyme in the brain, whereas in skeletal muscle hexokinase I and II are present, accounting for 70-75% and 25-30% of the isozymes, respectively.
Hexokinase IV, also known as glucokinase (EC, is mainly present in hepatocytes  and β cells of the pancreas, where it is the predominant isozyme. In the liver it catalyzes,  with glucose 6-phosphatase, the substrate cycle between glucose and glucose 6-phosphate.  Glucokinase differs from the other hexokinase isozymes in kinetic and regulatory properties.

Note: Isoenzymes or isozymes are different proteins that catalyze the same reaction, and that generally differ in kinetic and regulatory properties, subcellular distribution, or in the cofactors used. They may be present in the same species, in the same tissue or even in the same cell.

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Comparison of the kinetic properties of hexokinase isozymes

The kinetic properties of hexokinase I, II, and III are similar.
Hexokinase I and II have a Km for glucose of 0.03 mM and 0.1 mM, respectively. Therefore these isoenzymes work very efficiently at normal blood glucose levels, 4-5 mM.
Conversely, glucokinase has a high Km for glucose, approximately 10 mM; this means that the enzyme works efficiently only when blood glucose concentration is high, for example after a meal rich in carbohydrates with a high glycemic index.

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Regulation of the activity of hexokinases I-III

Hexokinases I-III are allosterically inhibited by glucose 6-phosphate, the product of their reaction. This ensures that glucose 6-phosphate does not accumulate in the cytosol when glucose is not needed for energy, for glycogen synthesis, for the pentose phosphate pathway, or as a source of precursors for biosynthetic pathways, leaving, at the same time, the monosaccharide in the blood, available for other organs and tissues. For example, when PFK-1 is inhibited, fructose 6-phosphate accumulates and then, due to phosphoglucose isomerase reaction, glucose 6-phosphate accumulates. Therefore, inhibition of PFK-1 leads to inhibition of hexokinases I-III.

In skeletal muscle, the activity of hexokinase I and II is coordinated with that of GLUT4, a low Km glucose transporter (5mM), whose translocation to the plasma membrane is induced by both insulin and physical activity. The combined action of GLUT4 on plasma membrane and hexokinase in the cytosol maintains a balance between glucose uptake and its phosphorylation. Because blood glucose concentration is between 4 and 5 mmol/L, its entry into the myocyte through GLUT4 may cause an increase in its concentration sufficient to saturate, or near saturate the enzyme, which therefore operates at or near its Vmax.

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Regulation of the activity of hepatic glucokinase

Glucokinase differs in three respects from hexokinases I-III, and is particularly suitable for the role that the liver plays in glycemic control. Why?

  • As previously said, glucokinase has a Km for glucose of about 10 mM, much higher than the Km for glucose of hexokinases I-III, and higher than the value of fasting blood glucose levels (4-5 mM) as well. In the liver, where it is the predominant hexokinase isoenzyme, its role is to provide glucose 6-phosphate for the synthesis of glycogen and fatty acids. The activity of glucokinase is linked to that of GLUT2, the major glucose transporter in hepatocytes, with a high Km for glucose, approximately 10 mM. Hence, GLUT2 is very active when blood glucose concentration is high, rapidly equilibrating sugar concentrations in cytosol of hepatocytes and blood. Under such conditions glucokinase is active and converts glucose to glucose 6-phosphate, and, due to high Km for glucose, its activity continues to increase even when the intracellular concentration of the monosaccharide reaches or exceeds 10 mM.  Therefore, the rate at which glucose uptake and phosphorylation occurs are determined by the value of blood glucose level itself. On the other hand, when glucose availability is low, its concentration in the cytosol of hepatocytes is just as low, much lower than the Km for glucose of glucokinase, so that glucose produced through gluconeogenesis and/or glycogenolysis is not phosphorylated and can leave the cell.
    A similar situation also occurs in pancreatic β cells, where the GLUT2/glucokinase system causes the intracellular G-6-P concentration to equalize with glucose concentration in the blood, allowing the cells to detect and respond to hyperglycemia.
  • Unlike hexokinases I-III, glucokinase is not inhibited by glucose 6-phosphate, that is, is not product inhibited, and catalyzes its synthesis even when it accumulates.
  • Glucokinase is inhibited by the reversible binding of glucokinase regulatory protein or GKRP, a liver-specific regulatory protein. The mechanism of inhibition by GKRP occurs via the anchorage of glucokinase inside the nucleus, where it is separated from the other glycolytic enzymes.
    Fig. 10 – Regulation of Hepatic Glucokinase

    The binding between glucokinase and GKRP is much tighter in the presence of fructose 6-phosphate, whereas it is weakened by glucose and fructose 1-phosphate.
    In the absence of glucose, glucokinase is in its super-opened conformation that has low activity. The rise in cytosolic glucose concentration causes a concentration dependent transition of glucokinase to its close conformation, namely, its active conformation that is not accessible for glucokinase regulatory protein. Hence, glucokinase is active and no longer inhibited.
    Notice that fructose 1-phosphate is present in the hepatocyte only when fructose is metabolized. Hence, fructose relieves the inhibition of glucokinase by glucokinase regulatory protein.
    After a meal rich in carbohydrates, blood glucose levels rise, glucose enters the hepatocyte through GLUT2, and then moves inside the nucleus through the nuclear pores. In the nucleus glucose determines the transition of glucokinase to its close conformation, active and not accessible to GKRP, allowing glucokinase to diffuse in the cytosol where it phosphorylates glucose.
    Conversely, when glucose concentration declines, such as during fasting when blood glucose levels may drop below 4 mM, glucose concentration in hepatocytes is low, and fructose 6-phosphate binds to GKRP allowing it to bind tighter to glucokinase. This results in a strong inhibition of the enzyme. This mechanism ensures that the liver, at low blood glucose levels, does not compete with other organs, primarily the brain, for glucose.
    In the cell, fructose 6-phosphate is in equilibrium with glucose 6-phosphate, due to phosphoglucose isomerase reaction. Through its association with GKRP, fructose 6-phosphate allows the cell to decrease glucokinase activity, so preventing the accumulation of intermediates.

To sum up, when blood glucose levels are normal, glucose is phosphorylated mainly by hexokinases I-III, whereas when blood glucose levels are high glucose can be phosphorylated by glucokinase as well.

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Regulation of phosphofructokinase 1 activity

Phosphofructokinase 1 is the key control point of carbon flow through the glycolytic pathway.
The enzyme, in addition to substrate binding sites, has several binding sites for allosteric effectors.
ATP, citrate, and hydrogen ions are allosteric inhibitors of the enzyme, whereas AMP, Pi and fructose 2,6-bisphosphate are allosteric activators.

Fig. 11 – Regulation of PFK 1 and Fructose 1,6-bisphosphatase

It should be noted that ATP, an end product of glycolysis, is also a substrate of phosphofructokinase 1. Indeed, the enzyme has two binding sites for the nucleotide: a low-affinity regulatory site, and a high affinity substrate site.
What do allosteric effectors signal?

  • ATP, AMP and Pi signal the energy status of the cell.
    The activity of PFK-1 increases when the energy charge of the cell is low, namely, when there is a need for ATP, whereas it decreases when the energy charge of the cell is high, namely when ATP concentration in the cell is high. How?
    When the nucleotide is produced faster than it is consumed, its cellular concentration is high. Under such condition ATP, binding to its allosteric site, inhibits PFK-1 by reducing the affinity of the enzyme for fructose 6-phosphate. From the kinetic point of view, the increase in ATP concentration modifies the relationship between enzyme activity and substrate concentration, chancing the hyperbolic fructose 6-phosphate velocity curve into a sigmoidal one, and then, increasing Km for the substrate. However, under most cellular conditions, ATP concentration does not vary much. For example, during a vigorous exercise ATP concentration in muscle may lower of about 10% compared to the resting state, whereas glycolysis rate varies much more than would be expected by such reduction.
    When ATP consumption exceeds its production, ADP and AMP concentrations rise, in particular that of AMP, due to the reaction catalyzed by adenylate kinase (EC, that form ATP from ADP.


The equilibrium constant, Keq, of the reaction is:

Keq = [ATP][AMP]/[ADP]2= 0.44

Under normal conditions, ADP and AMP concentrations are about 10% and often less than 1% of ATP concentration, respectively.  Therefore, considering that the total adenylate pool is constant over the short term, even a small reduction in ATP concentration leads, due to adenylate kinase activity, to a much larger relative increase in AMP concentration. In turn, AMP acts by reversing the inhibition due to ATP.
Therefore, the activity of phosphofructokinase 1 depends on the cellular energy status:

when ATP is plentiful, enzyme activity decreases;

when AMP levels increase and ATP levels fall, enzyme activity increases.

Why is not ADP a positive effector of PFK-1? There are two reasons.
When the energy charge of the cell falls, ADP is used to regenerate ATP, in the reaction catalyzed by adenylate kinase Moreover, as previously said, a small reduction in ATP levels leads to larger-percentage changes in ADP levels and, above all, in AMP levels.

  • Hydrogen ions inhibit PFK-1. Such inhibition prevents, by controlling the rate of glycolysis, excessive lactate buildup and the consequent fall of blood pH.
  • Citrate is an allosteric inhibitor of PFK-1 that acts by enhancing the inhibitory effect of ATP.
    It is the product of the first step of the citric acid cycle, a metabolic pathway that provides building blocks for biosynthetic pathways and directs electrons into mitochondrial electron transport chain for ATP synthesis via oxidative phosphorylation. High citrate levels in the cytosol mean that, in the mitochondria, an overproduction of building blocks is occurring and the current energy are met, namely, the citric acid cycle has reached saturation. Under such conditions glycolysis, that feeds the cycle under aerobic condition, can slow down, sparing glucose.
    So, it should be noted that PFK-1 couples glycolysis and the citric acid cycle.
  • In the liver, the central control point of glycolysis and gluconeogenesis is the substrate cycle between F-6-P and F-1,6-BP, catalyzed by PFK-1 and fructose 1,6-bisphosphatase.
    The liver plays a pivotal role in maintaining blood glucose levels within the normal range.
    When blood glucose levels drop, glucagon stimulates hepatic glucose synthesis, via both glycogenolysis and gluconeogenesis, and at the same time signals the liver to stop consuming glucose to meet its needs.
    Conversely, when blood glucose levels are high, insulin causes the liver to use glucose for energy, and to synthesize glycogen, and triglycerides.
    In this context, the regulation of glycolysis and gluconeogenesis is mediated by fructose 2,6-bisphosphate, a molecule that allows the liver to play a major role in regulating blood glucose levels, and whose levels are controlled by insulin and glucagon.
    As a result of binding to its allosteric site on PFK-1, fructose 2,6-bisphosphate increases the affinity of the enzyme for fructose 6-phosphate, its substrate, while decreases its affinity for the allosteric inhibitors citrate and ATP. It is remarkable to note that under physiological concentrations of the substrates and positive and negative allosteric effectors, phosphofructokinase 1 would be virtually inactive in the absence of fructose 2,6-bisphosphate.
    On the other hand, the binding of fructose 2,6-bisphosphate to fructose 1,6-bisphosphatase inhibits the enzyme, even in the absence of AMP, another allosteric inhibitor of the enzyme.
    Due to these effects,  fructose 2,6-bisphosphate increases the net flow of glucose through glycolysis.
    For an more in-depth analysis of fructose 2,6-bisphosphate metabolism, refer to the article on gluconeogenesis.
  • Another metabolite involved in the control of the flow of carbon through glycolysis and gluconeogenesis is xylulose 5-phosphate, whose concentration in hepatocytes rises after ingestion of a carbohydrate-rich meal. The molecule, by activating protein phosphatase 2A, finally leads to an increase in the concentration of fructose 2,6-bisphosphate, and then to an increase in the flow of carbon through glycolysis and to a reduction in the flow of carbon through gluconeogenesis.

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Regulation of pyruvate kinase activity

A further control point of carbon flow through glycolysis and gluconeogenesis is the substrate cycle between phosphoenolpyruvate and pyruvate, catalyzed by pyruvate kinase for glycolysis, and by the combined action of pyruvate carboxylase and phosphoenolpyruvate carboxykinase (EC for gluconeogenesis.
All isozymes of pyruvate kinase are allosterically inhibited by high concentrations of ATP, long-chain fatty acids, and acetyl-CoA, all signs that the cell is in an optimal energy status. Alanine, too, that can be synthesized from pyruvate through a transamination reaction, is an allosteric inhibitor of pyruvate kinase; its accumulation signals that building blocks for biosynthetic pathways are abundant.

Fig. 12 – Regulation of Hepatic Pyruvate Kinase

Conversely, pyruvate kinase is allosterically activated by fructose 1,6-bisphosphate, the product of the first committed step of glycolysis. Therefore, F-1,6-BP allows pyruvate kinase to keep pace with the flow of intermediates. It should be underlined that, at physiological concentration of PEP, ATP and alanine, the enzyme would be completely inhibited without the stimulating effect of F-1,6-BP.
The hepatic isoenzyme, but not the muscle isoenzyme, is also subject to regulation through phosphorylation by:

  • protein kinase A or PKA, activated by the binding of glucagon to the specific receptor or epinephrine to β-adrenergic receptors;
  • calcium/calmodulin dependent protein kinase or CAMK, activated by the binding of epinephrine to α1-adrenergic receptors.

Phosphorylation of the enzyme decreases its activity, by increasing the Km for phosphoenolpyruvate, and slows down glycolysis.
For example, when the blood glucose levels are low, glucagon-induced phosphorylation decreases pyruvate kinase activity. The phosphorylated enzyme is also less readily stimulated by fructose 1,6-bisphosphate but more readily inhibited by alanine and ATP. Conversely, the dephosphorylated form of pyruvate kinase is more sensitive to fructose 1,6-bisphosphate, and less sensitive to ATP and alanine. In this way, when blood glucose levels are low, the use of glucose for energy in the liver slows down, and the sugar is available for other tissues and organs, such as the brain. However, it should be noted that pyruvate kinase does not undergo glucagon-induced phosphorylation in the presence of fructose 1,6-bisphosphate.
An increase in the insulin/glucagon ratio, on the other hand, leads to dephosphorylation of the enzyme and then to its activation. The dephosphorylated enzyme is more readily stimulated by its allosteric activators F-1,6-BP, and less readily inhibited by allosteric inhibitors alanine and ATP.

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Berg J.M., Tymoczko J.L., and Stryer L. Biochemistry. 5th Edition. W. H. Freeman and Company, 2002

de la Iglesia N., Mukhtar M., Seoane J., Guinovart J.J., & Agius L. The role of the regulatory protein of glucokinase in the glucose sensory mechanism of the hepatocyte. J Biol Chem 2000;275(14):10597-603. doi: 10.1074/jbc.275.14.10597

Garrett R.H., Grisham C.M. Biochemistry. 4th Edition. Brooks/Cole, Cengage Learning, 2010

Kabashima T., Kawaguchi T., Wadzinski B.E., Uyeda K. Xylulose 5-phosphate mediates glucose-induced lipogenesis by xylulose 5-phosphate-activated protein phosphatase in rat liver. Proc Natl Acad Sci USA 2003;100:5107-12. doi:10.1073/pnas.0730817100

Kaminski M.T., Schultz J., Waterstradt R., Tiedge M., Lenzen S., Baltrusch S. Glucose-induced dissociation of glucokinase from its regulatory protein in the nucleus of hepatocytes prior to nuclear export. BBA – Molecular Cell Research 2014;1843(3):554-64. doi:10.1016/j.bbamcr.2013.12.002

Nelson D.L., M. M. Cox M.M. Lehninger. Principles of biochemistry. 6th Edition. W.H. Freeman and Company, 2012

Oslund R.C., Su X., Haugbro M., Kee J-M., Esposito M., David Y., Wang B., Ge E., Perlman D.H., Kang Y., Muir T.W., & Rabinowitz J.D. Bisphosphoglycerate mutase controls serine pathway flux via 3-phosphoglycerate. Nat Chem Biol 2017;13:1081-87. doi:10.1038/nchembio.2453

Rich P.R. The molecular machinery of Keilin’s respiratory chain. Biochem Soc Trans 2003;31(6):1095-105. doi:10.1042/bst0311095

Stipanuk M.H., Caudill M.A. Biochemical, physiological, and molecular aspects of human nutrition. 3rd Edition. Elsevier health sciences, 2013 [Google eBooks]

Van Schaftingen, E., and Hers, H-G. Inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphosphate. Proc Natl Acad Sci USA 1981;78(5):2861-63 [PDF]

Van Schaftingen E., Jett M-F., Hue L., and Hers, H-G. Control of liver 6-phosphofructokinase by fructose 2,6-bisphosphate and other effectors. Proc Natl Acad Sci USA 1981;78(6):3483-86 [PDF]


Gluconeogenesis: contents in brief

What is gluconeogenesis?

Gluconeogenesis is a metabolic pathway that leads to the synthesis of glucose from pyruvate and other non-carbohydrate precursors, even in non-photosynthetic organisms.
It occurs in all microorganisms, fungi, plants and animals, and the reactions are essentially the same, leading to the synthesis of one glucose molecule from two pyruvate molecules. Therefore, it is in essence glycolysis in reverse, which instead goes from glucose to pyruvate, and shares seven enzymes with it.

Fig. 1 – Gluconeogenesis and Glycolysis

Glycogenolysis is quite distinct from gluconeogenesis: it does not lead to de novo production of glucose from non-carbohydrate precursors, as shown by its overall reaction:

Glycogen or (glucose)n → n glucose molecules

The following discussion will focus on gluconeogenesis that occurs in higher animals, and in particular in the liver of mammals.

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Why is gluconeogenesis important?

Gluconeogenesis is an essential metabolic pathway for at least two reasons.

  • It ensures the maintenance of appropriate blood glucose levels when the liver glycogen is almost depleted and no carbohydrates are ingested.
  • Maintaining blood glucose within the normal range, 3.3 to 5.5 mmol/L (60 and 99 mg/dL), is essential because many cells and tissues depend, largely or entirely, on glucose to meet their ATP demands; examples are red blood cells, neurons, skeletal muscle working under low oxygen conditions, the medulla of the kidney, the testes, the lens and the cornea of the eye, and embryonic tissues. For example, glucose requirement of the brain is about 120 g/die that is equal to:

over 50% of the total body stores of the monosaccharide, about 210 g, of which 190 g are stored as muscle and liver glycogen, and 20 g are found in free form in body fluids;
about 75% of the daily glucose requirement, about 160 g.

During fasting, as in between meals or overnight, the blood glucose levels are maintained within the normal range due to hepatic glycogenolysis, and to the release of fatty acids from adipose tissue and ketone bodies by the liver. Fatty acids and ketone bodies are preferably used by skeletal muscle, thus sparing glucose for cells and tissues that depend on it, primarily red blood cells and neurons. However, after about 18 hours of fasting or during intense and prolonged exercise, glycogen stores are depleted and may become insufficient. At that point, if no carbohydrates are ingested, gluconeogenesis becomes important.
And, the importance of gluconeogenesis is further emphasized by the fact that if the blood glucose levels fall below 2 mmol/L, unconsciousness occurs.

  • The excretion of pyruvate would lead to the loss of the ability to produce ATP through aerobic respiration, i.e. more than 10 molecules of ATP for each molecule of pyruvate oxidized.

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Where does gluconeogenesis occurs?

In higher animals, gluconeogenesis occurs in the liver, kidney cortex and epithelial cells of the small intestine, that is, the enterocytes.
Quantitatively, the liver is the major site of gluconeogenesis, accounting for about 90% of the synthesized glucose, followed by kidney cortex, with about 10%. The key role of the liver is due to its size; in fact, on a wet weight basis, the kidney cortex produces more glucose than the liver.
In the kidney cortex, gluconeogenesis occurs in the cells of the proximal tubule, the part of the nephron immediately following the glomerulus. Much of the glucose produced in the kidney is used by the renal medulla, while the role of the kidney in maintaining blood glucose levels becomes more important during prolonged fasting and liver failure. It should, however, be emphasized that the kidney has no significant glycogen stores, unlike the liver, and contributes to maintaining blood glucose homeostasis only through gluconeogenesis and not through glycogenolysis.
Part of the gluconeogenesis pathway also occurs in the skeletal muscle, cardiac muscle, and brain, although at very low rate. In adults, muscle is about 18 the weight of the liver; therefore, its de novo synthesis of glucose might have quantitative importance. However, the release of glucose into the circulation does not occur because these tissues, unlike liver, kidney cortex, and enterocytes, lack glucose 6-phosphatase (EC, the enzyme that catalyzes the last step of gluconeogenesis (see below).
Therefore, the production of glucose 6-phosphate, including that from glycogenolysis, does not contribute to the maintenance of blood glucose levels, and only helps to restore glycogen stores, in the brain small and limited mostly to astrocytes. For these tissues, in particular for skeletal muscle due to its large mass, the contribution to blood glucose homeostasis results only from the small amount of glucose released in the reaction catalyzed by enzyme debranching (EC of glycogenolysis.
With regard to the cellular localization, most of the reactions occur in the cytosol, some in the mitochondria, and the final step) within the endoplasmic reticulum cisternae.

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Irreversible steps of gluconeogenesis

As previously said, gluconeogenesis is in essence glycolysis in reverse. And, of the ten reactions that constitute gluconeogenesis, seven are shared with glycolysis; these reactions have a ΔG close to zero, therefore easily reversible. However, under intracellular conditions, the overall ΔG of glycolysis is about -63 kJ/mol (-15 kcal/mol) and of gluconeogenesis about -16 kJ/mol (-3.83 kcal/mol), namely, both the pathways are irreversible.
The irreversibility of the glycolytic pathway is due to three strongly exergonic reactions, that cannot be used in gluconeogenesis, and listed below.

  • The phosphorylation of glucose to glucose 6-phosphate, catalyzed by hexokinase (EC or glucokinase (EC
    ΔG = -33.4 kJ/mol (-8 kcal/mol)
    ΔG°’ = -16.7 kJ/mol (-4 kcal/mol)
  • The phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate, catalyzed by phosphofructokinase-1 or PFK-1 (EC
    ΔG = -22.2 kJ/mol (-5.3 kcal/mol)
    ΔG°’ = -14.2 kJ/mol (-3.4 kcal/mol)
  •  The conversion of phosphoenolpyruvate or PEP to pyruvate, catalyzed by pyruvate kinase (EC
    ΔG = -16.7 kJ/mol (-4.0 kcal/mol)
    ΔG°’ = -31.4 kJ/mole (-7.5 kcal/mol)

In gluconeogenesis, these three steps are bypassed by enzymes that catalyze irreversible steps in the direction of glucose synthesis: this ensures the irreversibility of the metabolic pathway.
Below, such reactions are analyzed.

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From pyruvate to phosphoenolpyruvate

The first step of gluconeogenesis that bypasses an irreversible step of glycolysis, namely the reaction catalyzed by pyruvate kinase, is the conversion of pyruvate to phosphoenolpyruvate.
Phosphoenolpyruvate is synthesized through two reactions catalyzed, in order, by the enzymes:

  • pyruvate carboxylase (EC;
  • phosphoenolpyruvate carboxykinase or PEP carboxykinase (EC

Pyruvate → Oxaloacetate → Phosphoenolpyruvate

Fig. 2 – Phosphoenolpyruvate

Pyruvate carboxylase catalyzes the carboxylation of pyruvate to oxaloacetate, with the consumption of one ATP. The enzyme requires the presence of magnesium or manganese ions

Pyruvate + HCO3+ ATP → Oxaloacetate + ADP + Pi

The enzyme, discovered in 1960 by Merton Utter, is a mitochondrial protein composed of four identical subunits, each with catalytic activity. The subunits contain a biotin prosthetic group, covalently linked by amide bond to the ε-amino group of a lysine residue, that acts as a carrier of activated CO2 during the reaction. An allosteric binding site for acetyl-CoA is also present in each subunit.
It should be noted that the reaction catalyzed by pyruvate carboxylase, leading to the production of oxaloacetate, also provides intermediates for the citric acid cycle or Krebs cycle.
Phosphoenolpyruvate carboxykinase is present, approximately in the same amount, in mitochondria and cytosol of hepatocytes. The isoenzymes are encoded by separate nuclear genes.
The enzyme catalyzes the decarboxylation and phosphorylation of oxaloacetate to phosphoenolpyruvate, in a reaction in which GTP acts as a donor of high-energy phosphate. PEP carboxykinase requires the presence of both magnesium and manganese ions. The reaction is reversible under normal cellular conditions.

Oxaloacetate + GTP ⇄ PEP + CO2 + GDP

During this reaction, a CO2 molecule, the same molecule that is added to pyruvate in the reaction catalyzed by pyruvate carboxylase, is removed. Carboxylation-decarboxylation sequence is used to activate pyruvate, since decarboxylation of oxaloacetate facilitates, makes thermodynamically feasible, the formation of phosphoenolpyruvate.
More generally, carboxylation-decarboxylation sequence promotes reactions that would otherwise be strongly endergonic, and also occurs in the citric acid cycle, in the pentose phosphate pathway, also called the hexose monophosphate pathway, and in the synthesis of fatty acids.
The levels of PEP carboxykinase before birth are very low, while its activity increases several fold a few hours after delivery. This is the reason why gluconeogenesis is activated after birth.
The sum of the reactions catalyzed by pyruvate carboxylase and phosphoenolpyruvate carboxykinase is:

Pyruvate + ATP + GTP + HCO3 → PEP + ADP + GDP + Pi + CO2

ΔG°’ of the reaction is equal to 0.9 kJ/mol (0.2 kcal/mol), while standard free energy change associated with the formation of pyruvate from phosphoenolpyruvate by reversal of the pyruvate kinase reaction is + 31.4 kJ/mol (7.5 kcal/mol).
Although the ΔG°’ of the two steps leading to the formation of PEP from pyruvate is slightly positive, the actual free-energy change (ΔG), calculated from intracellular concentrations of the intermediates, is very negative, -25 kJ/mol (-6 kcal/mol). This is due to the fast consumption of phosphoenolpyruvate in other reactions, that maintains its concentration at very low levels. Therefore, under cellular conditions, the synthesis of PEP from pyruvate is irreversible.
It is noteworthy that the metabolic pathway for the formation of phosphoenolpyruvate from pyruvate depends on the precursor: pyruvate or alanine, or lactate.

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Phosphoenolpyruvate precursor: pyruvate or alanine

Fig. 3 – Conversion of Pyruvate to PEP

The bypass reactions described below predominate when alanine or pyruvate is the glucogenic precursor.
Pyruvate carboxylase is a mitochondrial enzyme, therefore pyruvate must be transported from the cytosol into the mitochondrial matrix. This is mediated by transporters located in the inner mitochondrial membrane, referred to as MPC1 and MPC2. These proteins, associating, form a hetero-oligomer that facilitates pyruvate transport.
Pyruvate can also be produced from alanine in the mitochondrial matrix by transamination, in the reaction catalyzed by alanine aminotransferase (EC
Since the enzymes involved in the later steps of gluconeogenesis, except glucose-6-phosphatase, are cytosolic, the oxaloacetate produced in the mitochondrial matrix is transported into the cytosol. However, there are no oxaloacetate transporters in the inner mitochondrial membrane. The transfer to the cytosol occurs as a result of its reduction to malate, that, on the contrary, can cross the inner mitochondrial membrane. The reaction is catalyzed by mitochondrial malate dehydrogenase (EC, an enzyme also involved in the citric acid cycle, where the reaction proceeds in the reverse direction. In the reaction NADH is oxidized to NAD+.

Oxaloacetate + NADH + H+ ⇄ Malate + NAD+

Although ΔG°’ of the reaction is highly positive, under physiological conditions, ΔG is close to zero, and the reaction is easily reversible.
Malate crosses the inner mitochondrial membrane through a component of the malate-aspartate shuttle, the malate-α-ketoglutarate transporter. Once in the cytosol, the malate is re-oxidized to oxaloacetate in the reaction catalyzed by cytosolic malate dehydrogenase. In this reaction NAD+ is reduced to NADH.

Malate + NAD+ → Oxaloacetate + NADH + H+

Note: Malate-aspartate shuttle is the most active shuttle for the transport of NADH-reducing equivalents from the cytosol into the mitochondria. It is found in mitochondria of liver, kidney, and heart.
The reaction enables the transport into the cytosol of mitochondrial reducing equivalents in the form of NADH. This transfer is needed for gluconeogenesis to proceed, as in the cytosolic the NADH, oxidized in the  reaction catalyzed by glyceraldehydes 3-phosphate dehydrogenase (EC, is present in very low concentration, with a [NADH]/[NAD+] ratio equal to 8×10-4, about 100,000 times lower than that observed in the mitochondria.
Finally, the oxaloacetate is converted to phosphoenolpyruvate in the reaction catalyzed by PEP carboxykinase.

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Phosphoenolpyruvate precursor: lactate

Lactate is one of the major gluconeogenic precursors. It is produced for example by:

  • red blood cells, that are completely dependent on anaerobic glycolysis for ATP production;
  • skeletal muscle during intense exercise, that is, under low oxygen condition, when the rate of glycolysis exceeds the rate of the citric acid cycle and oxidative phosphorylation.

When lactate is the gluconeogenic precursor, PEP synthesis occurs through a different pathway than that previously seen. In the hepatocyte cytosol NAD+ concentration is high and the lactate is oxidized to pyruvate in the reaction catalyzed by the liver isoenzyme of lactate dehydrogenase (EC In the reaction NAD+ is reduced to NADH.

Lactate + NAD+ → Pyruvate + NADH + H+

The production of cytosolic NADH makes unnecessary the export of reducing equivalents from the mitochondria.
Pyruvate enters the mitochondrial matrix to be converted to oxaloacetate in the reaction catalyzed by pyruvate carboxylase. In the mitochondria, oxaloacetate is converted to phosphoenolpyruvate in the reaction catalyzed by mitochondrial pyruvate carboxylase. Phosphoenolpyruvate exits the mitochondria through an anion transporter located in the inner mitochondrial membrane, and, once in the cytosol, continues in the gluconeogenesis pathway.
Note: The synthesis of glucose from lactate may be considered as the part of  the Cori cycle that takes place in the liver.

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From fructose 1,6-bisphosphate to fructose 6-phosphate

The second step of gluconeogenesis that bypasses an irreversible step of the glycolytic pathway, namely the reaction catalyzed by PFK-1, is the dephosphorylation of fructose 1,6-bisphosphate to fructose 6-phosphate.
This reaction, catalyzed by fructose 1,6-bisphosphatase or FBPasi-1 (EC, a Mg2+ dependent enzyme located in the cytosol, leads to the hydrolysis of the C-1 phosphate of fructose 1,6-bisphosphate, without production of ATP.

Fructose 1,6-bisphosphate + H2O → Fructose 6-phosphate + Pi

The ΔG°’ of the reaction is -16.3 kJ/mol (-3.9 kcal/mol), therefore an irreversible reaction.

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From glucose 6-phosphate to glucose

The third step of gluconeogenesis that bypasses an irreversible step of the glycolytic pathway, namely the reaction catalyzed by hexokinase or glucokinase, is the dephosphorylation of glucose 6-phosphate to glucose.
This reaction is catalyzed by the catalytic subunit of glucose 6-phosphatase, a protein complex located in the membrane of the endoplasmic reticulum of hepatocytes, enterocytes and cells of the proximal tubule of the kidney. Glucose 6-phosphatase complex is composed of a glucose 6-phosphatase catalytic subunit and a glucose 6-phosphate transporter called glucose 6-phosphate translocase or T1.
Glucose 6-phosphatase catalytic subunit has the active site on the luminal side of the organelle. This means that the enzyme catalyzes the release of glucose not in the cytosol but in the lumen of the endoplasmic reticulum.
Glucose 6-phosphate, both resulting from gluconeogenesis, produced in the reaction catalyzed by glucose 6-phosphate isomerase or phosphoglucose isomerase (EC, and glycogenolysis, produced in the reaction catalyzed by phosphoglucomutase (EC, is located in the cytosol, and must enter the lumen of the endoplasmic reticulum to be dephosphorylated. Its transport is mediated by glucose-6-phosphate translocase.

The catalytic subunit of glucose 6-phosphatase, a Mg2+-dependent enzyme, catalyzes the last step of both gluconeogenesis and glycogenolysis. And, like the reaction catalyzed by fructose 1,6-bisphosphatase, this reaction leads to the hydrolysis of a phosphate ester.

Glucose 6-phosphate + H2O → Glucose + Pi

It should also be underlined that, due to orientation of the active site, the cell separates this enzymatic activity from the cytosol, thus avoiding that glycolysis, that occurs in the cytosol, is aborted by enzyme action on glucose 6-phosphate.
The ΔG°’ of the reaction is -13.8 kJ/mol (-3.3 kcal/mol), therefore it is an irreversible reaction. If instead the reaction were that catalyzed by hexokinase/glucokinase in reverse, it would require the transfer of a phosphate group from glucose 6-phosphate to ADP. Such a reaction would have a ΔG equal to +33.4 kJ/mol (+8 kcal/mol), and then strongly endergonic. Similar considerations can be made for the reaction catalyzed by FBPase-1.
Glucose and Pi group seem to be transported into the cytosol via different transporters, referred to as T2 and T3, the last one an anion transporter.
Finally, glucose leaves the hepatocyte via the membrane transporter GLUT2, enters the bloodstream and is transported to tissues that require it. Conversely, under physiological conditions, as previously said, glucose produced by the kidney is mainly used by the medulla of the kidney itself.

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Gluconeogenesis: energetically expensive

Like glycolysis, much of the energy consumed is used in the irreversible steps of the process.
Six high-energy phosphate bonds are consumed: two from GTP and four from ATP. Furthermore, two molecules of NADH are required for the reduction of two molecules of 1,3-bisphosphoglycerate in the reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase. The oxidation of NADH causes  the lack of production of 5 molecules of ATP that are synthesized when the electrons of the reduced coenzyme are used in oxidative phosphorylation.
Also these energetic considerations show that gluconeogenesis is not simply glycolysis in reverse, in which case it would require the consumption of two molecules of ATP, as shown by the overall glycolytic equation.

Glucose + 2 ADP + 2 Pi + 2 NAD+ → 2 Pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O

Below, the overall equation for gluconeogenesis:

2 Pyruvate + 4 ATP + 2 GTP + 2 NADH+ + 2 H+ + 4 H2O → Glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+

At least in the liver, ATP needed for gluconeogenesis derives mostly from the oxidation of fatty acids or of the carbon skeletons of the amino acids, depending on the available “fuel”.

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Coordinated regulation of gluconeogenesis and glycolysis

If glycolysis and gluconeogenesis were active simultaneously at a high rate in the same cell, the only products would be ATP consumption and heat production, in particular at the irreversible steps of the two pathways, and nothing more.
For example, considering PFK-1 and FBPasi-1:

ATP + Fructose 6-phosphate → ADP + Fructose 1,6-bisphosphate

Fructose 1,6-bisphosphate + H2O → Fructose 6-phosphate + Pi

The sum of the two reactions is:

ATP + H2O → ADP + Pi + Heat

Two reactions that run simultaneously in opposite directions result in a futile cycle or substrate cycle. These apparently uneconomical cycles allow to regulate opposite metabolic pathways. In fact, a substrate cycle involves different enzymes, at least two, whose activity can be regulated separately. A such regulation would not be possible if a single enzyme would operate in both directions. The modulation of the activity of involved enzymes occurs through:

  • allosterical mechanisms;
  • covalent modifications, such as phosphorylation and dephosphorylation;
  • changes in the concentration of the involved enzymes, due to changes in their synthesis/degradation ratio.

Allosteric mechanisms are very rapid and instantly reversible, taking place in milliseconds. The others, triggered by signals from outside the cell, such as hormones, like insulin, glucagon, or epinephrine, take place on a time scale of seconds or minutes, and, for changes in enzyme concentration, hours.
This allows a coordinated regulation of the two pathways, ensuring that when pyruvate enters gluconeogenesis, the flux of glucose through the glycolytic pathway slows down, and vice versa.

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Regulation of gluconeogenesis

The regulation of gluconeogenesis and glycolysis involves the enzymes unique to each pathway, and not the common ones.
While the major control points of glycolysis are the reactions catalyzed by PFK-1 and pyruvate kinase, the major control points of gluconeogenesis are the reactions catalyzed by fructose 1,6-bisphosphatase and pyruvate carboxylase.
The other two enzymes unique to gluconeogenesis, glucose-6-phosphatase and PEP carboxykinase, are regulated at transcriptional level.

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Pyruvate carboxylase

Fig. 4 – Two Alternative Fates for Pyruvate

In the mitochondrion, pyruvate can be converted to:

          • acetyl-CoA, in the reaction catalyzed by pyruvate dehydrogenase complex, reaction that connects glycolysis to the Krebs cycle;
          • oxaloacetate, in the reaction catalyzed by pyruvate carboxylase, to continue in the gluconeogenesis pathway.

The metabolic fate of pyruvate depends on the availability of acetyl-CoA, that is, by the availability of fatty acids in the mitochondrion.
When fatty acids are available, their β-oxidation leads to the production of acetyl-CoA, that enters the Krebs cycle and leads to the production of GTP and NADH. When the energy needs of the cell are met, oxidative phosphorylation slows down, the [NADH]/[NAD+] ratio increases, NADH inhibits the citric acid cycle, and acetyl-CoA accumulates in the mitochondrial matrix. Acetyl-CoA is a positive allosteric effector of pyruvate carboxylase, and a negative allosteric effector of pyruvate kinase. Moreover, it inhibits pyruvate dehydrogenase both through end-product inhibition and phosphorylation through the activation of a specific kinase.
This means that when the energy charge of the cell is high, the formation of acetyl-CoA from pyruvate slows down, while the conversion of pyruvate to glucose is stimulated. Therefore acetyl-CoA is a molecule that signals that additional glucose oxidation for energy is not required and that glucogenic precursors can be used for the synthesis and storage of glucose.
Conversely, when acetyl-CoA levels decrease, the activity of pyruvate kinase and pyruvate dehydrogenase increases, and therefore also the flow of metabolites through the citric acid cycle. This supplies energy to the cell.
Summarizing, when the energy charge of the cell is high pyruvate carboxylase is active, and that the first control point of gluconeogenesis determines what will be the fate of pyruvate in the mitochondria.

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Fructose 1,6-bisphosphatase

The second major control point in gluconeogenesis is the reaction catalyzed by fructose 1,6-bisphosphatase. The enzyme is allosterically inhibited by AMP. Therefore, when AMP levels are high, and consequently ATP levels are low, gluconeogenesis slows down. This means that, as previously seen, FBPase-1 is active when the energy charge of the cell is sufficiently high to support de novo synthesis of glucose.
Conversely, PFK-1, the corresponding glycolytic enzyme, is allosterically activated by AMP and ADP and allosterically inhibited by ATP and citrate, the latter resulting from the condensation of acetyl-CoA and oxaloacetate.

Fig. 5 – Regulation of FBPase-1 and PFK-1


  • when AMP levels are high, gluconeogenesis slows down, and glycolysis accelerates;
  • when ATP levels are high or when acetyl-CoA or citrate are present in adequate concentrations, gluconeogenesis is promoted, while glycolysis slows down.
    The increase in citrate levels indicates that the activity of the citric acid cycle can slow down; in this way,  pyruvate can be used in glucose synthesis.

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PFK-1, FBPase-1 and fructose 2,6-bisphosphate

The liver plays a key role in maintaining blood glucose homeostasis: this requires regulatory mechanisms that coordinate glucose consumption and production. Two hormones are mainly involved: glucagon and insulin. They act intracellularly through fructose 2,6-bisphosphate or F26BP, an allosteric effector of PFK-1 and FBPase-1. This molecule is structurally related to fructose 1,6-bisphosphate, but is not an intermediate in glycolysis or gluconeogenesis.

Fig. 6 – Fructose 2,6-bisphosphate

It was discovered in 1980 by Emile Van Schaftingen and Henri-Gery Hers, as a potent activator of PFK-1. In the subsequent year, the same researchers showed that it is also a potent inhibitor of FBPase-1.
Fructose 2,6-bisphosphate, by binding to the allosteric site on PFK-1, reduces the affinity of the enzyme for ATP and citrate, allosteric inhibitors, and at the same time increases the affinity of the enzyme for fructose 6-phosphate, its substrate. PFK-1, in the absence of fructose 2,6-bisphosphate, and in the presence of physiological concentrations of ATP, fructose 6-phosphate, and of allosteric effectors AMP, ATP and citrate, is practically inactive. Conversely, the presence of fructose 2,6-bisphosphate activates PFK-1, thus stimulating glycolysis in the hepatocytes. At the same time fructose 2,6-bisphosphate slows down gluconeogenesis by inhibiting fructose 1,6-bisphosphatase, even in the absence of AMP. However, the effects of fructose-2,6-bisphosphate and AMP on FBPase-1 activity  are synergistic.

Fig. 7 – Role of F26BP in the Regulation of Gluconeogenesis and Glycolysis

Fructose-2,6-bisphosphate concentration is regulated by the relative rates of synthesis and degradation. It is synthesized from fructose 6-phosphate in the reaction catalyzed by phosphofructokinase-2 or PFK-2 (EC, and is hydrolyzed to fructose 6-phosphate in the reaction catalyzed by fructose 2,6-bisphosphatase or FBPasi-2 (EC These two enzymatic activities are located on a single bifunctional enzyme or tandem enzyme. In the liver, the balance of these two enzymatic activities is regulated by insulin and glucagon, as described below.

  • Glucagon
    It is released into the circulation when blood glucose levels drop, signaling the liver to reduce glucose consumption for its own needs and to increase de novo synthesis of glucose and its release from glycogen stores.
    After binding to specific membrane receptors, glucagon stimulates hepatic adenylate cyclase (EC to synthesize 3′,5′-cyclic AMP or cAMP, that activates cAMP-dependent protein kinase or protein kinase A or PKA (EC The kinase catalyzes the phosphorylation, at the expense of one molecule of ATP, of a specific serine residue (Ser32) of PFK-2/FBPase-2. As a result of the phosphorylation, phosphatase activity increases while kinase activity decreases. Such reduction, due to the increase in the Km for fructose 6-phosphate, causes a decrease in the levels of fructose 2,6-bisphosphate, that, in turn, inhibits glycolysis and stimulates gluconeogenesis. Therefore, in response to glucagon, hepatic production of glucose increases, enabling the organ to counteract the fall in blood glucose levels.
    Note: glucagon, like adrenaline, stimulates gluconeogenesis also by increasing the availability of substrates such as glycerol and amino acids.
  • Insulin
    After binding to specific membrane receptors, insulin activates a protein phosphatase, the phosphoprotein phosphatase 2A or PP2A, that catalyzes the removal of the phosphate group from PFK-2/FBPase-2, thus increasing PFK-2 activity and decreasing FBPase-2 activity. (At the same time, insulin also stimulates a cAMP phosphodiesterase that hydrolyzes cAMP to AMP). This increases the level of fructose 2,6-bisphosphate, that, in turn, inhibits gluconeogenesis and stimulates glycolysis.
    In addition, fructose 6-phosphate allosterically inhibits FBPase-2, and activates PFK-2. It should be noted that the activities of PFK-2 and FBPase-2 are inhibited by their reaction products. However, the main effectors are the level of fructose 6-phosphate and the phosphorylation state of the enzyme.

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Glucose 6-phosphatase

Unlike pyruvate carboxylase and fructose-1,6-bisphosphatase, the catalytic subunit of glucose-6-phosphatase is not subject to allosteric or covalent regulation. The modulation of its activity occurs at the transcriptional level. Low blood glucose levels and glucagon, namely, factors that lead to increased glucose production, and glucocorticoids stimulate its synthesis, that, conversely, is inhibited by insulin.
Also, the Km for glucose 6-phosphate is significantly higher than the range of physiological concentrations of glucose 6-phosphate itself. This is why it is said that the activity of the enzyme is almost linearly dependent on the concentration of the substrate, that is, enzyme is controlled by the level of substrate.

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PEP carboxykinase

The enzyme is regulated mainly at the level of synthesis and degradation. For example, high levels of glucagon or fasting increase protein production through the stabilization of its mRNA and the increase in its transcription rate. High blood glucose levels or insulin have opposite effects.

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Xylulose 5-phosphate

Xylulose 5-phosphate, a product of the hexose monophosphate shunt, is a recently discovered regulatory molecule. It stimulates glycolysis and inhibits gluconeogenesis by controlling the levels of fructose 2,6-bisphosphate in the liver.

Fig. 8 – Xylulose 5-phosphate

When blood glucose levels increase, e.g. after a meal high in carbohydrates, the activation of glycolysis and hexose monophosphate pathway occurs in the liver. Xylulose 5-phosphate produced activates protein phosphatase 2A, that, as previously said, dephosphorylates PFK-2/FBPase-2, thus inhibiting FBPase-2 and stimulating PFK-2. This leads to an increase in the concentration of fructose 2,6-bisphosphate, and then to the inhibition of gluconeogenesis and stimulation of glycolysis, resulting in increased production of acetyl-CoA, the main substrate for lipid synthesis. At the same time, an increase in flow through the hexose monophosphate shunt occurs, leading to the production of NADPH, a source of electrons for lipid synthesis. Finally, PP2A also dephosphorylates carbohydrate-responsive element-binding protein or ChREBP, a transcription factor that activates the expression of hepatic genes for lipid synthesis. Therefore, in response to an increase in blood glucose levels, lipid synthesis is stimulated.
It is therefore evident that xylulose 5-phosphate is a key regulator of carbohydrate and fat metabolism.

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Precursors of gluconeogenesis

Besides the aforementioned pyruvate, the major gluconeogenic precursors are lactate, glycerol, the majority of the amino acids, and, more generally, any compound that can be converted to pyruvate or oxaloacetate.

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Glycerol is released by hydrolysis of triglycerides in adipose tissue, and of glycerophospholipids. With the exception of propionyl-CoA, it is the only part of the lipid molecule that can be used for de novo synthesis of glucose in animals.
Glycerol enters gluconeogenesis, or glycolysis, depending on the cellular energy charge, as dihydroxyacetone phosphate or DHAP, whose synthesis occurs in two steps.

Fig. 9 – Conversion of Glycerol to DHAP

In the first step, glycerol is phosphorylated to glycerol 3-phosphate, in the reaction catalyzed by glycerol kinase (EC, with the consumption of one ATP. The enzyme is absent in adipocytes but present in the liver; this means that glycerol needs to reach the liver to be further metabolized.
Glycerol 3-phosphate is then oxidized to dihydroxyacetone phosphate, in the reaction catalyzed by glycerol 3-phosphate dehydrogenase (EC In this reaction NAD+ is reduced to NADH.
During prolonged fasting, glycerol is the major gluconeogenic precursor, accounting for about 20% of glucose production.

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Glucogenic amino acids

Pyruvate and oxaloacetate are the entry points for the glucogenic amino acids, i.e. those whose carbon skeleton or part of it can be used for de novo synthesis of glucose.
Amino acids result from the catabolism of proteins, both food and endogenous proteins, like those of skeletal muscle during the fasting state or during intense and prolonged exercise.
The catabolic processes of each of the twenty amino acids that made up the proteins converge to form seven major products, acetyl-CoA, acetoacetyl-CoA, α-ketoglutarate, succinyl-CoA, fumarate, oxaloacetate, and pyruvate.
Except acetyl-CoA, acetoacetyl-CoA , the other five molecules can be used for gluconeogenesis. This means that gluconeogenic amino acids may also be defined as those whose carbon skeleton or part of it can be converted to one or more of the above molecules.
Below, the entry points of the gluconeogenic amino acids are shown.

  • Pyruvate: alanine, cysteine, glycine, serine, threonine and tryptophan.
  • Oxaloacetate: aspartate and asparagine.
  • α-Ketoglutarate: glutamate, arginine, glutamine, histidine and proline.
  • Succinyl-CoA: isoleucine, methionine, threonine and valine.
  • Fumarate: phenylalanine and tyrosine.
Fig. 10 – Glucogenic and Ketogenic Amino Acids

α-Ketoglutarate, succinyl-CoA and fumarate, intermediates of the citric acid cycle, enter the gluconeogenic pathway after conversion to oxaloacetate.
The utilization of the carbon skeletons of the amino acids requires the removal of the amino group. Alanine and glutamate, the key molecules in the transport of amino groups from extrahepatic tissues to the liver, are major glucogenic amino acids in mammals. Alanine is the main gluconeogenic substrate for the liver; this amino acid is shuttled to the liver from muscle and other peripheral tissues through the glucose-alanine cycle.

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Ketogenic amino acids

Acetyl-CoA and acetoacetyl-CoA cannot be used for gluconeogenesis and are precursors of fatty acids and ketone bodies. The stoichiometry of the citric acid cycle elucidates why they cannot be used for de novo synthesis of glucose.
Acetyl-CoA, in the reaction catalyzed by citrate synthase, condenses with oxaloacetate to form citrate, a molecule with 6 carbon atoms instead of 4 as oxaloacetate. However, although the two carbon atoms from acetyl-CoA become part of the oxaloacetate molecule, two carbon atoms are oxidized and removed  as CO2, in the reactions catalyzed by isocitrate dehydrogenase (EC and α-ketoglutarate dehydrogenase complex. Therefore, acetyl-CoA does not yield any net carbon gain for the citric acid cycle.
Furthermore, the reaction leading to the formation of acetyl-CoA from pyruvate, catalyzed by the pyruvate dehydrogenase complex, that is the bridge between glycolysis and the Krebs cycle, is irreversible, and there is no other pathway to convert acetyl-CoA to pyruvate.

Pyruvate + NAD+ + CoASH → Acetyl-CoA + NADH + H+ + C02

For this reason, amino acids whose catabolism produces acetyl-CoA and/or acetoacetyl-CoA, are termed ketogenic.
Only leucine and lysine are exclusively ketogenic.

Note: Plants, yeasts, and many bacteria can use acetyl-CoA for de novo synthesis of glucose as they do have the glyoxylate cycle. This cycle has four reactions in common with the citric acid cycle, two unique enzymes, isocitrate lyase (EC and malate synthase (EC, but lacks the decarboxylation reactions. Therefore, organisms that have such pathway are able to use fatty acids for gluconeogenesis.

Five amino acids, isoleucine, phenylalanine, tyrosine, threonine and tryptophan, are both glucogenic and ketogenic, because part of their carbon backbone can be used for gluconeogenesis, while the other gives rise to ketone bodies.

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Propionate, a three carbon fatty acid, is a gluconeogenic precursor because, as propionyl-CoA, the active molecule, can be converted to succinyl-CoA.
Below, the different sources of propionate are analyzed.

  • It may arise from β-oxidation of odd-chain fatty acids such as margaric acid, a saturated fatty acid with 17 carbon atoms. Such fatty acids are rare compared to even-chain fatty acids, but present in significant amounts in the lipids of some marine organisms, ruminants, and plants. In the last pass through the β-oxidation sequence, the substrate is a five carbon fatty acid. This means that, once oxidized and cleaved to two fragments, it produces an acetyl-CoA and propionyl-CoA.
  • Another source is the oxidation of branched-chain fatty acids, with alkyl branches with an odd number of carbon atoms. An example is phytanic acid, produced in ruminants by oxidation of phytol, a breakdown product of chlorophyll.
  • In ruminants, propionate is also produced from glucose. Glucose is released from breakdown of cellulose by bacterial cellulase (EC in the rumen, one of the four chambers that make up the stomach of these animals. These microorganisms then convert, through fermentation, glucose to propionate, which, once absorbed, may be used for gluconeogenesis, synthesis of fatty acids, or be oxidized for energy.
    In ruminants, in which gluconeogenesis tends to be a continuous process, propionate is the major gluconeogenic precursor.
  • Propionate may also result from the catabolism of valine, leucine, and isoleucine (see above).

The oxidation of propionyl-CoA to succinyl-CoA involves three reactions that occur in the liver and other tissues.

Fig. 11 – Conversion of Propionyl-CoA to Succinyl-CoA

In the first reaction, propionyl-CoA is carboxylated to D-methylmalonyl-CoA in the reaction catalyzed by propionyl-CoA carboxylase (EC, a biotin-requiring enzyme. This reaction consumes one ATP. In the subsequent reaction, catalyzed by methylmalonyl-CoA epimerase (EC, D-methylmalonyl-CoA is epimerized to its L-stereoisomer. Finally, L-methylmalonyl-CoA undergoes an intramolecular rearrangement to succinyl-CoA, in the reaction catalyzed by methylmalonyl-CoA mutase (EC This enzyme requires 5-deoxyadenosylcobalamin or coenzyme B12, a derivative of cobalamin or vitamin B12, as a coenzyme.

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Bender D.A. Introduction to nutrition and metabolism. 3rd Edition. Taylor & Francis, 2004

Garrett R.H., Grisham C.M. Biochemistry. 4th Edition. Brooks/Cole, Cengage Learning, 2010

Kabashima T., Kawaguchi T., Wadzinski B.E., Uyeda K. Xylulose 5-phosphate mediates glucose-induced lipogenesis by xylulose 5-phosphate-activated protein phosphatase in rat liver. Proc Natl Acad Sci USA 2003;100:5107-12. doi:10.1073/pnas.0730817100

Kuriyama H. et all. Coordinated regulation of fat-specific and liver-specific glycerol channels, aquaporin adipose and aquaporin 9. Diabetes 2002;51(10):2915-21. doi:10.2337/diabetes.51.10.2915

McCommis K.S. and Finck B.N. Mitochondrial pyruvate transport: a historical perspective and future research directions. Biochem J 2015;466(3):443-54. doi:10.1042/BJ20141171

Nelson D.L., M. M. Cox M.M. Lehninger. Principles of biochemistry. 6th Edition. W.H. Freeman and Company, 2012

Rosenthal M.D., Glew R.H. Medical biochemistry – Human metabolism in health and disease. John Wiley J. & Sons, Inc., Publication, 2009

Soty M., Chilloux J., Delalande F., Zitoun C., Bertile F., Mithieux G., and Gautier-Stein A. Post-Translational regulation of the glucose-6-phosphatase complex by cyclic adenosine monophosphate is a crucial determinant of endogenous glucose production and is controlled by the glucose-6-phosphate transporter. J Proteome Res  2016;15(4):1342-49. doi:10.1021/acs.jproteome.6b00110

Stipanuk M.H., Caudill M.A. Biochemical, physiological, and molecular aspects of human nutrition. 3rd Edition. Elsevier health sciences, 2013 [Google eBooks]

Van Schaftingen, E., and Hers, H-G. Inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphosphate. Proc Natl Acad Sci USA 1981;78(5):2861-63 [PDF]

Van Schaftingen E., Jett M-F., Hue L., and Hers, H-G. Control of liver 6-phosphofructokinase by fructose 2,6-bisphosphate and other effectors. Proc Natl Acad Sci USA 1981;78(6):3483-86 [PDF]

Cori cycle: definition, function, biochemistry, involved tissues

Cori cycle: contents in brief

What is the Cori cycle?

The Cori cycle, or glucose-lactate cycle, was discovered by Carl Ferdinand Cori and Gerty Theresa Radnitz, a husband-and-wife team, in the ‘30s and ‘40s of the last century . They demonstrated the existence of a metabolic cooperation between the skeletal muscle working under low oxygen conditions and the liver. This cycle can be summarized as follows:

  • the conversion of glucose to lactic acid, or lactate, by anaerobic glycolysis in skeletal muscle cells;
  • the diffusion of lactate from muscle cells into the bloodstream, by which it is transported to the liver;
  • the conversion of lactate to glucose by hepatic gluconeogenesis;
  • the diffusion of glucose from the hepatocytes into the bloodstream, by which it is transported back to the skeletal muscle cells, thereby closing the cycle.

Summarizing, we have: part of the lactate produced in skeletal muscle is converted to glucose in the liver, and transported back to skeletal muscle, thus closing the cycle.

Glucose → Lactate →Glucose

The importance of this cycle is demonstrated by the fact that it may account for about 40% of plasma glucose turnover.

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Where does the Cori cycle occur?

In addition to skeletal muscle, this metabolic cooperation was also demonstrated between other extrahepatic tissues and liver.  Indeed, like the glucose-alanine cycle, the glucose-lactate cycle is active between the liver and all those tissues that do not completely oxidize glucose to CO2 and H2O, in which case pyruvate for conversion to lactate or, by transamination, to alanine would lack (see below).
In addition to skeletal muscle cells, examples of cells that continually produce lactic acid are red blood cells, immune cells in the lymph nodules,  proliferating cells in the bone marrow, and epithelial cells in the skin.
Notice that skeletal muscle produces lactate even at rest, although at low rate.

Cori Cycle
Fig. 1 – The Cori Cycle

From a biochemical point of view, the Cori cycle links gluconeogenesis with anaerobic glycolysis, using different tissues to compartmentalize opposing metabolic pathways. In fact, in the same cell, regardless of the cell type, these metabolic pathways are not very active simultaneously. Glycolysis is more active when the cell requires ATP; by contrast, when the demand for ATP is low, gluconeogenesis, in those cells where it occurs, is more active.
And it is noteworthy that, although traditionally the metabolic pathways, such as glycolysis, citric acid cycle, or gluconeogenesis, are considered to be confined within individual cells, the Cori cycle, as well as the glucose-alanine cycle, occurs between different cell types.
Finally, it should be underscored that the Cori cycle also involves the renal cortex, particularly the proximal tubules, another site where gluconeogenesis occurs.

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The steps of the Cori cycle

The analysis of the steps of the Cori cycle is made considering the lactate produced by red blood cells and skeletal muscle cells.
Mature red blood cells are devoid of mitochondria, nucleus and ribosomes, and obtain the necessary energy only by glycolysis. The availability of NAD+ is essential for glycolysis to proceed as well as for its rate: the oxidized form of the coenzyme is required for the oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate in the reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase (EC

Glyceraldehyde 3-phosphate + NAD+ → 1,3-Bisphosphoglycerate + NADH + H+

The accumulation of NADH is avoided by the reduction of pyruvate to lactate, in the reaction catalyzed by lactate dehydrogenase (EC, where NADH acts as reducing agent.

Pyruvate + NADH + H+ → Lactate + NAD+

The skeletal muscle, particularly fast-twitch fibers which contain a reduced number of mitochondria, under low oxygen condition, such as during intense exercise, produces significant amounts of lactate. In fact, in such conditions:

  • the rate of pyruvate production by glycolysis  exceeds the rate of its oxidation by the citric acid cycle, so that less than 10% of the pyruvate enters the citric acid cycle;
  • the rate at which oxygen is taken up by the cells is not sufficient to allow aerobic oxidation of all the NADH  produced.

And, like in red blood cells, the reaction catalyzed by lactate dehydrogenase, regenerating NAD+, allows glycolysis to proceed.
However, lactate is an end product of metabolism that must be converted back into pyruvate to be used.
The plasma membrane of most cells is freely permeable to both pyruvate and lactate that can thus reach the bloodstream. And, regarding for example the skeletal muscle, the amount of lactate that leaves the cell is greater than that of pyruvate due to the high NADH/NAD+ ratio in the cytosol and to the catalytic properties of the skeletal muscle isoenzyme of LDH.
Once into the bloodstream, lactate reaches the liver, which is its major user, where it is oxidized to pyruvate in the reaction catalyzed by the liver isoenzyme of lactate dehydrogenase.

Lactate + NAD+ → Pyruvate + NADH + H+

In the hepatocyte, this oxidation is favored by the low NADH/NAD+ ratio in the cytosol.
Then, pyruvate enters the gluconeogenesis pathway to be converted into glucose.
Glucose leaves the liver, enters into the bloodstream and is delivered to the muscle, as well as to other tissues and cells that require it, such as red blood cells and neurons, thus closing the cycle.

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Lactate dehydrogenase

The enzyme is a tetramer composed of two different types of subunits, designed as:

  • H subunit (heart) or B chain;
  • M subunit (muscle) or A chain.

The H subunit predominates in the heart, whereas the M subunit predominates in the  skeletal muscle and liver. Typically, tissues in which a predominantly or exclusively aerobic metabolism occurs, such as the heart, synthesize H subunits to a greater extent than M subunits, whereas tissues in which anaerobic metabolism is important, such as skeletal muscle, synthesize M subunits to a greater extent than H subunits.
The two subunits associate in 5 different ways to form homopolymers, that is, macromolecules formed by repeated, identical subunits, or heteropolymers, that is, macromolecules formed by different subunits. Different LDH  isoenzymes have different catalytic properties, as well as different distribution in various tissues, as indicated below:

  • H4, also called type 1, LDH1, or A4, a homopolymer of H subunits, is found in cardiac muscle, kidney, and red blood cells;
  • H3M1, also called type 2, LDH2, or A3B, has a tissue distribution similar to that of LDH1;
  • H2M2, also called type 3, LDH3, or A2B2, is found in the spleen, brain, white cells, kidney, and lung;
  • H1M3, also called type 4, LDH4, or AB3, is found in the spleen, lung, skeletal muscle, lung, red blood cells, and kidney;
  • M4, also called type 5, LDH5, or B4, a homopolymer of M subunits, is found in the liver, skeletal muscle, and spleen.

The H4 isoenzyme has a higher substrate affinity than the M4 isoenzyme.
The H4 isoenzyme is allosterically inhibited by high levels of pyruvate (its product), whereas the M4 isoenzyme is not.
The other LDH isoenzymes have intermediate properties, depending on the ratio between the two types of subunits.
It is thought that the H4 isoenzyme is the most suitable for catalyzing the oxidation of lactate to pyruvate that, in the heart, due to its exclusively aerobic metabolism, is then completely oxidized to CO2 and H2O. Instead, the M4 isoenzyme is the main isoenzyme found in skeletal muscle, most suitable for catalyzing the reduction of pyruvate to lactate, thus allowing glycolysis to proceed in anaerobic conditions.

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Other metabolic fates of lactate

From the above, it is clear that lactate is not a metabolic dead end, a waste product of glucose metabolism.
And it may have a different fate from that entering the Cori cycle.
For example, in skeletal muscle during recovery following an exhaustive exercise, that is, when oxygen is again available, or if the exercise is of low intensity, lactate is re-oxidized to pyruvate, due to NAD+ availability, and then completely oxidized to CO2 and H20, with a greater production of ATP than in anaerobic condition. In such conditions, the energy stored in NADH will be released, yielding on average 2.5 ATP per molecule of NADH.
In addition, lactate can be taken up by exclusively aerobic tissues, such as heart, to be oxidized to CO2 and H20.

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Energy cost of the Cori cycle

The Cori cycle results in a net consumption of 4 ATP.
The gluconeogenic leg of the cycle consumes 2 GTP and 4 ATP per molecule of glucose synthesized, that is, 6 ATP.
The ATP-consuming reactions are catalyzed by:

Since two molecules of lactate are required for the synthesis of one molecule of glucose, the net cost is 2 x 3 = 6 high energy bonds per molecule of glucose.
Conversely, the glycolytic leg of the cycle produces only 2 ATP per molecule of glucose.
Therefore, more energy is required to produce glucose from lactate than that obtained by anaerobic glycolysis in extrahepatic tissues. This explains why the Cori cycle cannot be sustained indefinitely.

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Is the Cori cycle a futile cycle?

The continuous breakdown and resynthesis of glucose, feature of the Cori cycle, might seem like a waste of energy. Indeed, this cycle allows the effective functioning of many extrahepatic cells at the expense of the liver and partly of the renal cortex. Below, two examples.

  • Red blood cells
    These cells, lacking a nucleus, ribosomes, and mitochondria, are smaller than most other cells. Their small size allows them to pass through tiny capillaries. However, the lack of mitochondria makes them completely dependent on anaerobic glycolysis for ATP production. Then, the lactate is partly disposed of by the liver and renal cortex.
  • Skeletal muscle
    Its cells, and particularly fast-twitch fibers contracting under low oxygen conditions, such as during intense exercise, produce much lactate.
    In such conditions, anaerobic glycolysis leads to the production of 2 ATP per molecule of glucose, 3 if the glucose comes from muscle glycogen, therefore, much lower than the 29-30 ATP produced by the complete oxidation of the monosaccharide. However, the rate of ATP production by anaerobic glycolysis is greater than that produced by the complete oxidation of glucose. Therefore, to meet the energy requirements of contracting muscle, anaerobic glycolysis is an effective means of ATP production. But this could lead to an intracellular accumulation of lactate, and a consequent reduction in intracellular pH. Obviously, such accumulation does not occur, due also to the Cori cycle, in which the liver pays the cost of the disposal of a large part of the muscle lactate, thereby allowing the muscle to use ATP for the contraction.
    And the oxygen debt, which always occurs after a strenuous exercise, is largely due to the increased oxygen demand of the hepatocytes, in which the oxidation of fatty acids, their main fuel, provides the ATP required for gluconeogenesis from lactate.
  • During trauma, sepsis, burns, or after major surgery, an intense cell proliferation occurs in the wound, that is a hypoxic tissue, and in bone marrow. This in turn results in greater production of lactate, an increase in the flux through the Cori cycle and an increase in ATP consumption in the liver, which, as previously said, is supported by an increase in fatty acid oxidation. Hence, the nutrition plan provided to these patients must be taken into account this increase in energy consumption.
  • A similar condition seems to occur also in cancer patients with progressive weight loss.
  • The Cori cycle is also important during overnight fasting and starvation.

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The Cori cycle and glucose-alanine cycle

These cycles are metabolic pathways that contribute to ensure a continuous delivery of glucose to tissues for which the monosaccharide is  the primary source of energy.
The main difference between the two cycles consists in the three carbon intermediate which is recycled: in the Cori cycle, carbon returns to the liver in the form of pyruvate, whereas in the glucose-alanine cycle in the form of alanine.
For more information, see: glucose-alanine cycle.

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Bender D.A. Introduction to nutrition and metabolism. 3rd Edition. Taylor & Francis, 2004

Berg J.M., Tymoczko J.L., and Stryer L. Biochemistry. 5th Edition. W. H. Freeman and Company, 2002

Iqbal S.A., Mido Y. Biochemistry. Discovery Publishing House, 2005 [Google eBook]

Nelson D.L., M. M. Cox M.M. Lehninger. Principles of biochemistry. 6th Edition. W.H. Freeman and Company, 2012

Newsholme E.A., Leech T.R. Functional biochemistry in health and disease. John Wiley J. & Sons, Inc., Publication, 2010 [Google eBook]

Rawn J.D. Biochimica. Mc Graw-Hill, Neil Patterson Publishers, 1990

Rosenthal M.D., Glew R.H. Medical biochemistry – Human metabolism in health and disease. John Wiley J. & Sons, Inc., Publication, 2009

Shils M.E., Olson J.A., Shike M., Ross A.C. Modern nutrition in health and disease. 9th Ed., by Lippincott, Williams & Wilkins, 1999

Stipanuk M.H., Caudill M.A. Biochemical, physiological, and molecular aspects of human nutrition. 3rd Edition. Elsevier health sciences, 2013 [Google eBooks]

Gluten: definition, structure, properties, wheat, cereal list

Gluten: contents in brief

What is gluten?

Fig. 1 – Wheat

Gluten is not a single protein but a mixture of cereal proteins, about 80% of its dry weight (for example gliadins and glutenins in wheat grains), lipids, 5-7%, starch, 5-10%, water, 5-8%, and mineral substances, <2%.
It forms when components naturally present in the grain of cereals, the caryopsis, and in their flours, are joined together by means of mechanical stress in aqueous environment, i.e. during the formation of the dough.
The term is also related to the family of proteins that cause problems for celiac patients (see below).
Isolated for the first time in 1745 from wheat flour by the Italian chemist Jacopo Bartolomeo Beccari, it can be extracted from the dough by washing it gently under running water: starch, albumins and globulins, that are water-soluble, are washed out, and a sticky and elastic mass remains, precisely the gluten (it means glue in Latin).

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Cereals containing gluten

It is present in:

  • wheat, such as:

durum wheat (Triticum durum); groats and semolina for dry pasta making are obtained from it;
common wheat or bread wheat (Triticum aestivum), so called because it is used in bread and fresh pasta making, and in bakery products;

  • rye (Secale cereale);
  • barley (Hordeum vulgare);
  • spelt, in the three species:

einkorn (Triticun monococcum);
emmer (Triticum dicoccum Schrank);
spelta (Triticum spelta);

  • khorasan wheat (Triticum turanicum); a variety of it is Kamut®;
  • triticale (× Triticosecale Wittmack), which is a hybrid of rye and common wheat;
  • bulgur, which is whole durum wheat, sprouted and then processed;
  • seitan, which is not a cereal, but a wheat derivative, also defined by some as “gluten steak”.

Given that most of the dietary intake of gluten comes from wheat flour, of which about 700 million tons per year are harvested, representing about 30% of the global cereal production, the following discussion will focus on wheat gluten, and mainly on its proteins.

Note: The term gluten is also used to indicate the protein fraction that remains after removal of starch and soluble proteins from the dough obtained with corn flour: however, this “corn gluten” is “functionally” different from that obtained from wheat flour.

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Cereal grain proteins

Fig. 2 – Cereal Grain Proteins

The study of cereal grain proteins, as seen, began with the work of Beccari. 150 years later, in 1924, the English chemist Osborne T.B., which can rightly be considered the father of plant protein chemistry, developed a classification based on their solubility in various solvents.
The classification, still in use today, divides plant proteins into 4 families.

  • Albumins, soluble in water.
  • Globulins, soluble in saline solutions; for example avenalin of oat.
  • Prolamins, soluble in 70% alcohol solution, but not in water or absolute alcohol.
    They include:

gliadins of wheat;
zein of corn;
avenin of oats;
hordein of barley;
secalin of rye.

They are the toxic fraction of gluten for celiac patients.

  • Glutelins, insoluble in water and neutral salt solutions, but soluble in acidic and basic solutions.
    They include glutenins of wheat.

Albumins and globulins are cytoplasmic proteins, often enzymes, rich in essential amino acids, such as lysine, tryptophan and methionine. They are found in the aleurone layer and embryo of the caryopsis.
Prolamins and glutelins are the storage proteins of cereal grains. They are rich in glutamine and proline, but very low in lysine, tryptophan and methionine. They are found in the endosperm, and are the vast majority of the proteins in the grains of wheat, corn, barley, oat, and rye.
Although Osborne classification is still widely used, it would be more appropriate to divide cereal grain proteins into three groups: structural and metabolic proteins, storage proteins, and defense proteins.

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Wheat gluten proteins

Proteins represent 10-14% of the weight of the wheat caryopsis (about 80% of its weight consists of carbohydrates).
According to the Osborne classification, albumins and globulins represent 15-20% of the proteins, while prolamins and glutelins are the remaining 80-85%, composed respectively of gliadins, 30-40%, and glutenins, 40-50%. Therefore, and unlike prolamins and glutelins in the grains of other cereals, gliadins and glutenins are present in similar amounts, about 40%.
Technologically, gliadins and glutenins are very important. Why?
These proteins are insoluble in water, and in the dough, that contains water, they bind to each other through a combination of intermolecular bonds, such as:

  • covalent bonds, i.e. disulfide bridges;
  • noncovalent bonds, such as hydrophobic interactions, van der Waals forces, hydrogen bonds, and ionic bonds.

Thanks to the formation of these intermolecular bonds, a three-dimensional lattice is formed. This structure entraps starch granules and carbon dioxide bubbles produced during leavening, and gives strength and elasticity to the dough, two properties of gluten widely exploited industrially.
In the usual diet of the European adult population, and in particular in Italian diet that is very rich in derivatives of wheat flour, gliadin and glutenin are the most abundant proteins, about 15 g per day. What does this mean? It means that gluten-free diet engages celiac patients both from a psychological and social point of view.

Note: The lipids of the gluten are strongly associated with the hydrophobic regions of gliadins and glutenins and, unlike what you can do with the flour, they are extracted with more difficulty (the lipid content of the gluten depends on the lipid content of the flour from which it was obtained).

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Gliadins: extensibility and viscosity

Fig. 3 – Wheat Grain Proteins

Gliadins are hydrophobic monomeric prolamins, of globular nature and with low molecular weight. On the basis of electrophoretic mobility in low pH conditions, they are separated into the following types:

  • alpha/beta, and gamma, rich in sulfur, containing cysteines, that are involved in the formation of intramolecular disulfide bonds, and methionines;
  • omega, low in sulfur, given the almost total absence of cysteine and methionine.

They have a low nutritional value and are toxic to celiac patients because of the presence of particular amino acid sequences in the primary structure, such as proline-serine-glutamine-glutamine and glutamine-glutamine-glutamine-proline.
Gliadins are associated with each other and with glutenins through noncovalent interactions; thanks to that, they act as “plasticizers” in dough making. Indeed, they are responsible for viscosity and extensibility of gluten, whose three-dimensional lattice can deform, allowing the increase in volume of the dough as a result of gas production during leavening. This property is important in bread-making.
Their excess leads to the formation of a very extensible dough.

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Glutenins: elasticity and toughness

Glutenins are polymeric proteins, that is, formed of multiple subunits, of fibrous nature, linked together by intermolecular disulfide bonds. The reduction of these bonds allows to divide them, by SDS-PAGE, into two groups.

  • High molecular weight (HMW) subunits, low in sulfur, that account for about 12% of total gluten proteins. The noncovalent bonds between them are responsible for the elasticity and tenacity of the gluten protein network, that is, of the viscoelastic properties of gluten, and so of the dough.
  • Low molecular weight (LMW) subunits, rich in sulfur (cysteine residues).
    These proteins form intermolecular disulfide bridges to each other and with HMW subunits, leading to the formation of a glutenin macropolymer.

Glutenins allow dough to hold its shape during mechanical (kneading) and not mechanical stresses (increase in volume due to both the leavening and the heat of cooking that increases the volume occupied by gases present) which is submitted. This property is important in pasta making.
If in excess, glutenins lead to the formation of a strong and rigid dough.

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Properties of wheat gluten

From the nutritional point of view, gluten proteins do not have a high biological value, being low in lysine, an essential amino acid. Therefore, a gluten-free diet does not cause any important nutritional deficiencies.
On the other hand, it is of great importance in food industry: the combination, in aqueous solution, of gliadins and glutenins to form a three-dimensional lattice, provides viscoelastic properties, that is, extensibility-viscosity and elasticity-tenacity, to the dough, and then, a good structure to bread, pasta, and in general, to all foods made with wheat flour.
It has a high degree of palatability.
It has a high fermenting power in the small intestine.
It is an exorphin: some peptides produced from intestinal digestion of gluten proteins may have an effect in central nervous system.

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Gluten-free cereals

The following is a list of gluten-free cereals, minor cereals, and pseudocereals used as foods.

  • Cereals

corn or maize (Zea mays)
rice (Oryza sativa)

  • Minor cereals
    They are defined “minor” not because they have a low nutritional value, but because they are grown in small areas and in lower quantities than wheat, rice and maize.

Fonio (Digitaria exilis)
Millet (Panicum miliaceum)
Panic (Panicum italicum)
Sorghum (Sorghum vulgare)
Teff (Eragrostis tef)
Teosinte; it is a group of four species of the genus Zea. They are plants that grow in Mexico (Sierra Madre), Guatemala and Venezuela.

  • Pseudocereals.
    They are so called because they combine in their botany and nutritional properties characteristics of cereals and legumes, therefore of another plant family.

Amaranth; the most common species are:

Amaranthus caudatus;
Amaranthus cruentus;
Amarantus hypochondriacus.

Buckwheat (Fagopyrum esculentum)
Quinoa (Chenopodium quinoa), a pseudocereal with excellent nutritional properties, containing fibers, iron, zinc and magnesium. It belongs to Chenopodiaceae family, such as beets.

  • Cassava, also known as tapioca, manioc, or yuca (Manihot useful). It is grown mainly in the south of the Sahara and South America. It is an edible root tuber from which tapioca starch is extracted.

It should be noted that naturally gluten-free foods may not be truly gluten-free after processing. Indeed, the use of derivatives of gliadins in processed foods, or contamination in the production chain may occur, and this is obviously important because even traces of gluten are harmful for celiac patients.

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Oats and gluten

Oats (Avena sativa) is among the cereals that celiac patients can eat. Recent studies have shown that it is tolerated by celiac patients, adult and child, even in subjects with dermatitis herpetiformis. Obviously, oats must be certified as gluten-free (from contamination).

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Beccari J.B. De Frumento. De bononiensi scientiarum et artium instituto atque Academia Commentarii, II. 1745:Part I.,122-127

Bender D.A. “Benders’ dictionary of nutrition and food technology”. 8th Edition. Woodhead Publishing. Oxford, 2006

Berdanier C.D., Dwyer J., Feldman E.B. Handbook of nutrition and food. 2th Edition. CRC Press. Taylor & Francis Group, 2007

Phillips G.O., Williams P.A. Handbook of food proteins. 1th Edition. Woodhead Publishing, 2011

Shewry P.R. and Halford N.G. Cereal seed storage proteins: structures, properties and role in grain utilization. J Exp Bot 2002:53(370);947-958. doi:10.1093/jexbot/53.370.947

Yildiz F. Advances in food biochemistry. CRC Press, 2009

Digestion of starch and alpha-amylase

Factors affecting relationship between starch and alpha-amylase

Fig. 1 – Spaghetti

Amylose and amylopectin, the two families of homopolysaccharides constituting starch, during their biosynthesis within vegetable cells, are deposited in highly organized particles called granules.
Granules have a partially crystalline structure and diameter ranging from 3 to 300 µm.
The access of the alpha-amylase, the enzyme that catalyzes the breakdown of amylose and amylopectin into maltose, maltotriose, and alpha-dextrins or alpha-limit dextrins, to carbohydrates making up granules varies as a function of:

  • amylose-amylopectin ratio;
  • temperature and packaging of amylose and amylopectin;
  • granules-associated proteins;
  • presence of fibers.

Amylose-amylopectin ratio

Starch for foodstuff use is obtained from various sources, the most important of which are corn (normal, waxy or high amylose content), potatoes, rice, tapioca and wheat.
Depending on botanical origin, molecular weight, degree of branching, and amylose-amylopectin ratio will vary.
Generally, there is 20-30% amylose and 70-80% amylopectin, even if there are starches with high amylose or amylopectin content (e.g. waxy corn). These differences justify the existence of starches with different chemical-physical characteristics and, to a certain extent, different digestibility.

  • corn: 24% amylose, 76% amylopectin;
  • waxy corn: 0,8% amylose, 99.2% amylopectin;
  • Hylon VII corn: 70% amylose, 30% amylopectin;
  • potatoes: 20% amylose, 80% amylopectin;
  • rice: 18.5 amylose, 81.5% amylopectin;
  • tapioca: 16.7% amylose, 83.3% amylopectin;
  • wheat: 25% amylose, 75% amylopectin.

Temperature and packaging of amylose and amylopectin

The chains of amylose, and to a lesser extent ramifications of amylopectin, thanks to the formation of hydrogen bonds with neighboring molecules and within the same molecules, have the tendency to aggregate. For this reason, pure amylose and amylopectin are poorly soluble in water at below 55 °C (131°F), and are more resistant to alpha-amylase action (resistant starch).
However, in aqueous solution, these granules hydrate increasing in volume of about 10%.
Above 55°C (131°F), the partially crystalline structure is lost, granules absorb further water, swell and pass to a disorganized structure, that is, starch gelatinization occurs, by which starch assumes an amorphous structure more easily attachable by alpha-amylase.

Granules-associated proteins

In granules, starch is present in association with proteins, many of which are hydrophobic, that means with low affinity for water. This association have the effect to hinder the interaction, in the intestinal lumen, between alpha-amylase, a polar protein, and the polysaccharides making up starch granules.
The physical processes to which cereals undergo before being eaten, such as milling or heating for several minutes, change the relationship between starch and the associated proteins, making it more available to α-amylase action.


Alpha-amylase activity may also be hindered by the presence of nondigestible polysaccharides, the fibers: cellulose, hemicellulose and pectin.


The presence of inhibitors, of both chemical and physical type, hinders starch digestion, even when pancreatic α-amylase secretion is normal. This means that a part of starch, ranging from 1% to 10%, may escape the action of the enzyme, being then metabolized by colonic bacteria.
Refined starch is instead hydrolyzed efficiently, even when there is an exocrine pancreatic insufficiency (EPI), condition in which alpha-amylase concentration in gut lumen may be reduced to 10% of the normal.


Arienti G. “Le basi molecolari della nutrizione”. Seconda edizione. Piccin, 2003

Belitz .H.-D., Grosch W., Schieberle P. “Food Chemistry” 4th ed. Springer, 2009

Bender D.A. “Benders’ Dictionary of Nutrition and Food Technology”. 8th Edition. Woodhead Publishing. Oxford, 2006

Cozzani I. and Dainese E. “Biochimica degli alimenti e della nutrizione”. Piccin Editore, 2006

Giampietro M. “L’alimentazione per l’esercizio fisico e lo sport”. Il Pensiero Scientifico Editore, 2005

Mahan LK, Escott-Stump S.: “Krause’s foods, nutrition, and diet therapy” 10th ed. 2000

Mariani Costantini A., Cannella C., Tomassi G. “Fondamenti di nutrizione umana”. 1th ed. Il Pensiero Scientifico Editore, 1999

Osorio-Dıaz P., Bello-Perez L.A., Agama-Acevedo E., Vargas-Torres A., Tovar J., Paredes-Lopez O. In vitro digestibility and resistant starch content of some industrialized commercial beans (Phaseolus vulgaris L.). Food Chem 2002;78:333-7 [Abstract]

Shils M.E., Olson J.A., Shike M., Ross A.C. “Modern nutrition in health and disease” 9th ed., by Lippincott, Williams & Wilkins, 1999

Stipanuk M.H.. “Biochemical and physiological aspects of human nutrition” W.B. Saunders Company-An imprint of Elsevier Science, 2000

Maltodextrin, fructose and endurance sports

Carbohydrate ingestion can improve endurance capacity and performance.
The ingestion of different types of carbohydrates, which use different intestinal transporters, can:

  • increase total carbohydrate absorption;
  • increase exogenous carbohydrate oxidation;
  • and therefore improve performance.

Glucose and fructose

When a mixture of glucose and fructose is ingested (in the analyzed literature, respectively 1.2 and 0.6 g/min, ratio 2:1, for total carbohydrate intake rate to 1.8 g/min), there is less competition for intestinal absorption compared with the ingestion of an iso-energetic amount of glucose or fructose,  two different intestinal transporters being involved. Furthermore, fructose absorption is stimulated by the presence of glucose.

This can:

The combined ingestion of glucose and fructose allows to obtain exogenous carbohydrate oxidation rate around 1,26 g/min, therefore, higher than the rate reported with glucose alone (1g/min), also in high concentration.
The observed difference (+0,26 g/min) can be fully attributed to the oxidation of ingested fructose.

Sucrose and glucose

The ingestion of sucrose and glucose, in the same conditions of the ingestion of glucose and fructose (therefore, respectively 1.2 and 0.6 g/min, ratio 2:1, for total carbohydrate intake rate to 1.8 g/min), gives similar results.

Glucose, sucrose and fructose

Very high oxidation rates are found with a mixture of glucose, sucrose, and fructose (in the analyzed literature, respectively 1.2, 0.6 and 0.6 g/min, ratio 2:1:1, for total carbohydrate intake rate to 2.4 g/min; however, note the higher amounts of ingested carbohydrates).

Maltodextrin and fructose

Maltodextrin and Fructose: Oxidation of Ingested Carbohydrates
Fig. 1 – Oxidation of Ingested Carbohydrates

High oxidation rates are also observed with combinations of maltodextrin and fructose, in the same conditions of the ingestion of glucose plus fructose (therefore, respectively 1.2 and 0.6 g/min, ratio 2:1, for total carbohydrate intake rate to 1.8 g/min).

Such high oxidation rates can be achieved with carbohydrates ingested in a beverage, in a gel or in a low-fat, low protein, low-fiber energy bar.

The best combination of carbohydrates ingested during exercise seems to be the mixture of maltodextrin and fructose in a 2:1 ratio, in a 5% solution, and in a dose around 80-90 g/h.

  • This mixture has the best ratio between amount of ingested carbohydrates and their oxidation rate and it means that smaller amounts of carbohydrates remain in the stomach or gut reducing the risk of gastrointestinal complication/discomfort during prolonged exercise (see brackets grafa in the figure).
  • A solution containing a combination of multiple transportable carbohydrates and a carbohydrate content not exceeding 5% optimizes gastric emptying rate and improves fluid delivery.

Example of a 5% carbohydrate solution containing around 80-90 g of maltodextrin and fructose in a 2:1 rate; ingestion time around 1 h.


During prolonged exercise, when high exogenous carbohydrate oxidation rates are needed, the ingestion of multiple transportable carbohydrates is preferred above that of large amounts of a single carbohydrate.
The best mixture seems to be maltodextrin and fructose, in a 2:1 ratio, in a 5% concentration solution, and at ingestion rate of around 80-90 g/h.


Burke L.M., Hawley J.A., Wong S.H.S., & Jeukendrup A. Carbohydrates for training and competition. J Sport Sci 2011;29:Sup1,S17-S27. doi:10.1080/02640414.2011.585473

Jentjens R.L.P.G., Moseley L., Waring R.H., Harding L.K., and Jeukendrup A.E. Oxidation of combined ingestion of glucose and fructose during exercise. J Appl Physiol 2004:96;1277-1284. doi:10.1152/japplphysiol.00974.2003

Jentjens R.L.P.G., Venables M.C., and Jeukendrup A.E. Oxidation of exogenous glucose, sucrose, and maltose during prolonged cycling exercise. J Appl Physiol 2004:96;1285-1291. doi:10.1152/japplphysiol.01023.2003

Jeukendrup A.E. Carbohydrate feeding during exercise. Eur J Sport Sci 2008:2;77-86. doi:10.1080/17461390801918971

Jeukendrup A.E. Nutrition for endurance sports: marathon, triathlon, and road cycling. J Sport Sci 2011:29;sup1, S91-S99. doi:10.1080/02640414.2011.610348

Prolonged exercise and carbohydrate ingestion

Prolonged Exercise: Open Water Swimming
Fig. 1 – Open Water Swimming

During prolonged exercise (>90 min), like marathon, Ironman, cross-country skiing, road cycling or open water swimming, the effects of supplementary carbohydrates on performance are mainly metabolic rather than central and include:

  • the provision of an additional muscle fuel source when glycogen stores become depleted;
  • muscle glycogen sparing;
  • the prevention of low blood glucose concentrations.

How many carbohydrates should an athlete take?

The optimal amount of ingested carbohydrate is that which results in the maximal rate of exogenous carbohydrate oxidation without causing gastrointestinal discomfort”. (Jeukendrup A.E., 2008).

Prolonged exercise: which carbohydrates should an athlete take?

Until 2004 it was believed that carbohydrates ingested during exercise (also prolonged exercise) could be oxidized at a rate no higher than 1 g/min, that is, 60 g/h, independent of the type of carbohydrate.
Exogenous carbohydrate oxidation is limited by their intestinal absorption and the ingestion of more than around 60 g/min of a single type of carbohydrate will not increase carbohydrate oxidation rate but it is likely to be associated with gastrointestinal discomfort (see later).
At intestinal level, the passage of glucose (and galactose) is mediated by a sodium dependent transporter called SGLT1. This transporter becomes saturated at a carbohydrate intake about 60 g/h and this (and/or glucose disposal by the liver that regulates its transport into the bloodstream) limits the oxidation rate to 1g/min or 60 g/h. For this reason, also when glucose is ingested at very high rate (>60 g/h), exogenous carbohydrate oxidation rates higher 1.0-1.1 g/min are not observed.

The rate of oxidation of ingested maltose, sucrose, maltodextrins and glucose polymer is fairly similar to that of ingested glucose.

Fructose uses a different sodium independent transporter called GLUT5. Compared with glucose, fructose has, like galactose, a lower oxidation rate, probably due to its lower rate of intestinal absorption and the need to be converted into glucose in the liver, again like galactose, before it can be oxidized.

Prolonged Exercise: Maltodextrin and Fructose: Oxidation of Ingested Carbohydrates
Fig. 1 – Oxidation of Ingested Carbohydrates

However, if the athlete ingests different types of carbohydrates, which use different intestinal transporters, exogenous carbohydrate oxidation rate can increase significantly.
It seems that the best mixture is maltodextrins and fructose.

Note: the high rates of carbohydrate ingestion may be associated with delayed gastric emptying and fluid absorption; this can be minimized by ingesting combinations of multiple transportable carbohydrates that enhance fluid delivery compared with a single carbohydrate. This also causes relatively little gastrointestinal distress.


The ingestion of different types of carbohydrates that use different intestinal transporters can:


Carbohydrate ingestion during exercise of relatively short duration and high intensity

Intermittent high intensity exercise and carbohydrate ingestion

High Intensity: During-Exercise Nutrition
Fig. 1- During-Exercise Nutrition

Carbohydrate ingestion during intermittent high intensity or prolonged (>90 min) sub-maximal exercise can:

  • increase exercise capacity;
  • improve exercise performance;
  • postpone fatigue.

The intake of very small amounts of carbohydrates or carbohydrate mouth rinsing (for example with a 6% maltodextrin solution) may improve exercise performance by 2-3% when the exercise is of relatively short duration (<1 h) and high intensity (>75% VO2max), that is, an exercise not limited by the availability of muscle glycogen stores, given adequate diet.
The underlying mechanisms for the ergogenic effect of carbohydrates during this type of activity are not metabolic but may reside in the central nervous system: it seems that carbohydrates are detected in the oral cavity by unidentified receptors, promoting an enhanced sense of well-being and improving pacing.
These effects are independent of taste or sweet and non-sweet of carbohydrates but are specific to carbohydrates.

It should be noted that performance effects with drink ingestion are similar to the mouth rinse; therefore athletes, when they don’t complain of gastrointestinal distress when ingesting too much fluid, may have an advantage taking the drink (in endurance sports, dehydration and carbohydrate depletion are the most likely contributors to fatigue).


It seems that during exercise of relatively short duration (<1 h) and high intensity (>75% VO2max) it is not necessary to ingest large amounts of carbohydrates: a carbohydrate mouth rinsing or the intake of very small amounts of carbohydrates may be sufficient to obtain a performance benefit.


Hydration before endurance sports

Dehydration and endurance sports

Fig. 1 – Pre-hydration

In endurance sports, like Ironman, open water swimming, road cycling, marathon, or cross-country skiing, the most likely contributors to fatigue are dehydration and carbohydrate (especially liver and muscle glycogen) depletion.


Due to sweat loss needed to dissipate the heat generated during exercise, dehydration can compromise exercise performance.
It is important to start exercising in a euhydrated state, with normal plasma electrolyte levels, and attempt to maintain this state during any activity.
When an adequate amount of beverages with meals are consumed and a protracted recovery period (8-12 hours) has elapsed since the last exercise, the athlete should be euhydrated.
However, if s/he has not had adequate time or fluids/electrolytes volume to re-establish euhydration, a pre-hydration program may be useful to correct any previously incurred fluid-electrolyte deficit prior to initiating the next exercise.

Pre-hydration program

If during exercise the nutritional target is to reduce sweat loss to less than 2–3% of body weight, prior to exercise the athlete should drink beverages at least 4 hours before the start of the activity, for example, about 5-7 mL/kg body weight.
But if the urine is still dark (highly concentrated) and/or is minimal, s/he should slowly drink more beverages, for example, another 3-5 mL/kg body weight, about 2 hours before the start of activity so that urine output normalizes before starting the event.

It is advisable to consume small amounts of sodium-containing foods or salted snacks and/or beverages with sodium that help to stimulate thirst and retain the consumed fluids.
Moreover, palatability of the ingested beverages is important to promote fluid consumption before, during, and after exercise. Fluid palatability is influenced by several factors, such as:

  • temperature, often between 15 and 21 °C;
  • sodium content;
  • flavoring.

And hyper-hydration?

Hyper-hydration, especially in the heat, could improve thermoregulation and exercise performance, therefore, it might be useful for those who lose body water at high rates, as during exercise in hot conditions or who have difficulty drinking sufficient amounts of fluid during exercise.
However there are several risks:

  • fluids that expand the intra- and extra-cellular spaces (e.g. glycerol solutions plus water) greatly increase the risk of having to void during exercise;
  • hyper-hydration may dilute and lower plasma sodium which increases the risk of dilutional hyponatraemia, if during exercise, fluids are replaced aggressively.

Finally, it must be noted that plasma expanders or hyper-hydrating agents are banned by the World Anti-Doping Agency (WADA).


“Pre-hydrating with beverages, if needed, should be initiated at least several hours before the exercise task to enable fluid absorption and allow urine output to return toward normal levels. Consuming beverages with sodium and/or salted snacks or small meals with beverages can help stimulate thirst and retain needed fluids” (Sawka et al., 2007).


Ingestion of solid, liquid or gel carbohydrates 60 min before exercise

Liquid Carbohydrate Ingestion
Fig. 1 – Liquid Carbohydrate Ingestion

The form of carbohydrates ingested before exercise may have different effects on both metabolism and performance. Moreover, the ingestion of solid foods slows gastric empty, digestion and absorption rates compared with liquid foods and this has a different impact on glycemia.
For these reasons, several studies have investigated the effects of the form of carbohydrates on glycemic responses, oxidation rates and performance.

  • Studies comparing solid versus liquid carbohydrates and solid versus gel carbohydrates have found no difference in glycemic responses between groups.
  • Studies that have investigated difference in performance effects have found no differences.
  • Furthermore, no differences are found in carbohydrate oxidation rates between the carbohydrate ingestion in the three forms during exercise.

Therefore, it seems not to be the form of ingested carbohydrates that can enhance or reduce performance (in addition, even glycogen synthesis doesn’t vary; study conducted with liquid or solid carbohydrates).


It is advisable that athlete ingests whichever form of carbohydrates best suits, based on his experience and cost-effectiveness of the product.