The pentose phosphate pathway

The pentose phosphate pathway: contents in brief

What is the pentose phosphate pathway?

The pentose phosphate pathway, also called the phosphogluconate pathway, is a metabolic pathway, common to all living organisms, for the oxidation of glucose alternative to glycolysis, from which it branches downstream of glucose 6-phosphate synthesis, and whose main functions are the production, in variable ratios, of NADPH, a reduced coenzyme, and ribose 5-phosphate, a five-carbon phosphorylated sugar, namely, a pentose phosphate, hence the name pentose phosphate pathway.

Pentose Phosphate Pathway
Fig. 1 – The Pentose Phosphate Pathway

In addition to the production of NADPH and ribose 5-phosphate, this pathway has other functions, both anabolic and catabolic.

  • In yeasts and many bacteria it is involved in the catabolism of the five carbon sugars ribose, xylose and arabinose.
    In humans too, it is involved in catabolism of the aforementioned pentoses and of the less common sugars with three, four and seven carbon atoms derived from diet, as well as of:

pentoses derived from the catabolism of structural carbohydrates;
ribose 5-phosphate derived from nucleotide catabolism.

  • In photosynthetic organisms it contributes to carbon dioxide (CO2) fixation during the Calvin cycle.
  • In addition to ribose 5-phosphate, it also provides other intermediates for various biosynthetic processes, such as:

erythrose 4-phosphate, used for the synthesis of phenylalanine, tryptophan, and tyrosine, the three aromatic amino acids;
ribulose 5-phosphate, used for riboflavin synthesis;
sedoheptulose 7-phosphate which, in Gram-negative bacteria, is used for the synthesis of heptose units in the lipopolysaccharide layer of the outer membrane.

The phosphogluconate pathway, branching from glycolysis, is also called the hexose monophosphate shunt.

It has been estimated that more than 10% of glucose is shuttled through this metabolic pathway that, noteworthy, although it oxidizes the monosaccharide, does not involve any direct production or consumption of ATP.

⇑ Back to the top ⇑

The elucidation of the pentose phosphate pathway

The first evidence of the existence of the phosphogluconate pathway was obtained in the 1930s by the studies of Otto Warburg, Nobel Prize in Physiology or Medicine in 1931, who discovered NADP during studies on the oxidation of glucose 6-phosphate to 6-phosphogluconate.
Further indications came from the observation that glucose continued to be metabolized in tissues even in the presence of glycolysis inhibitors, such as fluoride and iodoacetate ions, inhibitors of enolase (EC 4.2.1.11) and glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12), respectively.
However, the pathway was fully elucidated only in 1950s thanks to the work of several researchers and primarily of Efraim Racker, Fritz Lipmann, Nobel Prize in Physiology or Medicine in 1953 thanks to the discovery of coenzyme A, Bernard Horecker and Frank Dickens.

⇑ Back to the top ⇑

Functions of NADPH and ribose 5-phosphate

NADPH is needed for reductive biosynthesis, such as the synthesis of fatty acids, cholesterol, steroid hormones and of two non-essential amino acids, proline and tyrosine, from glutamate and phenylalanine, respectively, as well as for the reduction of oxidized glutathione. In such reactions the reduced coenzyme acts as an electron donor, or rather as a donor of a hydride ion (:H), namely, a proton and two electrons.

Pentose Phosphate Pathway
Fig. 2 – NADPH

Note: In vertebrates, about half of the NADPH necessary for the reductive steps of fatty acid synthesis derives from the pentose phosphate pathway, and the rest from the malic enzyme (EC 1.1.1.40) reaction.

Malate + NADP+ ↔ Pyruvate + NADPH + H+ + HCO3

Ribose 5-phosphate is used for the synthesis of nucleotides and nucleic acids, DNA and RNA, of ATP, coenzymes such as coenzyme A, NAD, NADP and FAD, and of the essential amino acids tryptophan and histidine.

Pentose Phosphate Pathway
Fig. 3 – Ribose 5-Phosphate

Ribose 5-phosphate is not used as such; it is activated to  5-phosphoribosyl 1-pyrophosphate (PRPP), in the reaction catalyzed by ribose phosphate pyrophosphokinase or PRPP synthase (EC 2.7.6.1).

Ribose 5-phosphate + ATP → 5-Phosphoribosyl 1-pyrophosphate + AMP

⇑ Back to the top ⇑

Where does the pentose phosphate pathway occur?

In animal cells and bacteria, the hexose monophosphate shunt, as well as glycolysis, fatty acid synthesis, and most of the reactions of gluconeogenesis, occurs in the cytosol. And, considering glycolysis, gluconeogenesis and the pentose phosphate pathway we can state that these three metabolic pathways are interconnected through several shared enzymes and/or intermediates.
In plant cells the phosphogluconate pathway occurs in plastids, and its intermediates can reach the cytosol through membrane pores of these organelles.

In humans, the level of expression of the enzymes of the pathway varies widely from tissue to tissue.
Relatively high levels are found in the liver, adrenal cortex, testicles and ovaries, thyroid, mammary glands during lactation, and in red blood cells. In all these sites, constant supply of NADPH is required to support reductive biosynthesis and/or to counteract the effects of reactive oxygen species (ROS) on sensitive cellular structures, such as DNA, membrane lipids, and proteins by the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH), in the reaction catalyzed by glutathione reductase (EC 1.8.1.7).

GSSG + NADPH + H+ → 2 GSH + NADP+

Note: Glutathione is a tripeptide, namely, γ-glutamyl-cysteinyl-glycine, that in the reduced state contains, in the cysteine residue, a sulfhydryl group (-SH), hence the abbreviation GSH. It is the major intracellular antioxidant in erythrocytes, as in most other cells.

The defense against ROS effects is particularly important in cells such as red blood cells and the cells of the cornea and crystalline lens that are directly exposed to oxygen.
High levels of the of the phosphogluconate pathway enzymes  are also present in rapidly dividing cells such as enterocytes, skin cells, bone marrow cells, those of the early embryo and, in pathological conditions, cancer cells. Indeed, these cell types require a constant supply of ribose 5-phosphate for nucleic acid synthesis.
Conversely, these enzymes are present in very low levels in skeletal muscle, in which the pentose phosphate pathway is virtually absent and glucose 6-phosphate is primarily used for energy production via glycolysis and the citric acid cycle.

⇑ Back to the top ⇑

The two phases of the pentose phosphate pathway

Conceptually, the hexose monophosphate shunt can be viewed as consisting of two phases.

  • In the first phase, the oxidative phase, glucose 6-phosphate, a six-carbon phosphorylated sugar, is converted to ribulose 5-phosphate, a five-carbon phosphorylated sugar, with the concomitant formation of two molecules of NADPH and the release of C-1 of glucose as CO2.
  • In the second phase, the nonoxidative phase, several phosphorylated carbohydrates are produced, whose fate depends on the relative needs for NADPH, ribose 5-phosphate, and ATP of the cell.

⇑ Back to the top ⇑

The steps of the oxidative phase of the pentose phosphate pathway

The oxidative phase of the phosphogluconate pathway consists of three steps, two irreversible oxidations, the first and third reactions, and a hydrolysis.

Pentose Phosphate Pathway
Fig. 4 – The Oxidative Phase of the Pentose Phosphate Pathway

Below, the reaction mechanisms of the involved enzymes are explained and, with regard to glucose 6-phosphate dehydrogenase or G6PD (EC 1.1.1.49), the regulation of the enzymatic activity too.

⇑ Back to the top ⇑

The oxidation of glucose 6-phosphate to 6-phosphoglucono-δ-lactone

In the first step of the oxidative phase, glucose 6-phosphate dehydrogenase catalyzes the oxidation of glucose 6-phosphate to 6-phosphoglucono-δ-lactone, an intramolecular ester, via the transfer of a hydride ion from carbon 1of glucose 6-phosphate to NADP+, that acts as oxidizing agent.

Pentose Phosphate Pathway
Fig. 5 – The Glucose-6-phosphate Dehydrogenase Reaction

Note: This reaction yields the first molecule of NADPH of the pentose phosphate pathway.

The reaction catalyzed by glucose 6-phosphate dehydrogenase is unique to the pathway. And, similarly to what happens in most metabolic pathways, also in this case the first reaction unique to  the pathway, generally known as a committed step, is an essentially irreversible step, with a ΔG in the liver of -17.6 kJ/mol (-4.21 kcal/mol), and is highly allosterically regulated.  And the enzyme is indeed the major control point for the flow of metabolites through the pathway.
In humans, the highest levels of G6PD are found in neutrophils and macrophages, phagocytic cells in which, during inflammation, NADPH is used for to produce superoxide radicals (O2-.) from molecular oxygen in the reaction catalyzed by NADPH oxidase (EC 1.6.3.1).

2 O2 + NADPH → 2 O2-. + NADP+ + H+

In turn, superoxide radicals can be used for the synthesis for defensive purposes, namely, to kill phagocytized microorganisms, of other ROS but also of reactive nitrogen species (RNS), such as:

  • hydrogen peroxide (H2O2), in the reaction catalyzed by superoxide dismutase or SOD (EC 1.15.1.1)

2 O2-. + 2 H+ → H2O2 + O2

  • peroxynitrite (O=N–O–O), in the reaction with nitric oxide (·NO)

O2. + ·NO → O=N–O–O

  • hydroperoxide radical (HOO·)

O2-. + H+ → HOO·

⇑ Back to the top ⇑

Catalytic mechanism  of glucose 6-phosphate dehydrogenase

The catalytic mechanism of the enzyme has been studied in great detail in the microorganism Leuconostoc mesenteroides, whose glucose 6-phosphate dehydrogenase has the peculiar characteristic of being able to use NAD+ and/or NADP+ as coenzyme.

Pentose Phosphate Pathway,
Fig. 6 – G6PD: Catalytic Mechanism

The enzyme does not require metal ions for its activity; one of the amino acids in the active site acts as a general base being able to abstract a hydride ion from the hydroxyl group bound to C1 of glucose 6-phosphate.
In the bacterial enzyme this is carried out by the atom Nɛ2 of the imidazole ring of a histidine side chain. This nitrogen atom has a lone pair of electrons able to make a nucleophilic attack. This causes glucose 6-phosphate, a cyclic hemiacetal with carbon 1 in the aldehyde oxidation state, to be oxidized to a cyclic ester, namely, a lactone. This allows the transfer of an hydride ion from C1 of glucose to C4 of the nicotinamide ring of NADP+ to form NADPH.
Because such histidine is conserved in many of the glucose 6-phosphate dehydrogenases sequenced, it is likely that this catalytic mechanism can be generalized to all glucose 6-phosphate dehydrogenases.

⇑ Back to the top ⇑

Regulation of glucose 6-phosphate dehydrogenase activity

Glucose 6-phosphate dehydrogenase is the major control point of carbon flow through the pentose phosphate pathway, and then the major control point for the rate of NADPH synthesis.
In humans, the enzyme exists in two forms: the inactive monomeric form, and the active form that exists in a dimer-tetramer equilibrium.
One of the main modulators of its activity is the cytosolic NADP+/NADPH ratio. High levels of NADPH inhibit enzyme activity, because NADPH is a potent competitive inhibitor of G6PD, whereas NADP+ is required for the catalytic activity and for the maintenance of the active conformation. In fact, the binding of the oxidized coenzyme to a specific site close to the dimer interface, but distant from the active site, is required to maintain its dimeric conformation.

Pentose Phosphate Pathway
Fig. 7 – Regulation of G6PD activity

Under most metabolic conditions the NADP+/NADPH ratio is low, less NADP+ is available to bind to the enzyme, and hence enzyme activity is reduced, regardless of gene expression levels. Under these conditions the oxidative phase is virtually inactive.
Conversely, in cells in which metabolic pathways and/or reactions using NADPH are particularly active, the reduction of cytosolic NADPH concentration, and hence the increase in NADP+ concentration occurs. This leads to an increase in glucose 6-phosphate dehydrogenase activity, and to the activation of the oxidative phase of the hexose monophosphate pathway.
Therefore it is possible to state that the fate of glucose 6-phosphate, an intermediate common to both glycolysis and the phosphogluconate pathway, also depends on the current needs for NADPH.
A second mechanism for the regulation of glucose 6-phosphate dehydrogenase activity calls into question the accumulation of acyl-CoAs, intermediates in fatty acid synthesis. These molecules, by binding to the dimeric form of the enzyme, lead to dissociation into the constitutive monomers, and then to the loss of the catalytic activity.
Insulin up-regulates the expression of the genes for glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Therefore, in the well-fed state, the hormone increases carbon flow through the pentose phosphate pathway and then the production of NADPH.
Note: Insulin also promotes the synthesis of fatty acids.

⇑ Back to the top ⇑

Hydrolysis of 6-phosphoglucono-δ-lactone to 6-phosphogluconate

In the second step of the oxidative phase 6-phosphoglucono-δ-lactone is hydrolyzed to 6-phosphogluconate, a linear molecule.
6-Phosphoglucono-δ-lactone is hydrolytically unstable and undergoes a nonenzymatic ring-opening, a reaction that occurs at a significant rate. However, in the cell this ring-opening reaction, an hydrolysis, is accelerated by the catalytic action of 6-phosphogluconolactonase (EC 3.1.1.31).

Pentose Phosphate Pathway
Fig. 8 – The 6-Phosphogluconolactonase Reaction

⇑ Back to the top ⇑

Oxidative decarboxylation of 6-phosphogluconate to ribulose 5-phosphate

In the third step of the oxidative phase, 6-phosphogluconate undergoes an oxidative decarboxylation to form ribulose 5-phosphate, a keto pentose, CO2, and a molecule of NADPH. The reaction is catalyzed by 6-phosphogluconate dehydrogenase (EC 1.1.1.44), enzyme that requires the presence of magnesium ions, Mg2+.

Pentose Phosphate Pathway
Fig. 9 – The 6-Phosphogluconate Dehydrogenase Reaction

Note: This reaction yields the second molecule of NADPH of the pentose phosphate pathway.

⇑ Back to the top ⇑

Catalytic mechanism of 6-phosphogluconate dehydrogenase

The catalytic mechanism of the enzyme is similar to that of isocitrate dehydrogenase (EC 1.1.1.41), an enzyme of the citric acid cycle. It consists of an acid-base catalysis proceeding through a three step mechanism in which two strictly conserved residues, a lysine (Lys), and a glutamate (Glu), are involved; in humans, Lys185 and the Glu192. Lysine acts as acid/base group, whereas glutamate as an acid.

Pentose Phosphate Pathway
Fig. 10 – 6-Phosphogluconate Dehydrogenase: Catalytic Mechanism

In the first step, the oxidative step, 6-phosphogluconate is oxidized to a β-keto acid, the 3-keto-6-phosphogluconate.
In this step the ε-amino group of the aforementioned lysine acts as a general base, as a nucleophile, abstracting a proton from the hydroxyl group bound to C-3. Then, the transfer of a hydride ion from C-3 to C-4 of the nicotinamide ring of the NADP+occurs. This leads to the formation of the 3-keto intermediate and a molecule of NADPH that leaves the active site.
In the second step, the decarboxylation step, 3-keto-6-phosphogluconate, that is very susceptible to decarboxylation, is converted to the cis-1,2-enediol of ribulose 5-phosphate, a high energy intermediate. In this step the aforementioned lysine acts as a general acid donating an H+ at the C-3 carbonyl oxygen, and the C-1 of glucose 6-phosphate is lost as CO2.
Finally,  6-phosphogluconate dehydrogenase catalyzes a stereospecific keto-enol conversion leading to the formation of ribulose 5-phosphate. In this step, the aforementioned glutamic acid residue acts as a general acid donating an H+ to the C-1 of cis-1,2-enediol intermediate, while the ε-amino group of the lysine accepts a proton from the hydroxyl group bound to the C-2. The result is the formation of ribulose 5-phosphate.

Note: An enediol is an organic compound containing two carbon atoms linked by a double bond and an hydroxyl group (-OH) bound to both carbon atoms. The enediol can have cis or trans configuration.

Pentose Phosphate Pathway
Fig. 11 – Enediol Configuration

For example, in the plant world many polyphenols possess enediol structures.

Therefore, the oxidative phase of the pentose phosphate pathway ends with the production of ribulose 5-phosphate, namely, the substrate for the reactions of the non-oxidative phase.
The overall equation of the oxidative phase is:

3 Glucose 6-phosphate + 6 NADP+ + H2O → 6 NADPH + 6 H+ + 3 CO2 + 3 Ribulose 5-phosphate

⇑ Back to the top ⇑

The steps of the nonoxidative phase of the pentose phosphate pathway

The nonoxidative phase of the pathway consists of five steps, all freely reversible, in which a series of interconversions of phosphorylated sugars occurs.
This phase begins with two reactions: the isomerization and epimerization of ribulose 5-phosphate to form ribose 5-phosphate and xylulose 5-phosphate, respectively.

Note: Enzymatic isomerizations and epimerizations play an important role in carbohydrate metabolism.
Epimerases (EC 5.1), a subclass of Isomerases (EC 5.), catalyze the configurational reversal at an asymmetric carbon atom, usually by a deprotonation/protonation mechanism.
In isomerization reactions, the interchange of chemical groups occurs between carbon atoms.

⇑ Back to the top ⇑

Isomerization of ribulose 5-phosphate to ribose 5-phosphate

In the isomerization reaction, ribulose 5-phosphate, a ketose, is converted to the corresponding aldose, ribose 5-phosphate. This reaction is catalyzed by phosphopentose isomerase or ribose 5-phosphate isomerase (EC 5.3.1.6).

Pentose Phosphate Pathway
Fig. 12 – The Phosphopentose Isomerase Reaction

⇑ Back to the top ⇑

Catalytic mechanism of phosphopentose isomerase or ribose 5-phosphate isomerase

The catalytic mechanism of the enzyme is similar to that of phosphohexose isomerase (EC 5.3.1.9), an enzyme of the glycolytic pathway, and leads to the formation of the high energy intermediate cis-1,2-enediol of ribulose 5-phosphate. The formation of the cis-1,2-enediol intermediate occurs via a proton-transfer mechanism common to the aldose-ketose isomerizations.
The proposed catalytic mechanism for phosphopentose isomerase from E. coli, in the direction of ribulose 5-phosphate formation from ribose 5-phosphate, as in the Calvin cycle of photosynthesis, is described below.

Pentose Phosphate Pathway
Fig. 13 – Phosphopentose Isomerase: Catalytic Mechanism

In the first step, the furanose ring of the substrate is opened, opening induced by the interaction with an aspartic acid residue (Asp81) that accepts a proton from the hydroxyl group bound to C-1, whereas it is likely that water is the proton donor.
Note: The opening of the furanose ring is quite rare in solution (<0.5%).
Once the chain is opened, a glutamic acid residue (Glu103) acts as a general base, as a nucleophile, abstracting a proton bound to the C-2, whereas Asp81 donates a proton. As a result, cis-1,2-enediol intermediate is produced.
Finally, the protonated Glu103 acts as a general acid and donates an H+ at C-1 of the cis-1,2-enediol intermediate, while Asp81 acts as a general base accepting a proton from the hydroxyl group bound to C-2. The result is the formation of ribulose 5-phosphate.

During the synthesis of ribose 5-phosphate from ribulose 5-phosphate phosphopentose isomerase works in reverse.

⇑ Back to the top ⇑

Epimerization of ribulose 5-phosphate to xylulose 5-phosphate

The other metabolic fate of ribulose 5-phosphate in the pentose phosphate pathway is to be epimerized to xylulose 5-phosphate, a ketose like ribulose 5-phosphate, in the reaction catalyzed by phosphopentose epimerase (EC 5.1.3.1).

Pentose Phosphate Pathway
Fig. 14 – The Phosphopentose Epimerase Reaction

Note: Xylulose 5-phosphate is a regulatory molecule that inhibits gluconeogenesis and stimulates glycolysis by controlling the levels of fructose 2,6-bisphosphate in the liver.

⇑ Back to the top ⇑

Catalytic mechanism of phosphopentose epimerase

Also this reaction, like those catalyzed by 6-phosphogluconate dehydrogenase and ribose 5-phosphate isomerase, proceeds through the formation of an enediol intermediate, but with the double bond between C-2 and C-3 and not between C-1 and C-2.
During the reaction an amino acid residue present in the active site of the enzyme acts as a general base, as a nucleophile, and abstracts a proton bound to the C-3, leading to the formation of the cis-2,3-enediol intermediate. Then, an acidic amino acid residue donates a proton to C-3, but from the opposite side, hence, with an inversion at C-3 to form xylulose 5-phosphate.

Pentose Phosphate Pathway
Fig. 15 – Phosphopentose Epimerase: Catalytic Mechanism

To this point, the hexose monophosphate shunt has generated for each molecule of glucose 6-phosphate metabolized:

  • a pool of three pentose 5-phosphates, namely, ribulose 5-phosphate, ribose 5-phosphate and xylulose 5-phosphate, that coexist at equilibrium;
  • 2 molecules of NADPH.

In the following three steps, from the sixth to the eighth, transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2), two enzymes unique to the pentose phosphate pathway, catalyze a series of rearrangements of the carbon skeletons leading to the formation of three-, four-, six-, and seven carbon units, that can be used for various metabolic purposes, depending on the needs of the cell.
Analyzing the flow of metabolites through the different metabolic pathways, the concerted action of transketolase and transaldolase allows the interaction of the pentose phosphate pathway, in particular of its non-oxidative phase, with glycolysis, and gluconeogenesis, as well as with the pathways leading to the formation of numerous vitamins, coenzymes and nucleic acid precursors.

⇑ Back to the top ⇑

Transketolase: step 6 and 8

Transketolase is the rate-limiting enzyme of the non-oxidative phase of the pentose phosphate pathway, and the first enzyme that acts downstream of ribose 5-phosphate isomerase and phosphopentose epimerase.
Discovered independently in 1953 by Horecker and Racker, and named by Racker, it catalyzes in the sixth and eighth steps, the transfer of a two carbon unit from a ketose, the donor substrate, namely, xylulose 5-phosphate, sedoheptulose 7-phosphate or fructose 6-phosphate, to an aldose, the acceptor substrate, ribose 5-phosphate, glyceraldehyde 3-phosphate or erythrose 4-phosphate.

Pentose Phosphate Pathway
Fig. 16 – Transketolase: the General Reaction

Taking as an example the forward reactions, in the sixth step, the ketose donor is xylulose 5-phosphate, whereas the aldose acceptor is ribose 5-phosphate, to form glyceraldehyde 3-phosphate, the remaining three-carbon fragment from xylulose 5-phosphate, and sedoheptulose 7-phosphate, a seven-carbon sugar that will be used in the next step, the seventh.

Pentose Phosphate Pathway
Fig. 17 – Transketolase: Step 6

In the eighth step, the ketose donor is xylulose 5-phosphate, whereas the aldose acceptor is erythrose 4-phosphate, to form another glyceraldehyde 3-phosphate and a fructose 6-phosphate.

Pentose Phosphate Pathway
Fig. 18 – Transketolase: Step 8

It should be noted that three of the four products of the reactions catalyzed by this enzyme, two molecules of glyceraldehyde 3-phosphate and one of fructose 6-phosphate, are also intermediates of glycolysis.

⇑ Back to the top ⇑

Transketolase and thiamine pyrophosphate

Transketolase is an enzyme that requires thiamine pyrophosphate (TPP) as a cofactor.
Thiamine pyrophosphate is the biologically active form of thiamin or vitamin B1, and is tightly bound to the enzyme.

Pentose Phosphate Pathway
Fig. 19 – Thiamine Pyrophosphate

Other enzymes that require TPP as a cofactor are:

  • pyruvate decarboxylase (EC 4.1.1.1), that is involved in alcoholic fermentation;
  • pyruvate dehydrogenase or E1 component (EC 1.2.4.1) of the pyruvate dehydrogenase complex;
  • alpha-keto acid dehydrogenase or E1 component (EC 1.2.4.4) of the branched-chain alpha-ketoacid dehydrogenase complex;
  • alpha-ketoglutarate dehydrogenase or E1 component (EC 1.2.4.2) of the alpha-ketoglutarate dehydrogenase complex, an enzyme of the citric acid cycle.

Thiamine pyrophosphate is involved in the transfer of activated aldehyde intermediates by stabilizing the two-carbon carbanion formed during the reaction.

⇑ Back to the top ⇑

Catalytic mechanism of transketolase

The carbon atom between the sulfur and nitrogen atoms of the thiazolium ring of thiamine pyrophosphate, namely, the C-2 atom, is much more acidic than most =CH groups found in other molecules because of adjacent positively charged nitrogen atom that electrostatically stabilizes the carbanion resulting from dissociation of the proton. This causes the C-2 proton to be easily dissociable to form a carbanion, i.e. a carbon atom with a negative charge. Such proton abstraction is catalyzed by transketolase.
The carbanion attacks the carbonyl carbon of the substrate, in the step 6, xylulose 5-phosphate or, in the reverse reaction, sedoheptulose 7-phosphate, whereas in the step 8, xylulose 5-phosphate or, in the reverse reaction, fructose 6-phosphate.
Taking as an example the forward reaction of step 6, the covalent adduct between thiamine pyrophosphate and xylulose 5-phosphate undergoes fragmentation, via the cleavage of the C2-C3 bond of xylulose 5-phosphate, to form glyceraldehyde 3-phosphate, that is released, and a two carbon unit, a negatively charged hydroxyethyl group, that remains bound to C-2 of the thiazolium ring.

Pentose Phosphate Pathway
Fig. 20 – Transketolase: Catalytic Mechanism

The negative charge on the hydroxyethyl intermediate, that is, the carbanion intermediate, is stabilized by the thiazolium ring of thiamine pyrophosphate because of the positively charged nitrogen atom that acts as an electron trap or electron sink. Therefore, thiazolium ring provides an electron deficient or electrophilic structure that can delocalize by resonance the carbanion electrons.
Then, the condensation occurs between the hydroxyethyl group and the ribose 5-phosphate, the acceptor aldehyde substrate, via carbanion attack on the aldehyde carbon of ribose 5-phosphate, to form a covalent adduct bound to thiamine pyrophosphate.
Finally, the cleavage of the adduct leads to the release of sedoheptulose 7-phosphate, and regenerates the TPP carbanion.

Note: In addition to xylulose 5-phosphate, sedoheptulose 7-phosphate and fructose 6-phosphate, transketolase can use as substrates other 2-keto sugars in a similar way, as well as a variety of different aldose phosphates.

⇑ Back to the top ⇑

Carbanions and carbocations

A carbanion is a species containing a negatively charged,trivalent carbon.
It is an highly reactive reaction intermediate, resulting from the heterolytic cleavage of a bond between a carbon atom and another atom or group.
The carbanions, having an unshared electron pair, are strong nucleophiles and bases, and attack a proton or an electrophilic center, like a polarized or positively charged center, to form a covalent bond. Due to their reactivity, and with few exceptions, they are transient intermediates in organic reactions, like free radicals and carbocations.
A carbocation is a species containing a positively charged,trivalent carbon. Like free radicals, carbocations are species characterized by an electron deficiency, having not eight but only six electrons in their valence shell. Free radicals have seven electrons in their valence shell. Because of this electronic deficiency, free radicals and carbocations are strong electrophiles, and, like carbanions, are highly reactive reaction intermediates. During the reactions they accept electrons, one the free radicals, two the carbocations, to achieve the stable octet configuration.

The cleavage of a covalent bond between two carbon atom, and more generally between atom A and B, can take place via two different mechanisms: homolytic or heterolytic cleavage.

  • In homolytic bond cleavage, each atom takes one of the two electrons holding the atoms together to form two species with an odd number of electrons, namely, with one unpaired electron, without charge, called free radicals.
  • In heterolytic bond cleavage, two charged species, namely, a cation and an anion, are produced, because of one atom retains both bonding electrons.
Pentose Phosphate Pathway
Fig. 21 – Homolytic and Heterolytic Cleavage

Note: Heterolytic cleavage is more common than homolytic cleavage.

⇑ Back to the top ⇑

Transaldolase: step 7

Discovered in 1953 by Horecker and Smyrniotis in the brewer’s yeast, assigned to the species Saccharomyces cerevisiae, it catalyzes, in the seventh step of the pentose phosphate pathway, the transfer of a three carbon unit from a donor substrate, sedoheptulose 7-phosphate, to an acceptor substrate, glyceraldehyde 3-phosphate, to form fructose 6-phosphate and erythrose 4-phosphate.

Pentose Phosphate Pathway
Fig. 22 – The Transaldolase Reaction

Note: Like in transketolase catalyzed reactions, the carbon unit donor is a ketose while the acceptor is an aldose.
In the reverse reaction, the donor substrate is fructose 6-phosphate, while the acceptor substrate  is erythrose 4-phosphate.

⇑ Back to the top ⇑

Catalytic mechanism of transaldolase

Unlike transketolase, transaldolase does not require a cofactor for activity.
The reaction occurs in two step, an aldol cleavage and an aldol condensation.  Below, the catalytic mechanism of E. coli transaldolase B is analyzed, taking as an example the forward reaction leading to erythrose 4-phosphate and fructose 6-phosphate synthesis.
In the first step an ε-amino group of a lysine residue (Lys132) in the active site, after a proton transfer to a glutamic acid residue (Glu96) mediated by a water molecule, performs a nucleophilic attack on the carbonyl carbon of sedoheptulose 7-phosphate, that is, on the C-2 atom. The result is the formation of a carbinolamine with sedoheptulose 7-phosphate.

Pentose Phosphate Pathway
Fig. 23 – Transaldolase: Catalytic Mechanism

In the second step, the removal of a water molecule from carbinolamine leads to the formation of an enzyme-bound imine or Schiff base intermediate; this step, too, involves the transfer of a proton from Glu96 to the “catalytic” water molecule.
Note: This enzyme-substrate covalent intermediate is quite similar to that formed in the reaction catalyzed by aldolase (EC 4.1.2.13) in the fourth step of glycolysis.
In the next step, the carboxylic group of an aspartic acid residue (Asp17) extracts a proton from the hydroxyl group bound to C-4, leading to the cleavage of the C–C bond between C-3 and C-4. This reaction is an aldol cleavage and releases the first product, erythrose 4-phosphate, an aldose, whereas a three-carbon carbanion remains bound to the enzyme and is stabilized by resonance, like in transketolase catalyzed reactions. In fact, like the nitrogen atom in the thiazolium ring of thiamine pyrophosphate, the nitrogen atom with a positive charge of the Schiff base acts as an electron trap stabilizing the negative charge carried by the carbanion.
Once the acceptor substrate glyceraldehyde 3-phosphate is in the active site, the carbanion performs a nucleophilic attack on the carbonyl carbon of glyceraldehyde 3-phosphate to form, by aldol condensation, a new C–C bond and an enzyme-bound ketose.
Then, the hydrolysis of the Schiff base releases fructose 6-phosphate, a ketose and the second product of the reaction. At this point, a new reaction cycle can start.

Finally, as seen previously, in the eighth step of the pentose phosphate pathway, transketolase catalyzes the synthesis of fructose 6-phosphate and glyceraldehyde 3-phosphate from erythrose 4-phosphate and xylulose 5-phosphate.

⇑ Back to the top ⇑

Regulation of the pentose phosphate pathway

Glucose 6-phosphate is a metabolite that can enter glycolysis or the pentose phosphate pathway depending on the cell’s need for ATP, NADPH and ribose 5-phosphate.
In case of increased need for ATP, glucose 6-phosphate is mostly channelled into glycolysis.
Conversely, if the need for NADPH and/or ribose 5-phosphate increases, most of the phosphorylated sugar is channeled into the pentose phosphate pathway.
From the molecular point of view, the fate of glucose 6-phosphate depends, to a large extent, on the relative activities of the enzymes that metabolize it in glycolysis, namely, phosphofructokinase 1 (PFK-1) (EC 2.7.1.11), and in the hexose monophosphate shunt, namely, glucose 6-phosphate dehydrogenase, activities that are highly regulated.

Note: In the glycolytic pathway, glucose 6-phosphate is a substrate of phosphohexose isomerase that catalyzes the reversible isomerization to fructose 6-phosphate, which, in turn, is a  substrate of phosphofructokinase 1.

PFK-1 is inhibited when ATP and/or citrate concentrations increase, namely, when the energy charge of the cell is high, whereas it is activated when AMP and/or fructose 2,6-bisphosphate concentrations increase, namely, when the energy charge of the cell is low. Thus, when the energy charge of the cell is high, the carbon flow, and therefore the flow of glucose 6-phosphate through the glycolytic pathway decreases.
Glucose 6-phosphate dehydrogenase is inhibited by NADPH and acyl-CoAs, intermediates in fatty acid biosynthesis. Thus, when the cytosolic levels of NADPH increases, the flow of glucose 6-phosphate through the pentose phosphate pathway is inhibited, whereas if NADPH levels drop, the inhibition disappears, the pathway switches on again, and NADPH and ribose 5-phosphate are synthesized.

However, even when glucose 6-phosphate dehydrogenase is active, the cell is still able to respond to the relative needs of NADPH, ribose 5-phosphate and ATP, regulating accordingly the carbon flow through the phosphogluconate pathway. And, depending on the cell’s need for ATP, NADPH and ribose 5-phosphate, some reactions of glycolysis, gluconeogenesis, and the pentose phosphate pathway can be combined in novel ways to emphasize the synthesis of needed metabolites, also exploiting the fact that the non-oxidative phase  of the hexose monophosphate shunt is essentially controlled by the availability of the substrates.
The four principal possibilities are described below.

⇑ Back to the top ⇑

The need for NADPH is much greater than that for ribose 5-phosphate and ATP

When much more NADPH than ribose 5-phosphate is needed, and there is no need for additional ATP to be produced, namely, the energy charge of the cell is high, glucose 6-phosphate enters the pentose phosphate pathway and is completely oxidized to CO2. Such metabolic conditions are found, for example, in the adipose tissue during fatty acid synthesis.
In the oxidative phase of the pathway, two molecules of NADPH are produced for each molecule of glucose 6-phosphate oxidized to ribulose 5-phosphate. Through a combination of the reactions of the non-oxidative phase and of some reactions of gluconeogenesis, namely, those catalyzed by triose phosphate isomerase (EC 5.3.1.1), aldolase (EC 4.1.2.13), phosphohexose isomerase (EC 5.3.1.9), and fructose 1,6-bisphosphatase (EC 3.1.3.11), six molecules of ribulose 5-phosphate are converted into five molecules of glucose 6-phosphate. Thus, it is possible to state that the reactions of the non-oxidative phase allow the reactions of the oxidative phase to proceed.
Three groups of reactions can be identify.

  • In the first group there are the reactions catalyzed by the enzymes of the oxidative phase, leading to the formation of two molecules of NADPH and one molecule of ribulose 5-phosphate.

6 Glucose 6-phosphate + 12 NADP+ + 6 H20 → 6 Ribulose 5-phosphate + 6 CO2 + 12 NADPH + 12 H+

6 Ribulose 5-phosphate → 4 Fructose 6-phosphate + 2 Glyceraldehyde 3-phosphate

  • Finally, fructose 6-phosphate and glyceraldehyde 3-phosphate can be recycled to glucose 6-phosphate via some reactions of gluconeogenesis, so that the cycle can begin again.

4 Fructose 6-phosphate + 2 Glyceraldehyde 3-phosphate + H2O → 5 Glucose 6-phosphate + Pi

The sum of the last two reactions shows that six molecules of ribulose 5-phosphate are converted to five molecules of glucose 6-phosphate.

6 Ribulose 5-phosphate+ H2O → 5 Glucose 6-phosphate + Pi

The sum of the reactions of the first, second and third group gives the overall reaction:

Glucose 6-phosphate + 12 NADP+ + 7 H20 → 6 CO2 + 12 NADPH + 12 H+ + Pi

Therefore, one molecule of glucose 6-phosphate, via six cycles of the pentose phosphate pathway coupled with some reactions of gluconeogenesis, is converted to six molecules of CO2, with the concomitant production of 12 molecules of NADPH, and without net production of ribose-5-phosphate.

⇑ Back to the top ⇑

The need for NADPH and ATP is much greater than that for ribose 5-phosphate

When much more NADPH than ribose 5-phosphate is needed, and the energy charge of the cell is low, that is, there is a need for ATP, ribulose 5-phosphate formed in the oxidative phase is converted to fructose 6-phosphate and glyceraldehyde 3-phosphate through the reactions of the non-oxidative phase. These two intermediates, through the reactions of glycolysis, are oxidized to pyruvate with concomitant ATP production.
The net reaction is:

3 Glucose 6-phosphate + 6 NADP+ + 5 NAD+ + 5 Pi + 8 ADP → 5 Pyruvate + 3 CO2 + 6 NADPH + 5 NADH + 8 ATP + 2 H2O + 8 H+

If the cell requires more ATP, the pyruvate produced can be oxidized through the citric acid cycle.
Conversely, if there is no need for additional ATP to be produced, the carbon skeleton of pyruvate can be used as a building block in several biosynthetic pathways.

Note: As in the previous case, there is no net production of ribose 5-phosphate.

⇑ Back to the top ⇑

The need for ribose 5-phosphate is much greater than that for NADPH

When much more ribose 5-phosphate than NADPH is needed, as in rapidly dividing cells in which there is a high rate of synthesis of nucleotides, precursors of DNA, the reactions of the oxidative phase of the pentose phosphate pathway are bypassed, and there is no synthesis of NADPH. Conversely, because the reactions of the non-oxidative phase are easily reversible, the drop in ribose 5-phosphate levels, due to its rapid use, stimulates its synthesis.
What happens is that, through the glycolytic pathway, most of the glucose 6-phosphate is converted to fructose 6-phosphate and glyceraldehyde 3-phosphate. Then, transaldolase and transketolase lead to the synthesis of ribose 5-phosphate and xylulose 5-phosphate. Xylulose 5-phosphate, through the reactions catalyzed by phosphopentose epimerase and ribose 5-phosphate isomerase, is converted to ribose 5-phosphate.
The net reaction is:

6 Glucose 6-phosphate + ATP → 6 Ribose 5-phosphate + ADP + H+

Under this metabolic conditions therefore, what happens is an interplay between reactions of glycolysis and of the non-oxidative phase of the phosphogluconate pathway, with the latter in the direction of ribose 5-phosphate synthesis.
It should be noted no metabolites return to glycolysis.

⇑ Back to the top ⇑

The needs for ribose 5-phosphate and NADPH are balanced

If one molecule of ribose 5-phosphate and two molecules of NADPH per molecule of glucose 6-phosphate metabolized satisfy the metabolic needs of the cell, the reactions that predominate are those of the oxidative phase and that catalyzed by ribose 5-phosphate isomerase.
The net reaction is:

Glucose 6-phosphate + 2 NADP+ + H2O → Ribose 5-phosphate + 2 NADPH + 2 H+ + CO2

Under this metabolic conditions, too, no metabolites return to glycolysis.

⇑ Back to the top ⇑

References

Au S.W.N., Gover S., Lam V.M.S. and Adams M.J. Human glucose-6-phosphate dehydrogenase: the crystal structure reveals a structural NADP+ molecule and provides insights into enzyme deficiency. Structure 2000;8(3):293-303 doi:10.1016/S0969-2126(00)00104-0

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

Cosgrove M.S., Naylor C., Paludan S., Adams M.J., and Richard Levy H. On the mechanism of the reaction catalyzed by glucose 6-phosphate dehydrogenase. Biochemistry 1998;37(9);2759-67. doi:10.1021/bi972069y

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

Hanau S., Montin K., Cervellati C., Magnani M., and Dallocchio F.  6-Phosphogluconate dehydrogenase mechanism: evidence for allosteric modulation by substrate. J Biol Chem 2010;285(28):21366-71. doi:10.1074/jbc.M110.105601

Harvey R.A., Ferrier D.R. Lippincott’s illustrated reviews: biochemistry. 5th Edition. Lippincott Williams & Wilkins, 2011

Horecker B.L. The pentose phosphate pathway. J Biol Chem 2002;277(50):47965-71. doi:10.1074/jbc.X200007200

Jelakovic S., Kopriva S., Süss K-H and Schulz G.E. Structure and catalytic mechanism of the cytosolic D-ribulose-5-phosphate 3-epimerase from rice. J Mol Biol 2003;326:127-35 doi:10.1016/S0022-2836(02)01374-8

Michal G., Schomburg D. Biochemical pathways. An atlas of biochemistry and molecular biology. 2th Edition. John Wiley J. & Sons, Inc. 2012

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

Patra K.C. and Hay N. The pentose phosphate pathway and cancer. Trends Biochem Sci 2014;39(8):347-54 doi:10.1016/j.tibs.2014.06.005

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., 2009

Kochetov G.A., Solovjeva O.N. Structure and functioning mechanism of transketolase. Biochim Biophys Acta 2014;1844(9):1608-18 doi:10.1016/j.bbapap.2014.06.003

Samland A.K., Sprenger G.A. Transaldolase: from biochemistry to human disease. Int J Biochem Cell Biol 2009;41(7):1482-94 doi:10.1016/j.biocel.2009.02.001

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

Voet D. and Voet J.D. Biochemistry. 4th Edition. John Wiley J. & Sons, Inc. 2011

Wang J. and Yang W. Concerted proton transfer mechanism of Clostridium thermocellum ribose-5-phosphate isomerase. J Phys Chem B 2013;117:9354-61 doi:10.1021/jp404948c

Zhang R., Andersson C.E., Savchenko A., Skarina T., Evdokimova E., Beasley S., Arrowsmith C.H., Edwards A.M., Joachimiak A. and Mowbray S.L. Structure of Escherichia coli ribose-5-phosphate isomerase: a ubiquitous enzyme of the pentose phosphate pathway and the Calvin cycle. Structure 2003;11(1):31-42 doi: 10.1016/S0969-2126(02)00933-4

Glycolysis

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.

Glycolysis
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.

⇑ Back to the top ⇑

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.

Glycolsysis
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 Buchner was awarded the Nobel Prize in Chemistry 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.

⇑ Back to the top ⇑

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.

Glycolysis
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).

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

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 2.7.1.1), 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.

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

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;
galactose;
glucosamine and other sugars used for protein glycosylation.

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.

⇑ Back to the top ⇑

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 5.3.1.9).

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.

Glycolysis
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.
Why?

  • 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.

⇑ Back to the top ⇑

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 2.7.1.11) 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 2.7.1.105), 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.

⇑ Back to the top ⇑

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 4.1.2.13), 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.

⇑ Back to the top ⇑

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 5.3.1.1).

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 1.1.1.8).

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.

⇑ Back to the top ⇑

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 1.2.1.12) 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 4.2.1.11). 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+

⇑ Back to the top ⇑

Reaction 7: phosphoglycerate kinase and the first ATP forming reaction

In the seventh step of the glycolytic pathway, phosphoglycerate kinase (EC 2.7.2.3) 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.

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

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 5.4.2.1). 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 1.1.1.95). 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.

⇑ Back to the top ⇑

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 5.4.2.4.

Glycolisys
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 3.1.3.13. 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.

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

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 2.7.1.40) 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.

Glycolysis
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.

⇑ Back to the top ⇑

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.

Glycolysis
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.

⇑ Back to the top ⇑

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 1.1.1.27).

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.

⇑ Back to the top ⇑

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 4.1.1.1), 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 1.1.1.1), 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.

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

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).

⇑ Back to the top ⇑

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.

Glycolysis
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.

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

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:

Glycolysis
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.

⇑ Back to the top ⇑

Glycogen and starch

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

⇑ Back to the top ⇑

Fructose

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 2.7.1.4), 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 2.7.1.28), 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+

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

Sorbitol

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 1.1.99.21) 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.

⇑ Back to the top ⇑

Galactose

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 5.4.2.2).

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.

⇑ Back to the top ⇑

Mannose

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 5.3.1.8.).

Mannose 6-phosphate ⇄ Fructose 6-phosphate

⇑ Back to the top ⇑

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 6.4.1.1) and fructose 1,6-bisphosphatase (EC 3.1.3.11).

⇑ Back to the top ⇑

Hexokinase

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 2.7.1.2), 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.

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

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.
    Glycolysis
    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.
    Example
    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.

⇑ Back to the top ⇑

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.

Glycolysis
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 2.7.4.3), that form ATP from ADP.

ADP + ADP ⇄ ATP + AMP

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, a product of the pentose phosphate pathway, 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.

⇑ Back to the top ⇑

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 4.1.1.32) 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.

Glycolysis
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.

⇑ Back to the top ⇑

References

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

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.

Gluconeogenesis
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.

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

Where does gluconeogenesis occur?

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 3.1.3.9), 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 3.2.1.33) 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.

⇑ Back to the top ⇑

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 2.7.1.1) or glucokinase (EC 2.7.1.2).
    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 2.7.1.11)
    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 2.7.1.40)
    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.

⇑ Back to the top ⇑

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 6.4.1.1);
  • phosphoenolpyruvate carboxykinase or PEP carboxykinase (EC 4.1.1.32).

Pyruvate → Oxaloacetate → Phosphoenolpyruvate

Gluconeogenesis
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.

⇑ Back to the top ⇑

Phosphoenolpyruvate precursor: pyruvate or alanine

Gluconeogenesis
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 2.6.1.2).
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 1.1.1.37), 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 1.2.1.12), 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.

⇑ Back to the top ⇑

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 1.1.1.27). 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.

⇑ Back to the top ⇑

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 3.1.3.11), 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.

⇑ Back to the top ⇑

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 5.3.1.9), and glycogenolysis, produced in the reaction catalyzed by phosphoglucomutase (EC 5.4.2.2), 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.

⇑ Back to the top ⇑

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”.

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

Pyruvate carboxylase

Gluconeogenesis
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.

⇑ Back to the top ⇑

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.

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

Therefore:

  • 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.

⇑ Back to the top ⇑

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.

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.

Gluconeogenesis
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 2.7.1.105), and is hydrolyzed to fructose 6-phosphate in the reaction catalyzed by fructose 2,6-bisphosphatase or FBPasi-2 (EC 3.1.3.46). 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 4.6.1.1) to synthesize 3′,5′-cyclic AMP or cAMP, that activates cAMP-dependent protein kinase or protein kinase A or PKA (EC 2.7.11.11). 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.

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

Xylulose 5-phosphate

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

Gluconeogenesis
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.

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

Glycerol

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.

Gluconeogenesis
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 2.7.1.30), 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 1.1.1.8). In this reaction NAD+ is reduced to NADH.
During prolonged fasting, glycerol is the major gluconeogenic precursor, accounting for about 20% of glucose production.

⇑ Back to the top ⇑

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.
Gluconeogenesis
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.

⇑ Back to the top ⇑

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 1.1.1.42) 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 4.1.3.1) and malate synthase (EC 2.3.3.9), 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.

⇑ Back to the top ⇑

Propionate

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 3.2.1.4) 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.

Gluconeogenesis
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 6.4.1.3), a biotin-requiring enzyme. This reaction consumes one ATP. In the subsequent reaction, catalyzed by methylmalonyl-CoA epimerase (EC 5.1.99.1), 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 5.4.99.2). This enzyme requires 5-deoxyadenosylcobalamin or coenzyme B12, a derivative of cobalamin or vitamin B12, as a coenzyme.

⇑ Back to the top ⇑

References

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]

Glucose-alanine cycle

Glucose-alanine cycle: contents in brief

What is the glucose-alanine cycle?

The glucose-alanine cycle, or Cahill cycle, proposed for the first time by Mallette, Exton and Park, and Felig et al. between 1969 and 1970, consists of a series of steps through which extrahepatic tissues, for example the skeletal muscle, export pyruvate and amino groups as alanine to the liver, and  receive glucose from the liver via the bloodstream.
The main steps of the glucose-alanine cycle are summarized below.

  • When in extrahepatic tissues amino acids are used for energy, pyruvate, derived from the glycolytic pathway, is used as amino group acceptor, forming alanine, a nonessential amino acid.
  • Alanine diffuses into the bloodstream and reaches the liver.
  • In the liver, the amino group of alanine is transferred to α-ketoglutarate to form pyruvate and glutamate, respectively.
  • The amino group of glutamate mostly enters the urea cycle, and in part acts as a nitrogen donor in many biosynthetic pathways.
    Pyruvate enters the gluconeogenesis pathway and is used for glucose synthesis.
  • The newly formed glucose diffuses into the bloodstream and reaches the peripheral tissues where, due to glycolysis, is converted into pyruvate that can accept amino groups from the free amino acids, thus closing the cycle.

Therefore, the glucose-alanine cycle provides a link between carbohydrate and amino acid metabolism, as schematically described below.

Glucose → Pyruvate → Alanine → Pyruvate → Glucose

Glucose-Alanine Cycle
Fig. 1 – Glucose-Alanine Cycle

The glucose-alanine cycle occurs not only between the skeletal muscle, the first tissue in which it was observed, and the liver, but involves other cells and extrahepatic tissues including cells of the immune system, such as lymphoid organs.

⇑ Back to the top ⇑

The steps of the glucose-alanine cycle

The analysis of the steps of the glucose-alanine cycle is made considering the cycle between skeletal muscle and the liver.
Both intracellular and extracellular proteins are continuously hydrolyzed to the constituent amino acids and resynthesized, and the rate at which these processes occur is balanced precisely, thereby preventing loss of fat free mass.
However, under catabolic conditions, such as intense and prolonged exercise or fasting, the rate of muscle protein breakdown exceeds synthesis. This leads to the liberation of amino acids, some of which are used for energy and others for gluconeogenesis. And the oxidation of the carbon skeletons of amino acids, in particular branched chain amino acids or BCAA (leucine, isoleucine  and valine), may be a significant source of energy for the muscle. For example, after about 90 minutes of strenuous exercise, amino acid oxidation in muscle provides 10-15% of the energy needed for contraction.
The utilization of the carbon skeletons of amino acids for energy involves the removal of the amino group, and then the excretion of amino nitrogen in a non-toxic form.
The removal of the α-amino group occurs by transamination, that can be summarized as follows:

α-Keto acid + Amino acid ⇄ New amino acid + New α-keto acid

Such reactions, catalyzed by enzymes called aminotransferases or transaminases (EC 2.6.1) are freely reversible.
Branched chain amino acids, for example, transfer the amino group to α-ketoglutarate or 2-oxoglutaric acid, to form glutamate and the α-keto acid derived from the original amino acid, in a reaction catalyzed by branched chain aminotransferase or BCAT (EC 2.6 .1.42).

⇑ Back to the top ⇑

The glucose-alanine cycle in skeletal muscle

In skeletal muscle, the newly formed glutamate may react with ammonia to form glutamine, for many tissues and organs, such as the brain, the major vehicle for interorgan transport of nitrogen. The reaction is catalyzed by the cytosolic enzyme glutamine synthetase (EC 6.3.1.2), and consumes an ATP.

Glutamate + NH4+ + ATP → Glutamine + ADP + Pi

In this case, glutamate leaves the Cahill cycle.
Alternatively, and in contrast to what happens in most of the other tissues, the newly formed glutamate may transfer the amino group to pyruvate, derived from glycolysis, to form alanine and α-ketoglutarate. This transamination is catalyzed by alanine aminotransferase or ALT (EC 2.6.1.2), an enzyme found in most animal and plant tissues.

Glutamate + Pyruvate ⇄ Alanine + α-Ketoglutarate

The alanine produced and that derived directly from protein breakdown, and muscle proteins are rich in alanine, can leave the cell and be carried by the bloodstream to the liver; in this way the amino group reaches the liver. And the rate at which alanine formed by transamination of pyruvate is transferred into the circulation is proportional to the intracellular pyruvate production.
Note: Alanine and glutamine are the major sources of nitrogen and carbon in interorgan amino acid metabolism.

⇑ Back to the top ⇑

The glucose-alanine cycle in the liver

Once in the liver, a hepatic alanine aminotransferase catalyzes a transamination in which alanine, the major gluconeogenic amino acid, acts as an amino group donor and α-ketoglutarate as an α-keto acid acceptor. The products of the reaction are pyruvate, i.e. the carbon skeleton of alanine, and glutamate.

Alanine + α-Ketoglutarate ⇄ Glutamate + Pyruvate

Glutamate, in the reaction catalyzed by glutamate dehydrogenase (EC 1.4.1.2), an enzyme present in the mitochondrial matrix, forms ammonium ion, which enters the urea cycle, and α-ketoglutarate, which can enter the Krebs cycle. This reaction is an anaplerotic reaction that links amino acid metabolism with the Krebs cycle.

Glucose-Alanine Cycle

However, glutamate can also react  with oxaloacetate to form aspartate and α-ketoglutarate, in a reaction catalyzed by aspartate aminotransferase (EC 2.6.1.1). Aspartate is involved in the formation of urea as well as in the synthesis of purines and pyrimidines.

Glutamate + Oxaloacetate ⇄ Aspartate + α-Ketoglutarate

Also the pyruvate produced may have different metabolic fates: it can be oxidized for ATP production, and then leave the glucose-alanine cycle, or enter the gluconeogenesis pathway, and thus continue in the cycle.
The glucose produced is released from the liver into the bloodstream and delivered to various tissues that require it, as the skeletal muscle, in which it is used for pyruvate synthesis. In turn, the newly formed pyruvate may react with glutamate, thus closing the cycle.

⇑ Back to the top ⇑

Transaminases

As previously mentioned, the removal of the amino group from amino acids occurs through transamination (see above for the general reaction). These reactions are catalyzed by enzymes called aminotransferases or transaminases.
They are cytosolic enzymes, present in all cells and particularly abundant in the liver, kidney, intestine and muscle; they require pyridoxal phosphate or PLP, the active form of vitamin B6 or pyridoxine, as a coenzyme, which is tightly bound to the active site.
In transamination reactions, the amino group of free amino acids, except of threonine and lysine, is channeled towards a small number of α-keto acids, notably pyruvate, oxaloacetate and α-ketoglutarate.
Cells contain different types of aminotransferases: many are specific for α-ketoglutarate as α-keto acid acceptor, but differ in specificity for the amino acid, from which they are named. Examples are the aforementioned alanine aminotransferase, also called alanine transaminase and glutamic pyruvic transferase or GPT, and aspartate aminotransferase or AST, also called glutamic-oxaloacetic transaminase or GOT.
It should be underlined that there is no net deamination in these reactions, no loss of amino groups, as the α-keto acid acceptor is aminated and the amino acid deaminated.

⇑ Back to the top ⇑

Functions of the glucose-alanine cycle

This cycle has various functions.

  • It transports nitrogen in a non-toxic form from peripheral tissues to the liver.
  • It transports pyruvate, a gluconeogenic substrate, to the liver.
  • It removes pyruvate from peripheral tissues.  This leads to a higher production of ATP from glucose in these tissues. In fact, the NADH produced during glycolysis can enter the mitochondria and be oxidized through oxidative phosphorylation.
  • It allows to maintain a relatively high concentration of alanine in hepatocytes, sufficient to inhibit protein degradation.
  • It may play a role in host defense against infectious diseases.

Finally, it is important to underline that there is no net synthesis of glucose in the glucose-alanine cycle.

⇑ Back to the top ⇑

Energy cost of the glucose-alanine cycle

Like the Cori cycle, also the glucose-alanine cycle has an energy cost, equal to 3-5 ATP.
The part of the cycle that takes place in peripheral tissues involves the production of 5-7 ATP per molecule of glucose:

  • 2 ATP are produced by glycolysis;
  • 3-5 ATP derive from NADH/FADH2 (see below).

Instead in the liver, gluconeogenesis and the urea cycle cost 10 ATP:

  • 6 ATP are consumed in the during gluconeogenesis per molecule of glucose synthesized;
  • 4 ATP are consumed in the urea cycle per molecule of urea synthesized.

The glucose-alanine cycle, like the Cori cycle, shifts part of the metabolic burden from extrahepatic tissues to the liver. However, the energy cost paid by the liver is justified by the advantages that the cycle brings to the whole body, as it allows, in particular conditions, an efficient breakdown of proteins in extrahepatic tissues (especially skeletal muscle), which in turn allows to obtain gluconeogenic substrates as well as the use of amino acids for energy in extrahepatic tissues.

⇑ Back to the top ⇑

Similarities and differences between glucose-alanine cycle and Cori cycle

There are some analogies between the two cycles, which are listed below.

Glucose-Alanine Cycle
Fig. 2 – Glucose-Alanine Cycle and Cori Cycle

Below, some differences between the two cycles.

  • The main difference concerns the three carbon intermediate that from peripheral tissues reach the liver: lactate in the Cori cycle, and alanine in the glucose-alanine cycle.
  • Another difference concerns the fate of the NADH produced by glycolysis in peripheral tissues.
    In the Cori cycle, the coenzyme acts as reducing agent to reduce pyruvate to lactate, in the reaction catalyzed by lactate dehydrogenase (EC 1.1.1.27).
    In the glucose-alanine cycle, this reduction does not occur and the electrons of NADH can be transported into the mitochondria via the malate-aspartate and glycerol 3-phosphate shuttles, generating NADH, the first shuttle, and FADH2, the other shuttle. And the yield of ATP from NADH and FADH2 is 2.5 and 1.5, respectively.
  • Finally, from the previous point, it is clear that, unlike the Cori cycle, the Cahill cycle requires the presence of oxygen and mitochondria in the peripheral tissues.

⇑ Back to the top ⇑

References

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

Felig P., Pozefsk T., Marlis E., Cahill G.F. Alanine: key role in gluconeogenesis. Science 1970;167(3920):1003-4. doi:10.1126/science.167.3920.1003

Gropper S.S., Smith J.L., Groff J.L. Advanced nutrition and human metabolism. Cengage Learning, 2009 [Google eBooks]

Lecker S.H., Goldberg A.L. and Mitch W.E. Protein degradation by the ubiquitin–proteasome pathway in normal and disease states. J Am Soc Nephrol 2006;17(7):1807-19. doi:10.1681/ASN.2006010083

Mallette L. E., Exton J. H., and Park C. R. Control of gluconeogenesis from amino acids in the perfused  rat liver. J Biol Chem 1969;244(20):5713-23 [PDF]

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

Raju S.M., Madala B. Illustrated medical biochemistry. Jaypee Brothers Publishers, 2005 [Google eBooks]

Wu G. Amino acids: biochemistry and nutrition. CRC Press, 2013 [Google eBooks]


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.

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

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 1.2.1.12).

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 1.1.1.27), 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.

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

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.

⇑ Back to the top ⇑

References

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]

Bile salts: definition, functions, enterohepatic circulation, synthesis

Bile salts: contents in brief

What are bile salts

Bile salts and bile acids are polar cholesterol derivatives, and represent the major route for the elimination of the steroid from the body.
They are molecules with similar but not identical structures, and diverse physical and biological characteristics.
They are synthesized in the liver, stored in the gallbladder, secreted into the duodenum, and finally, for the most part, reabsorbed in the ileum.
Because at physiological pH these molecules are present as anions, the terms bile acid and bile salts are used herein as synonyms.

⇑ Back to the top ⇑

Chemical structure of bile salts

Bile Salts
Fig. 1 – Chemical Structures of the Most Abundant Bile Acids

Bile salts have similarities and differences with cholesterol molecule.
Like the steroid, they have a nucleus composed of four fused rings: three cyclohexane rings, labeled A, B and C, and a cyclopentane ring, labeled D. This structure is the perhydrocyclopentanophenanthrene, more commonly known as steroid nucleus.
In higher vertebrates, they have 24 carbon atoms, as the side chain is three carbons shorter than the original. In lower vertebrates, bile acids have 25, 26, or 27 carbon atoms. The side chain ends with a carboxyl group, ionized at pH 7, that can be linked to the amino acid glycine or taurine (see below).
In addition to the hydroxyl group at position 3, they have hydroxyl groups at positions 7 and/or 12.
All this makes them much more polar than cholesterol.

Bile Salts
Fig. 2 – Cholic Acid Structure

Since A and B rings are fused in cis configuration, the planar structure of the steroid nucleus is curved, and it is possible to identify:

  • a concave side, which is hydrophilic because the hydroxyl groups and the carboxyl group of the side chain, with or without the linked amino acid, are oriented towards it;
  • a convex side, which is hydrophobic because the methyl groups present at position 18 and 19 are orientated towards it.

Therefore, having both polar and nonpolar groups, they are amphiphilic molecules and excellent surfactants. However, their chemical structure makes them different from many other surfactants, often composed of a polar head region and a nonpolar tail.

⇑ Back to the top ⇑

Primary, conjugated and secondary bile salts

Bile Salts
Fig. 3 – Conjugated Bile Acids

Primary bile acids are those synthesized directly from cholesterol in the hepatocytes. In humans, the most important are cholic acid and chenodeoxycholic acid, which make up 80% of all bile acids. Before being secreted into the biliary tree, they are almost completely conjugated, up to 98%, with the glycine or taurine, to form glycoconjugates and tauroconjugates, respectively. In particular, approximately 75% of cholic acid and chenodeoxycholic acid are conjugated with glycine, to form glycocholic acid  and glycochenodeoxycholic acid, the remaining 25% with taurine, to form taurocholic acid and taurochenodeoxycholic.
Conjugated bile acids are molecules with more hydrophilic groups than unconjugated bile acids, therefore with a increased emulsifying capacity. In fact, conjugation decreases the pKa of bile acids, from about 6, a value typical of non-conjugated molecules, to about 4 for glycocholic acid, and about 2 for taurocholic acid. This makes that conjugated bile acids are ionized in a broader range of pH to form the corresponding salts.
The hydrophilicity of the common acid and bile salts decreases in the following order: glycine-conjugated < taurine-conjugated < lithocholic acid  < deoxycholic acid  < chenodeoxycholic acid < cholic acid <ursodeoxycholic acid.
Finally, conjugation also decreases the cytotoxicity of primary bile acids.

Secondary bile acids  are formed from primary bile acids which have not been reabsorbed from the small intestine. Once they reach the colon, they can undergo several modifications by colonic microbiota to form secondary bile acids (see below). They make up the remaining 20% of the body’s bile acid pool.

Another way of categorizing bile salts is based on their conjugation with glycine and taurine and their degree of hydroxylation. On this basis, three categories are identified.

  • Trihydroxy conjugates, such as taurocholic acid and glycocholic acid.
  • Dihydroxy conjugates, such as glycodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, and taurodeoxycholic acid. They account for about 60% of bile salts present in the bile.
  • Unconjugated forms, such as cholic acid, deoxycholic acid, chenodeoxycholic acid, and lithocholic acid.

⇑ Back to the top ⇑

Function of bile acids

All their physiological functions are performed in the conjugated form.

  • They are the major route for the elimination of cholesterol from human body.
    Indeed, humans do not have the enzymes to break open the cyclohexane rings or  the cyclopentane ring of the steroid nucleus, nor to oxidize cholesterol to CO2 and water.
    The other mechanism to eliminate the steroid from the body is as cholesterol per se in the bile.
  • Bile salts are strong surfactants. And in particular, di- and trihydroxy conjugates are the best surfactants among bile acids, much more effective than unconjugated counterparts, since they have more polar groups.
    Once in contact with apolar lipids in the lumen of the small intestine, the convex apolar surface interacts with the apolar lipids, such as triglycerides, cholesterol esters, and ester of fat-soluble vitamins, whereas the concave polar surface interacts with the surrounding aqueous medium. This increases the dispersion of apolar lipids in the aqueous medium, as it allows the formation of tiny lipid droplets, increasing the surface area for:

lipase activity, mainly pancreatic lipase, (bile salts also play a direct role in the activation of this enzyme);

intestinal esterase activity.

Subsequently, they facilitate the absorption of lipid digestion products, as well as of fat soluble vitamins by the intestinal mucosa thanks to the formation of mixed micelles.
Bile acids perform a similar function in the gallbladder where, forming mixed micelles with phospholipids, they prevent the precipitation of cholesterol.
Note: As a consequence of the arrangement of polar and nonpolar groups, bile acids form micelles in aqueous solution, usually made up of less than 10 monomers, as long as their concentration is above the so-called critical micellar concentration or CMC.

  • At the intestinal level, they modulate the secretion of pancreatic enzymes and cholecystokinin.
  • In the small and large intestine, they have a potent antimicrobial activity, mainly deoxycholic acid, in particular against Gram-positive bacteria. This activity may be due to oxidative DNA damage, and/or to the damage of the cell membrane. Therefore, they play an important role in the prevention of bacterial overgrowth, but also in the regulation of gut microbiota composition.
  • In the last few years, it becomes apparent their regulatory role in the control of energy metabolism, and in particular for the hepatic glucose handling.

⇑ Back to the top ⇑

Enterohepatic circulation of bile salts

After fat intake, enteroendocrine cells of the duodenum secrete cholecystokinin into the blood stream. Hormone binding to receptors on smooth muscle cells of the gallbladder promotes their contraction; the hormone also causes the relaxation of the sphincter of Oddi. All this results in the secretion of the bile, and therefore of bile acids into the duodenum.
Under physiological conditions, human bile salt pool is constant, and equal to about 3-5 g. This is made possible by two processes:

  • their intestinal reabsorption;
  • their de novo synthesis (see below).

Up to 95% of the secreted bile salts is reabsorbed from the gut, not together with the products of lipid digestion, but through a process called enterohepatic circulation.
It is an extremely efficient recycling system, which seems to occur at least two times for each meal, and includes the liver, the biliary tree, the small intestine, the colon, and the portal circulation through which reabsorbed molecules return to the liver. Such recirculation is necessary since liver’s capacity to synthesize bile acids is limited and insufficient to satisfy intestinal needs if the bile salts were excreted in the feces in high amounts.
Most of the bile salts are reabsorbed into the distal ileum, the lower part of the small intestine, by a sodium-dependent transporter within the brush border of the enterocytes, called sodium-dependent bile acid transporter or ASBT, which carries out the cotransport of a molecule of bile acid and two sodium ions.
Within the enterocyte, it is thought that bile acids are transported across the cytosol to the basolateral membrane by the ileal bile acid-binding protein or IBABP. They cross the basolateral membrane by the organic solute transporter alpha-beta or OSTα/OSTβ, pass into the portal circulation, and, bound to albumin, reach the liver.
It should be noted that a small percentage of bile acids reach the liver through the hepatic artery.
A hepatic level, their extraction is very efficient, with a first-pass extraction fraction ranging from 50 to 90%, a percentage that depends on bile acid structure. The uptake of conjugated bile acids is mainly mediated by a Na+-dependent active transport system, that is, the sodium-dependent taurocholate cotransporting polypeptide or NTCP. However, a sodium-independent uptake can also occur, carried out by proteins of the family of organic anion transporting polypeptides or OATP, mainly OATP1B1 and OATP1B3.
The rate limiting step in the enterohepatic circulation is their canalicular secretion, largely mediated by the bile salt export pump or BSEP, in an ATP-dependent process. This pump carries monoanionic bile salts, which are the most abundant. Bile acids conjugated with glucuronic acid or sulfate, which are dianionic, are transported by different carriers, such as MRP2 and BCRP.

Note: Serum levels of bile acids vary on the basis of the rate of their reabsorption, and therefore they are higher during meals, when the enterohepatic circulation is more active.

⇑ Back to the top ⇑

Intestinal metabolism of bile acids

Bile Salts
Fig. 4 – Intestinal Bile Acid Metabolism

Bile acids which escape ileal absorption pass into the colon where they partly undergo modifications by intestinal microbiota and are converted to secondary bile acids.
The main reactions are listed below.

  • Deconjugation
    On the side chain, hydrolysis of the C24 N-acyl amide bond can occur, with release of unconjugated bile acids and glycine or taurine. This reaction is catalyzed by bacterial hydrolases present both in the small intestine and in the colon.
  • 7α-Dehydroxylation
    Quantitatively, it is the most important reaction, carried out by colonic bacterial dehydratases that remove the hydroxyl group at position 7 to form 7-deoxy bile acids. In particular, deoxycholic acid is formed from cholic acid, and lithocholic acid, a toxic secondary bile acid, from chenodeoxycholic acid.
    It should be noted that 7α-dehydroxylation, unlike oxidation and epimerization (see below), can only occur on unconjugated bile acids, and therefore, deconjugation is an essential prerequisite.
  • Oxidation and epimerization
    They are reactions involving the hydroxyl groups at positions 3, 7 and 12, catalyzed by bacterial hydroxysteroid dehydrogenases. For example, ursodeoxycholic acid derives from the epimerization of chenodeoxycholic acid.

Some of the secondary bile acids are then reabsorbed from the colon and return to the liver. In the hepatocytes, they are reconjugated, if necessary, and resecreted. Those that are not reabsorbed, are excreted in the feces.
Whereas oxidations and deconjugations are carried out by a broad spectrum of anaerobic bacteria, 7α-dehydroxylations is carried out by a limited number of colonic anaerobes.
7α-Dehydroxylations and deconjugations increase the pKa of the bile acids, and therefore their hydrophobicity, allowing a certain degree of passive absorption across the colonic wall.
The increase of hydrophobicity is also associated with an increased toxicity of these molecules. And a high concentration of secondary bile acids in the bile, blood, and feces has been associated to the pathogenesis of colon cancer.

⇑ Back to the top ⇑

Soluble fibers and reabsorption of bile salts

The reabsorption of bile salts can be reduced by chelating action of soluble fibers, such as those found in fresh fruits, legumes, oats and oat bran, which bind them, decreasing their uptake. In turn, this increases bile acid de novo synthesis, up-regulating the expression of the 7α-hydroxylase and sterol 12α-hydroxylase (see below), and thereby reduces hepatocyte cholesterol concentration.
The depletion of hepatic cholesterol increases the expression of the LDL receptor, and thus reduces plasma concentration of LDL cholesterol. On the other hand, it also stimulates the synthesis of HMG-CoA reductase, the key enzyme in cholesterol biosynthesis.
Note: Some anti-cholesterol drugs act by binding bile acids in the intestine, thereby preventing their reabsorption.

⇑ Back to the top ⇑

Synthesis of primary bile acids

Bile Salts
Fig. 5 – Primary Bile Acid Biosynthesis

Quantitatively, bile acids are the major product of cholesterol metabolism.
As previously said, enterohepatic circulation and their de novo synthesis maintain a constant bile acid pool size. In particular, de novo synthesis allows the replacement of bile salts excreted in the faces, about 5-10% of the body pool, namely ~ 0.5 g/day.
Below, the synthesis of cholic acid and chenodeoxycholic acid, and their conjugation with the amino acids taurine and glycine, is described.
There are two main pathways for bile acid synthesis: the classical pathway and the alternative pathway. In addition, some other minor pathways will also be described.

⇑ Back to the top ⇑

The classical or neutral pathway

In humans, up to 90% of bile salts are produced via the classical pathway (see fig. 5), also referred to as “neutral” pathway since intermediates are neutral molecules.
It is a metabolic pathway present only in the liver, that consists of reactions catalyzed by enzymes localized in the cytosol, endoplasmic reticulum, peroxisomes, and mitochondria, and whose end products are the conjugates of cholic acid and chenodeoxycholic acid.

  • The first reaction is the hydroxylation at position 7 of cholesterol, to form 7α-hydroxycholesterol. The reaction is catalyzed by cholesterol 7α-hydroxylase or CYP7A1 (E.C. 1.14.14.23). It is an enzyme localized in the endoplasmic reticulum, and catalyzes the rate-limiting step of the pathway.

Cholesterol + NADPH + H+ + O2 → 7α-Hydroxycholesterol + NADP+ + H2O

  • 7α-Hydroxycholesterol undergoes oxidation of the 3β-hydroxyl group and the shift of the double bond from the 5,6 position to the 4,5 position, to form 7α-hydroxy-4-cholesten-3-one. The reaction is catalyzed by 3β-hydroxy-Δ5-C27-steroid oxidoreductase or HSD3B7 (E.C. 1.1.1.181), an enzyme localized in the endoplasmic reticulum.
  • 7α-Hydroxy-4-cholesten-3-one can follow two routes:

to enter the pathway that leads to the synthesis of cholic acid, through the reaction catalyzed by 7α-hydroxy-4-cholesten-3-one 12α-monooxygenase or sterol 12α-hydroxylase or CYP8B1 (E.C. 1.14.18.8), an enzyme localized in the endoplasmic reticulum;

to enter the pathway that leads the synthesis of chenodeoxycholic acid, through the reaction catalyzed by 3-oxo-Δ4-steroid 5β-reductase or AKR1D1 (E.C. 1.3.1.3), a cytosolic enzyme.

It should be underlined that the activity of sterol 12α-hydroxylase determines the ratio of cholic acid to chenodeoxycholic acid, and, ultimately, the detergent capacity of bile acid pool. And in fact, the regulation of sterol 12α-hydroxylase gene transcription is one of the main regulatory step of the classical pathway.

Therefore, if 7α-hydroxy-4-cholesten-3-one proceeds via the reaction catalyzed by sterol 12α-hydroxylase, the following reactions will occur.

  • 7α-Hydroxy-4-cholesten-3-one is hydroxylated at position 12 by sterol 12α-hydroxylase, to form 7α,12α-dihydroxy-4-cholesten-3-one.
  • 7α,12α-Dihydroxy-4-cholesten-3-one undergoes reduction of the double bond at 4,5 position, in the reaction catalyzed by 3-oxo-Δ4-steroid 5β-reductase, to form 5β-cholestan-7α,12α-diol-3-one.
  • 5β-Cholestan-7α,12α-diol-3-one undergoes reduction of the hydroxyl group at position 4, in the reaction catalyzed by 3α-hydroxysteroid dehydrogenase or AKR1C4 (EC 1.1.1.213), a cytosolic enzyme, to form 5β-cholestan-3α,7α,12α-triol.
  • 5β-Cholestan-3α,7α,12α-triol undergoes oxidation of the side chain via three reactions catalyzed by sterol 27-hydroxylase or CYP27A1 (EC 1.14.15.15). It is a mitochondrial enzyme also present in extrahepatic tissues and macrophages, which introduces a hydroxyl group at position 27. The hydroxyl group is oxidized to aldehyde, and then to carboxylic acid, to form 3α,7α,12α-trihydroxy-5β-cholestanoic acid.
  • 3α,7α,12α-Trihydroxy-5β-cholestanoic  acid is activated to its coenzyme A ester, 3α,7α,12α-trihydroxy-5β-cholestanoyl-CoA, in the reaction catalyzed by either very long chain acyl-CoA synthetase or VLCS (EC 6.2.1.-), or bile acid CoA synthetase or BACS (EC 6.2.1.7), both localized in the endoplasmic reticulum.
  • 3α,7α,12α-Trihydroxy-5β-cholestanoyl-CoA is transported to peroxisomes where it undergoes five successive reactions, each catalyzed by a different enzyme. In the last two reactions, the side chain is shortened to four carbon atoms, and finally cholylCoA is formed.
  • In the last step, the conjugation, via amide bond, of the carboxylic acid group of the side chain with the amino acid glycine or taurine occurs. The reaction is catalyzed by bile acid-CoA:amino acid N-acyltransferase or the BAAT (EC 2.3.1.65), which is predominantly localized in peroxisomes.
    The reaction products are thus the conjugated bile acids: glycocholic acid and taurocholic acid.

If 7α-hydroxy-4-cholesten-3-one does not proceed via the reaction catalyzed by sterol 12α-hydroxylase, it enters the pathway that leads to the synthesis of chenodeoxycholic acid conjugates, through the reactions described below.

  • 7α-Hydroxy-4-cholesten-3-one is converted to 7α-hydroxy-5β-cholestan-3-one in the reaction catalyzed by 3-oxo-Δ4-steroid 5β-reductase.
  • 7α-Hydroxy-5β-cholestan-3-one is converted to 5β-cholestan-3α,7α-diol in the reaction catalyzed by 3α-hydroxysteroid dehydrogenase.

Then, the conjugated bile acids glycochenodeoxycholic acid and taurochenodeoxycholic acid are formed by modifications similar to those seen for the conjugation of cholic acid, and catalyzed mostly by the same enzymes.

Note: Unconjugated bile acids formed in the intestine must reach the liver to be reconjugated.

⇑ Back to the top ⇑

The alternative or acidic pathway

It is prevalent in the fetus and neonate, whereas in adults it leads to the synthesis of less than 10% of the bile salts.
This pathway  (see fig. 5) differs from the classical pathway in that:

  • the intermediate products are acidic molecules, from which the alternative name “acidic pathway”;
  • the oxidation of the side chain is followed by modifications of the steroid nucleus, and not vice versa;
  • the final products are conjugates of chenodeoxycholic acid.

The first step involves the conversion of cholesterol into 27-hydroxycholesterol in the reaction catalyzed by sterol 27-hydroxylase.
27-Hydroxycholesterol can follow two routes.

Route A

  • 27-hydroxycholesterol is converted to 3β-hydroxy-5-cholestenoic acid in a reaction catalyzed by sterol 27-hydroxylase.
  • 3β-Hydroxy-5-cholestenoic acid is hydroxylated at position 7 in the reaction catalyzed by oxysterol 7α-hydroxylase or CYP7B1 (EC 1.14.13.100), an enzyme localized in the endoplasmic reticulum, to form 3β-7α-dihydroxy-5-colestenoic acid.
  • 3β-7α-Dihydroxy-5-cholestenoic acid is converted to 3-oxo-7α-hydroxy-4-cholestenoic acid, in the reaction catalyzed by 3β-hydroxy-Δ5-C27-steroid oxidoreductase.
  • 3-Oxo-7α-hydroxy-4-cholestenoic acid, as a result of side chain modifications, forms chenodeoxycholic acid, and then its conjugates.

Route B

  • 27-Hydroxycholesterol is converted to 7α,27-dihydroxycholesterol in the reaction catalyzed by oxysterol 7α-hydroxylase and cholesterol 7α-hydroxylase.
  • 7α,27-Dihydroxycholesterol is converted to 7α,26-dihydroxy-4-cholesten-3-one in the reaction catalyzed by 3β-hydroxy-Δ5-C27-steroid oxidoreductase;

7α, 26-Dihydroxy-4-cholesten-3-one can be transformed directly to conjugates of chenodeoxycholic acid, or can be converted to 3-oxo-7α-hydroxy-4-colestenoic acid,  and then undergo side chain modifications and other reactions that lead to the synthesis of the conjugates of chenodeoxycholic acid.

⇑ Back to the top ⇑

Minor pathways

There are also minor pathways (see fig. 5) that contribute to bile salt synthesis, although to a lesser extent than classical and alternative pathways.

For example:

  • A cholesterol 25-hydroxylase (EC 1.14.99.38) is expressed in the liver.
  • A cholesterol 24-hydroxylase or CYP46A1 (EC 1.14.14.25) is expressed in the brain, and therefore, although the organ cannot export cholesterol, it exports oxysterols.
  • A nonspecific 7α-hydroxylase has also been discovered. It is  expressed in all tissues and appears to be involved in the generation of oxysterols, which may be transported to hepatocytes to be converted to chenodeoxycholic acid.

Additionally, sterol 27-hydroxylase is expressed in various tissues, and therefore its reaction products must be transported to the liver to be converted to bile salts.

⇑ Back to the top ⇑

Bile salts: regulation of synthesis

Regulation of bile acid synthesis occurs via a negative feedback mechanism, particularly on the expression of cholesterol 7α-hydroxylase and sterol 12α-hydroxylase.
When an excess of bile acids, both free and conjugated, occurs, these molecules bind to the nuclear receptor farnesoid X receptor or FRX, activating it: the most efficacious bile acid is chenodeoxycholic acid, while others, such as ursodeoxycholic acid, do not activate it.
FRX induces the expression of the transcriptional repressor small heterodimer partner or SHP, which in turn interacts with other transcription factors, such as liver receptor homolog-1 or LRH-1, and hepatocyte nuclear factor-4α or HNF-4α. These transcription factors bind to a sequence in the promoter region of 7α-hydroxylase and 12α-hydroxylase genes, region called bile acid response elements or BAREs, inhibiting their transcription.
One of the reasons why bile salt synthesis is tightly regulated is because many of their metabolites are toxic.

⇑ Back to the top ⇑

References

Chiang J.Y.L. Bile acids: regulation of synthesis. J Lipid Res 2009;50(10):1955-66. doi:10.1194/jlr.R900010-JLR200

Gropper S.S., Smith J.L. Advanced nutrition and human metabolism. 6h Edition. Cengage Learning, 2012 [Google eBook]

Moghimipour E., Ameri A., and Handali S. Absorption-enhancing effects of bile salts. Molecules 2015;20(8); 14451-73. doi:10.3390/molecules200814451

Monte M.J., Marin J.J.G., Antelo A., Vazquez-Tato J. Bile acids: Chemistry, physiology, and pathophysiology. World J Gastroenterol 2009;15(7):804-16. doi:10.3748/wjg.15.804

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

Sundaram S.S., Bove K.E., Lovell M.A. and Sokol R.J. Mechanisms of Disease: inborn errors of bile acid synthesis. Nat Clin Pract Gastroenterol Hepatol 2008;5(8):456-68. doi:10.1038/ncpgasthep1179

Human gut microbiota: definition, composition, and the effect of diet

Human gut microbiota: contents in brief

Definition and composition of the human gut microbiota

Gut Microbiota
Fig. 1 – Lactobacillus acidophilus

The human gastrointestinal tract is one of the most fierce and competitive ecological niches. It harbors viruses, eukaryotes, bacteria, and one member of Archaebacteria, Methanobrevibacter smithii.
Bacteria vary in proportion and amount all along the gastrointestinal tract; the greatest amount is found in the colon, which contains over 400 different species belonging to 9 phyla or divisions (of the 30 recognized phyla), and hereafter you refer to them as gut microbiota.
These are the phyla and some of their most represented genera.

  • Actinobacteria (Gram-positive bacteria); Bifidobacterium, Collinsella, Eggerthella, and Propionibacterium.
  • Bacteroidetes (Gram-negative bacteria); more than 20 genera including Bacteroides, Prevotella and Corynebacterium.
  • Cyanobacteria (Gram-negative bacteria).
  • Firmicutes (Gram-positive bacteria); at least 250 genera, including Mycoplasma, Bacillus, Clostridium, Dorea, Faecalibacterium, Ruminococcus, Eubacterium, Staphylococcus, Streptococcus, Lactobacillus, Lactococcus, Enterococcus, Sporobacter, and Roseburia.
  • Fusobacteria (Gram-negative bacteria);
  • Lentisphaerae (Gram-negative bacteria).
  • Proteobacteria (Gram-negative bacteria); Escherichia, Klebsiella, Shigella, Salmonella, Citrobacter, Helicobacter, and Serratia.
  • Spirochaeates (Gram-negative bacteria).
  • Verrucomicrobia (Gram-negative bacteria).

The presence of a small subset of the bacterial world in the colon is the result of a strong selective pressure which acted, during evolution, on both the microbial colonizers, selecting organisms very well adapted to this environment, and the intestinal niche. And nevertheless, each individual harbors an unique bacterial community in his gut.
Despite the high variability existing both with regard to taxa and between individuals, it has been proposed, but not accepted by all researchers, that in most adults the bacterial gut microbiota can be classified into variants or “enterotypes”, on the basis of the ratio of the abundance of the genera Bacteroides and Prevotella. This seems to indicate that there is a limited number of well balanced symbiotic states, which could respond differently to factors such as diet, age, genetics, and drug intake (see below).

Adult’s gut harbors a large and diverse community of DNA and RNA viruses made up of about 2,000 different genotypes, none of which is dominant. Indeed, the most abundant virus accounts for only about 6% of the community, whereas in infants the most abundant virus accounts over 40% of the community. The majority of DNA viruses are bacteriophages or phages, that is, viruses that infect bacteria (they are the most abundant biological entity on earth, with an estimated population of about 1031 units), whereas the majority of RNA viruses are plant viruses.

⇑ Back to the top ⇑

Factors affecting gut microbiota composition and development

The intestinal bacterial community is regulated by several factors, most of which are listed below.

  • The diet of the host.
    It seems to be the most important factor.
    Traditionally considered sterile, mother’s milk harbors a rich microbiota consisting of more than 700 species, dominated by staphylococci, streptococci, bifidobacteria and lactic acid bacteria. Therefore, it is a major source for the colonization of the breastfed infant gut, and it was suggested that this mode of colonization is closely correlated with infant’s health status, because, among other functions, it could protect against infections and contribute to the maturation of the immune system. Breast milk affects intestinal microbiota also indirectly, through the presence of oligosaccharides with prebiotic activity that stimulate the growth of specific bacterial groups including staphylococci and bifidobacteria.
    A recent study has compared the intestinal microbiota of European and African children (respectively from Florence and a rural village in Burkina Faso) between the ages of 1 and 6 years old. It has highlighted the dominant role of diet over variables such as climate, geography, hygiene and health services (it was also observed the absence of significant differences in the expression of key genes regulating the immune function, which suggests a functional similarity between the two groups). Indeed infants, as long as they are breastfed, have a very similar gut microbiota, rich in Actinobacteria, mainly Bifidobacterium (see below). The subsequent introduction of solid foods in the two groups, a Western diet rich in animal fat and protein in European children, and low in animal protein but rich in complex carbohydrates in African children, leads to a differentiation in the Firmicutes/Bacteroidetes ratio between the two groups. Gram-positive bacteria, mainly Firmicutes, were more abundant than Gram-negative bacteria in European children, whereas Gram-negative bacteria, mainly Bacteroidetes, prevailed over Gram-positive bacteria in African children.
    And the long-term diets are strongly associated to the enterotype partitioning. Indeed, it has been observed that:

a diet high in animal fat and protein, i.e. a Western-type diet, leads to a gut microbiota dominated by the Bacteroides enterotype;
a diet high in complex carbohydrates, typical of agrarian societies, leads to the prevalence of the Prevotella enterotype.

Similar results emerged from the aforementioned study on children. In the Europeans, gut microbiota was dominated by taxa typical of Bacteroides enterotype, whereas in the Burkina Faso children, Prevotella enterotype dominates.
With short-term changes in the diet (10 days), such as the switch from a low-fat and high-fiber diet to a high-fat and low-fiber diet and vice versa, changes were observed in the composition of the microbiome (within 24 hours), but no stable change in the enterotype partitioning. And this underlines as a long-term diet is needed for a change in the enterotypes of the gut microbiota.
Dietary interventions can also result in changes in the gut virome, which moves to a new state, that is, changes occur in the proportions of the pre-existing viral populations, towards which subjects on the same diet converge.

  • pH, bile salts and digestive enzymes.
    The stomach, due to its low pH, is a hostile environment for bacteria, which are not present in high numbers, about 102-103 bacterial cells/gram of tissue. In addition to Helicobacter pylori, able to cause gastritis and gastric ulcers, microorganisms of the genus Lactobacillus are also present.
    Reached the duodenum, an increase in bacterial cell number occurs, 104-105 bacterial cells/gram of tissue; and similar bacterial concentrations are present in the jejunum and proximal ileum. The low number of microorganisms present in the small intestine is due to the inhospitable environment, consequent to the fact that there is the opening of the ampulla of Vater in the descending part of the duodenum, which pours pancreatic juice and bile into the duodenum, that is, pancreatic enzymes and bile salts, which damage microorganisms.
    In the terminal portion of the ileum, where the activities of pancreatic enzymes and bile salts are lower, there are about 107 bacterial cells/gram of tissue, and up to 1012-1014 bacterial cells/gram of tissue in the colon, so that bacteria represent a large proportion, about 40%, of the fecal mass.
    The distribution of bacteria along the intestine is strategic. In the duodenum and jejunum, the amount of available nutrients is much higher than that found in the terminal portion of the ileum, where just water, fiber, and electrolytes remain. Therefore, the presence of large number of bacteria in the terminal portion of the ileum, and even more in the colon, is not a problem. The problem would be to find a high bacterial concentration in the duodenum, jejunum, and proximal parts of the ileum; and there is a disease condition, called small intestinal bacterial overgrowth or SIBO, in which the number of bacteria in the small intestine increases by about 10-15 times. This puts them in a position to compete with the host for nutrients and give rise to gastrointestinal disturbances such as diarrhea.
  • The geographical position and the resulting differences in lifestyle, diet, religion etc.
    For example, a kind of geographical gradient occurs in the microbiota of European infants, with a higher number of Bifidobacterium species and some of Clostridium in Northern infants, whereas Southern infants have higher levels of Bacteroides, Lactobacillus and Eubacterium.
  • The mode of delivery (see below).
  • The genetics of the host.
  • The health status of the infant and mother.
    For example, in mothers with inflammatory bowel disease or IBD, Faecalibacterium prausnitzii, a bacterium that produces butyrate (an important source of energy for intestinal cells), and with anti-inflammatory activity is depleted, whereas there is an increase in the number of adherent Escherichia coli.
  • The treatment with antibiotics.
  • Bacterial infections and predators.
    Bacteriocins, i.e. proteins with antibacterial activity, and bacteriophages.
    Phages play an important role in controlling the abundance and composition of the gut microbiota. In particular, they could play a major role in the colonization of the newborn, infecting the dominant bacteria thus allowing to another bacterial strain to become abundant.
    This model of predator-prey dynamics, called “kill the winner”, suggests that the blooms of a specific bacterial species would lead to blooms of their corresponding bacteriophages, followed by a decline in their abundance. Therefore, the most abundant bacteriophage genotype will not be the same at different times. And although some the gene sequences present in the infant gut virome are stable over the first three months of life, dramatic changes occur in the overall composition of the viral community between the first and second week of life. During this time period also the bacterial community is extremely dynamic (see below).
  • The competition for space and nutrients.

⇑ Back to the top ⇑

The composition of the gut microbiota throughout life

Gut Microbiota
Fig. 2 – Development of Intestinal Microflora

The development of the intestinal microbial ecosystem is a complex and crucial event in human life, highly variable from individual to individual, and influenced by the factors outlined above.
In utero, the gut is considered sterile, but is rapidly colonized by microbes at birth, as the infant is born with an immunological tolerance instructed by the mother.
However, recent studies show the presence of bacteria in the placental tissue, umbilical cord blood, fetal membranes and amniotic fluid from healthy newborns without signs of infection or inflammation. And for example, the meconium of premature infants, born to healthy mothers, contains a specific microbiota, with Firmicutes as the main phylum, and predominance of staphylococci, whereas Proteobacteria, in particular species such as Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, but also enterococci are more abundant in the faeces.
Note: The meconium is free of detectable viruses.
It seems that both vaginal and gut bacteria may gain access to the fetus, although via different route of entry: by ascending entry the vaginal ones, by dendritic cells of the immune system the gut ones. Therefore, there could exist a fetal microbiota.

Colonization occurs during delivery by a maternal inoculum, generally composed of aerobic and facultative bacteria (the newborn’s gut initially contains oxygen), then replaced by obligate anaerobes,  bacteria typically present in adulthood, to which they have created a hospitable environment.
Furthermore, there is a small number of different taxa, with a relative dominance of the phyla Actinobacteria and Proteobacteria, that remains unchanged during the first month of life, but not in the subsequent ones as there is a large increase in variability and new genetic variants. Many studies underline that the initial exposure is important in defining the “trajectories” which will lead to the adult ecosystems. Additionally, these initial communities may act as a source of protective or pathogenic microorganisms.

Mother’s vaginal and fecal microbiotas are the main sources of inoculum in vaginally delivered infants. Indeed, infants harbor microbial communities dominated by species of the genera Lactobacillus (the most abundant genus in the vaginal microbiota and early gut microbiota) Bifidobacterium, Prevotella, or Sneathia. And it seems likely that anaerobes, such as members of the phyla Firmicutes and Bacteroidetes, not growing outside of their host, rely on the close contact between mother and offspring for transmission. Finally, due to the presence of oxygen in infant gut, the transmission of strict anaerobes could occur not directly at birth but at a later stage by means of spores.
The first bacteria encountered by infants born by caesarean section are those of the skin and hospital environment, and gut microbiota is dominated by species of the genera Corynebacterium, Staphylococcus and Propionibacterium, with a lower bacterial count and diversity in first weeks of life than infants born vaginally.
Further evidence supporting the hypothesis of vertical transmission is the similarity between the microbiota of meconium and samples obtained from possible sites of contamination.
These “maternal bacteria” do not persist indefinitely, and are replaced by other populations within the first year of life.
Objects, animals, mouths and skin of relatives, and breast milk are secondary sources of inoculum; and breast milk (see below) seems to have a primary role in determining the microbial succession in the gut.
The variation and diversity among children reflect instead the individuality of these microbial exposures.
Note: The delivery mode seems also to influence the immune system during the first year of life, perhaps via the influence on the development of gut microbiota. Infants born by cesarean section have:

  • a lower bacterial count in stool samples at one month of age, mainly due to the higher number of bifidobacteria in infants born vaginally;
  • a higher number of antibody secreting cells, which could reflect an excessive antigen exposure (the intestinal barrier would be more vulnerable to the passage of antigens).

Within a days after birth, a thriving community is established. This community is less stable over time and more variable in composition than that of adults. Very soon, it will be more numerous than that of the child’s cells, evolving according to a temporal pattern highly variable from individual to individual.
Viruses, absent at birth, reach about 108 units/gram wet weight of faeces by the end of the first week of life, therefore representing a dynamic and abundant component of the developing gut microbiota. However, viral community has an extremely low diversity, like bacteria, and is dominated by phages, which probably influence the abundance and diversity of co-occurring bacteria, as seen above. The initial source of the viruses is unknown; of course, maternal and/or environmental inocula are among the possibilities. Notably, the earliest viruses could be the result of induction of prophages from the “newborn” gut bacterial flora, hypothesis supported by the observation that more than 25% of the phage sequences seem to be very similar to those of phages infecting bacteria such as Lactococcus, Lactobacillus, Enterococcus, and Streptococcus, which are abundant in breast milk.

By the end of the first month of life it is thought that the initial phase of rapid acquisition of microorganism is over.
In 1-month-old-infants, the most abundant bacteria belong to the genera Bacteroides and Escherichia, whereas Bifidobacterium, along with Ruminococcus, appear and grow to become dominant in the gastrointestinal tract of the breastfed infants between 1 and 11 months. Bifidobacteria such as Bifidobacterium longum subspecies infantis:

  • are known to be closely related to breastfeeding;
  • are among the best characterized commensal bacteria;
  • are considered probiotics, that is, microorganisms which can confer health benefits to the host.

Their abundance confers also benefits through competitive exclusion, that is, they are an obstacle to colonization by pathogens. And indeed, Escherichia and Bacteroides can become preponderant if Bifidobacterium is not adequately present in the gut.
In contrast, bacteria of the genera Escherichia (e.g. E. coli), Clostridium (e.g. C. difficile), Bacteroides (e.g. B. fragilis) and Lactobacillus are present in higher levels in formula-fed infants than in breastfed infants.
Although breast-fed infants receive only breast milk until weaning, their microbiota can show a large variability in the abundances of bacterial taxa, with differences between individuals also with regard to the temporal patterns of variation. These variations may be due to diseases, treatments with antibiotics, changes in host lifestyle, random colonization events, as well as differences in immune responses to the gut colonizing microbes. However, it is not yet clear how these factors contribute to shape infant gut microbiota.
It seems that also the virome changes rapidly after birth, as the majority of the viral sequences present in the first week of life are not found after the second week. Moreover, the repertoire expands rapidly in number and diversity during the first three months. This is in contrast with the stability observed in the adult virome, where 95% of the sequences are conserved over time.

In normal condition, towards the end of the first year of life, babies have consumed an adult-like diet for a significant time period and should have developed a microbial community with characteristics similar to those found in the adult gut, such as:

  • a more stable composition, phylogenetically more complex, and progressively more similar among different subjects;
  • a preponderance of Firmicutes and Bacteroidetes, followed by Verrucomicrobia and a very low abundance of Proteobacteria;
  • an increase in short-chain fatty acid (SCFA) levels and bacterial load in the feces;
  • an increase of genes associated with xenobiotic degradation, vitamin biosynthesis, and carbohydrate

Interestingly, the significant turnover of taxa occurring from birth to the end of the first year is accompanied by a remarkable constancy in the overall functional capabilities.
Towards the end of the first year of life also the early viral colonizers were replaced by a community specific to the child.

The gut microbiota reaches maturity at about 2.5 years of age, fully resembling the adult gut microbiota.
The selection of the most adapted bacteria is the result of various factors.

  • The transition to an adult diet.
  • An increased fitness to the intestinal environment of the taxa that typically dominate the adult gut microbiota than the early colonizers.
  • The significant changes in the intestinal environment, result of the developmental changes in the intestinal mucosa.
  • The effects of the microbiota itself.

Therefore, the first 2-3 years of life are the most critical period in which you can intervene to shape the microbiota as best as possible, and so optimize child growth and development.

From a chaotic beginning, all this leads to the establishment of the gut ecosystem typical of the young adult, which is relatively stable over time until old age (viral, archaeal and eukaryotic components included), and dominated, at least in the western population, by members of the phyla Firmicutes, about 60% of the bacterial communities, Bacteroidetes and Actinobacteria (mainly belonging to the Bifidobacterium genus), each comprising about 10% of the bacterial community, followed by Proteobacteria and Verrucomicrobia. The genera Bacteroides, Clostridium, Faecalibacterium, Ruminococcus and Eubacterium make up, together with Methanobrevibacter smithii, the large majority of the adult gut microbial community.
It should be noted that different data were obtained from analysis of populations of African rural areas, as seen above.
And the gut microbiota is sufficiently similar among subjects to allow the identification of a shared core microbiome.
Stability and resilience, however, are subject to numerous variables among which, as previously said, diet seems to be one of the most important. Therefore, in order to maintain the stability of the gut microbiota, the variables have to be kept constant, or in the case of diseases prevented (also through vaccinations). However, the stability and resilience could be harmful if the dominant community is pathogenic.

The gut microbiota undergoes substantial changes in the elderly. In a study conducted in Ireland on 161 healthy people aged 65 years and over, the gut microbiota is distinct from that of younger adults in the majority of subjects, with a composition that seems to be dominated by the phyla Bacteroidetes, the main ones, and Firmicutes, with almost inverted percentages than those found in younger adults (although large variations across subjects were observed). And there are Faecalibacterium, about 6% of the main genera, followed by species of the genera Ruminococcus, Roseburia and Bifidobacterium (the latter about 0.4%) among the most abundant genera.
Also the variability in the composition of the community is greater than in younger adults; this could be due to the increase in morbidities associated with aging and the subsequent increased intake of medications, as well as to changes in the diet.

⇑ Back to the top ⇑

References

Breitbart M., Haynes M., Kelley S., Angly F., Edwards R.A., Felts B., Mahaffy J.M., Mueller J., Nulton J., Rayhawk S., Rodriguez-Brito B., Salamon P., Rohwer F. Viral diversity and dynamics in an infant gut. Res Microbiol 2008;159:367-73. doi:10.1016/j.resmic.2008.04.006

Claesson M.J., Cusack S., O’Sullivan O., Greene-Diniz R., de Weerd H., Flannery E., Marchesi J.R., Falush D., Dinan T., Fitzgerald G., et al. Composition, variability, and temporal stability of the intestinal microbiota of the elderly. Proc Natl Acad Sci USA 2011;108(Suppl 1);4586-91. doi:10.1073/pnas.1000097107

Clemente J.C., Ursell L.K., Wegener Parfrey L., and Knight R. The impact of the gut microbiota on human health: an integrative view. Cell 2012;148:1258-70. doi:10.1016/j.cell.2012.01.035

De Filippo c., Cavalieri D., Di Paola M., Ramazzotti M., Poullet J.B., Massart S., Collini S., Pieraccini G., and Lionetti P. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc Natl Acad Sci 2010;107(33):14691-6. doi:10.1073/pnas.1005963107

Dominguez-Bello M.G., Costello E.K., Contreras M., Magris M., Hidalgo G., Fierer N., and Knight R. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc Natl Acad Sci 2010;107:11971-5. doi:10.1073/pnas.1002601107

Fernández L., Langa S., Martín V., Maldonado A., Jiménez E., Martín R., Rodríguez J.M. The human milk microbiota: origin and potential roles in health and disease. Pharmacol Res 2013;69(1):1-10. doi:10.1073/pnas.1002601107

Huurre A., Kalliomäki M., Rautava S., Rinne M., Salminen S., and Isolauri E. Mode of delivery-effects on gut microbiota and humoral immunity. Neonatology 2008;93:236-40. doi:10.1159/000111102

Koenig J.E., Spor A., Scalfone N., Fricker A.D., Stombaugh J., Knight R., Angenent L.T., and Ley R.E. Succession of microbial consortia in the developing infant gut microbiome. Proc Natl Acad Sci 2011;108(1):4578-85. doi:10.1073/pnas.1000081107

Ley R.E., Peterson D.A., and Gordon J.I. Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell 2006;124(4):837-48. doi:10.1016/j.cell.2006.02.017

Minot S., Sinha R., Chen J., Li H., Keilbaugh S.A., Wu G.D., Lewis J.D., and Bushman F.D. The human gut virome: inter-individual variation and dynamic response to diet. Genome Res 2011;21:1616-1625. doi:10.1101/gr.122705.111

Moreno-Indias I.M., Cardona F., Tinahones F.J. and Queipo-Ortuño M.I. Impact of the gut microbiota on the development of obesity and type 2 diabetes mellitus. Front Microbiol 2014;5(190):1-10 . doi:10.3389/fmicb.2014.00190

Newburg D.S. & Morelli L. Human milk and infant intestinal mucosal glycans guide succession of the neonatal intestinal microbiota. Pediatr Res 2015;77:115-120. doi:10.1038/pr.2014.178

Palmer C., Bik E.M., DiGiulio D.B., Relman D.A., and Brown P.O. Development of the human infant intestinal microbiota. PLoS Biol 2007;5(7):e177. doi:10.1371/journal.pbio.0050177

Rodrıguez J.M., Murphy K., Stanton C., Ross R.P., I. Kober O.I., Juge N., Avershina E., Rudi K., Narbad A., Jenmalm M.C., Marchesi J.R. and Collado M.C. The composition of the gut microbiota throughout life, with an emphasis on early life. Microb Ecol Health Dis 2015;26:26050. doi:10.3402/mehd.v26.26050

Wu G.D., Chen J., Hoffmann C., Bittinger K., Chen Y.Y., Keilbaugh S.A., Bewtra M., Knights D., Walters W.A., Knight R., et al. Linking long-term dietary patterns with gut microbial enterotypes. Science 2011;334:105-8. doi:10.1126/science.1208344

Human microbiota: definition, composition, functions, antibiotics

Human microbiota: contents in brief

What is the human microbiota?

Human Microbiota
Fig. 1 – Lactobacillus casei

It has been known for almost a century that humans harbor a microbial ecosystem, known as human microbiota, remarkably dense and diverse, made up of a  number of viruses and cells much higher than those of the human body, and that accounts for one to three percent of body weight. All the genes encoded by the human body’s microbial ecosystem, which are about 1,000 times more numerous than those of our genome, make up the human microbiome. Microorganisms colonize all the surfaces of the body that are exposed to the environment. Indeed, distinct microbial communities are found on the skin, in the vagina, in the respiratory tract, and along the whole intestinal tract, from the mouth up to rectum, the last part of the intestine.

⇑ Back to the top ⇑

Composition of the human microbiota

Human Microbiota
Fig. 2 – Escherichia coli

It is composed of organisms from all taxa.

  • Bacteria, at least 100 trillion (1014) cells, a number ten times greater than that of the human body. They are found in very high concentration in the intestinal tract, up to 1012-1014/gram of tissue, where they form one of the most densely populated microbial habitats on Earth. In the gut, bacteria mainly belong to the Firmicutes, Bacteroidetes and Actinobacteria phyla. Fusobacteria (oropharynx), Tenericutes, Proteobacteria, and Verrucomicrobia are other phyla present in our body.
    Note: Bacterial communities in a given body region resemble themselves much more across individuals than those from different body regions of the same individual; for example, bacterial communities of the upper respiratory tract are much more similar across individuals than those of the skin or intestine of the same individual.
  • Viruses, by far the most numerous organisms, about quadrillion units. The genomes of all the viruses harbored in the human body make up the human virome. In the past, viruses and eukaryotes (see below) have been studied focusing on pathogenic microorganisms, but in recent years the attention has also shifted on many non-pathogenic members of these groups. And many of the viral gene sequences found are new, which suggests that there is still much to learn about the human virome. Finally, just like for bacteria, there is considerable interpersonal variability.
  • Archaebacteria, primarily those belonging to the order Methanobacteriales, with Methanobrevibacter smithii predominant in the human gut (up to 10% of all anaerobes).
  • Eukaryotes, and the parasites of the genera Giardia and Entamoeba have probably been among the first to be identified. But there is also a great abundance and diversity of fungal species, belonging to genera such as Candida, Penicillium, Aspergillus, Hemispora, Fusarium, Geotrichum, Hormodendrum, Cryptococcus, Saccharomyces, and Blastocystis.
Human Microbiota
Fig. 3 – Candida albicans

Based on the relationships with the human host, microorganisms may be classified as commensals or pathogens.

  • Commensals cause no harm to the host, with which they establish a symbiotic relationship that generally brings benefits to both.
  • On the contrary, pathogens are able to cause diseases, but fortunately represent a small percentage of the human microbiota. These microorganisms establish a symbiosis with the human host and benefit from it at the expense of the host. They can cause disease:

if they move from their niche, such as the intestine, into another one where they do not usually reside, such as the vagina or bladder (as in the case of Candida albicans, normally present in the intestine, but in very small quantities);
in patients with impaired immunological defenses, such as after an immunosuppressive therapy.

⇑ Back to the top ⇑

Functions of the human microbiota

Human Microbiota
Fig. 4 – Bifidobacterium longum

Sometimes referred to as “the forgotten organ“, human microbiota, mainly with its intestinal bacterial members, plays many important functions that can lead to nutritional, immunological, and developmental benefits, but can also cause diseases. Here are some examples.

  • It is involved in the development of the gastrointestinal system of the newborn, as shown by experiments carried out on germ-free animals in which, for example, the thickness of the intestinal mucosa is thinner than that of colonized animals, therefore more easily subject to rupture.
  • It contributes to energy harvest from nutrients, due to its ability to ferment indigestible carbohydrates, promote the absorption of monosaccharides and the storage of the derived energy. This has probably been a very strong evolutionary force that has played a major role in favor of the fact that these bacteria became our symbionts.
  • It contributes to the maintenance of the acidic pH of the skin and in the colon.
  • It is involved in the metabolism of xenobiotics and several polyphenols.
  • It improves water and mineral absorption in the colon.
  • It increases the speed of intestinal transit, slower in germ-free animals.
  • It has an important role in resistance to colonization by pathogens, primarily in the vagina and gut.
  • It is involved in the biosynthesis of isoprenoids and vitamins through the methylerythritol phosphate pathway.
  • It stimulates angiogenesis.
  • In the intestinal tract, it interacts with the immune system, providing signals for promoting the maturation of immune cells and the normal development of immune functions. And this is perhaps the most important effect of the symbiosis between the human host and microorganisms. Experiments carried out on germ-free animals have shown, for example, that:

macrophages, the cells that engulf pathogens and then present their antigens to the immune system, are found in much smaller amounts than those present in the colonized intestine, and if placed in the presence of bacteria they fail to find and therefore engulf them, unlike macrophages extracted from a colonized intestine;
there is not the chronic non-specific inflammation, present in the normal intestine as a result of the presence of bacteria (and of what we eat).

  • Changes in its composition can contribute to the development of obesity and metabolic syndrome.
  • It protects against the development of type I diabetes.
  • Many diseases, both in children and adults, such as stomach cancer, lymphoma of mucosa-associated lymphoid tissue, necrotizing enterocolitis (an important cause of morbidity and mortality in premature babies) or chronic intestinal diseases, are, and others seem to be, related to the gut microbiota.

In conclusion, it seems very likely that the human body represents a superorganism, result of years of evolution and made up of human cells, and the resulting metabolic and physiological capacities, as well as an additional organ, the microbiota.

⇑ Back to the top ⇑

Human Microbiome Project

Human Microbiota
Fig. 5- Human Microbiome Project

The bacterial component of the human microbiota is the subject of most studies including a large-scale project started in 2008 called “Human Microbiome Project“, whose aim is to characterize the microbiome associated with multiple body sites, such as the skin, mouth, nose, vagina and intestine, in 242 healthy adults. These studies have shown a great variability in the composition of the human microbiota; for example, twins share less than 50% of their bacterial taxa at the species level, and an even smaller percentage of viruses. The factors that shape the composition of bacterial communities begin to be understood: for example, the genetic characteristics of the host play an important, although this is not true for the viral community. And metagenomic studies have shown that, despite the great interpersonal variability in microbial community composition, there is a core of shared genes encoding signaling and metabolic pathways. It appears namely that the assembly and the structure of the microbial community does not occur according to the species but the more functional set of genes. Therefore, disease states of these communities might be better identified by atypical distribution of functional classes of genes.

⇑ Back to the top ⇑

Effects of antibiotics on the human microbiota

Human Microbiota
Fig. 6 – Clostridium difficile

The microbiota in healthy adult humans is generally stable over time. However, its composition can be altered by factors such as dietary changes, urbanization, travel, and especially the use of broad-spectrum antibiotics. Here are some examples of the effects of antibiotic treatments.

  • There is a long-term reduction in microbial diversity.
  • The taxa affected vary from individual to individual (even up to a third of the taxa).
  • Several taxa do not recover even after 6 months from treatment.
  • Once the bacterial communities have reshaped, a reduced resistance to colonization occurs. This allows foreign and/or pathogen bacteria, able to grow more than the commensals, to cause permanent changes in human microbiota structure, as well as acute diseases, such as the dangerous pseudomembranous colitis, and chronic diseases, as it is suspected for asthma following the use and abuse of antibiotics in childhood. Moreover, their repeated use has been suggested to increase the pool of antibiotic-resistance genes in our microbiome. In support of this hypothesis, a decrease in the number of antibiotic-resistant pathogens has been observed in some European countries following the reduction in the number of antibiotics prescribed.

Finally, you must not underestimate the fact that the intestinal microflora is involved in many chemical transformations, and its alteration could be implicated in the development of cancer and obesity. However, regarding use of antibiotics, you should be underlined that if western population has a life expectancy higher than in the past is also because you do not die of infectious diseases!

⇑ Back to the top ⇑

References

Burke C., Steinberg P., Rusch D., Kjelleberg S., and Thomas T. Bacterial community assembly based on functional genes rather than species. Proc Natl Acad Sci USA 2011;108:14288-93. doi:10.1073/pnas.1101591108

Clemente J.C., Ursell L.K., Wegener Parfrey L., and Knight R. The impact of the gut microbiota on human health: an integrative view. Cell 2012;148:1258-70. doi:10.1016/j.cell.2012.01.035

Gill S.R., Pop M., Deboy R.T., Eckburg P.B., Turnbaugh P.J., Samuel B.S., Gordon J.I., Relman D.A., Fraser-Liggett C.M., and Nelson K.E. Metagenomic analysis of the human distal gut microbiome. Science 2006;312:1355-59. doi:10.1126/science.1124234

Palmer C., Bik E.M., DiGiulio D.B., Relman D.A., and Brown P.O. Development of the human infant intestinal microbiota. PLoS Biol 2007;5(7):e177. doi:10.1371/journal.pbio.0050177

The Human Microbiome  Project

Turnbaugh P.J., Gordon J.I. The core gut microbiome, energy balance and obesity. J Physiol 2009;587:4153-58. doi:10.1113/jphysiol.2009.174136

Zhang, T., Breitbart, M., Lee, W., Run, J.-Q., Wei, C., Soh, S., Hibberd, M., Liu, E., Rohwer, F., Ruan, Y. Prevalence of plant viruses in the RNA viral community of human feces. PLoS Biol 2006;4(1):e3. doi:10.1371/journal.pbio.0040003

Flavonoid biosynthesis pathway

Flavonoid biosynthesis pathway: contents in brief

The flavonoid biosynthetic pathway in plants

Flavonoid Biosynthesis
Fig. 1 – Biosynthesis of Flavonoids

The biosynthesis of flavonoids, probably the best characterized pathway of plant secondary metabolism, is part of the phenylpropanoid pathway that, in addition to flavonoids, leads to the formation of a wide range of phenolic compounds, such as hydroxycinnamic acids, stilbenes, lignans and lignins.
Flavonoid biosynthesis is linked to primary metabolism through both mitochondria- and plastid-derived molecules. Since it seems that most of the involved enzymes characterized to date operate in protein complexes located in the cell cytosol, these molecules must be exported to the cytoplasm to be used.
The end products are transported to different intracellular or extracellular locations, with flavonoids involved in pigmentation usually transported into the vacuoles.
The biosynthesis of this group of polyphenols requires one p-coumaroyl-CoA and three malonyl-CoA molecules as initial substrates.

⇑ Back to the top ⇑

Biosynthesis of p-coumaroyl-CoA

p-Coumaroyl-CoA is the pivotal branch-point metabolite in the phenylpropanoid pathway, being the precursor of a wide variety of phenolic compounds, both flavonoid and non-flavonoid polyphenols.

Flavonoid Biosynthesis
Fig. 2 – p-Cumaroyl-CoA

It is produced from phenylalanine via three reactions catalyzed by cytosolic enzymes collectively called group I or early-acting enzymes, in order of action:

  • phenylalanine ammonia lyase (EC 4.3.1.24);
  • cinnamate 4-hydroxylase or cinnamic acid 4-hydroxylase (EC 1.14.13.11);
  • 4-cumarato: CoA ligase or hydroxycinnamic: CoA ligase (EC 6.2.1.12).

They seems to be associated in a multienzyme complex anchored to the endoplasmic reticulum membrane. The anchoring is probably ensured by cinnamate 4-hydroxylase that inserts its N-terminal domain into the membrane of the endoplasmic reticulum itself. These complexes, referred to as “metabolons”, allow the product of a reaction to be channeled directly as substrate to the active site of the enzyme that catalyzes the consecutive reaction in the metabolic pathway.
With the exception of cinnamate 4-hydroxylase, the enzymes which act downstream of phenylalanine ammonia lyase are encoded by small gene families in all species analyzed so far.
The different isoenzymes show distinct temporal, tissue, and elicitor-induced patterns of expression. It seems, in fact, that each member of each family can be used mainly for the synthesis of a specific compound, thus acting as a control point for carbon flux among the metabolic pathways leading to lignan, lignin, and flavonoid biosynthesis.

Note: Phenylalanine is a product of the shikimic acid pathway, which converts simple precursors derived from carbohydrate metabolism, phosphoenolpyruvate and erythrose-4-phosphate, into the aromatic amino acids phenylalanine, tyrosine and tryptophan. Unlike plants and microorganisms, animals do not possess the shikimic acid pathway, and are not able to synthesize the three above-mentioned amino acids, which are therefore essential nutrients.

⇑ Back to the top ⇑

Phenylalanine ammonia lyase (PAL)

It is one of the most studied and best characterized enzymes of plant secondary metabolism. It requires no cofactors and catalyzes the reaction that links primary and secondary metabolism: the deamination of phenylalanine to trans-cinnamic acid, with the release of nitrogen as ammonia and introduction of a trans double bond between carbon atoms 7 and 8 of the side chain.

Flavonoid Biosynthesis
Fig. 3 – Phenylalanine Ammonia Lyase

Therefore, it directs the flow of carbon from the shikimic acid pathway to the different branches of the phenylpropanoid pathway. The released ammonia is probably fixed in the reaction catalyzed by glutamine synthetase.
The enzyme from monocots is also able to act as tyrosine ammonia lyase (EC 4.3.1.25), converting tyrosine to p-coumaric acid directly, (therefore without the 4-hydroxylation step), but with a lower efficiency.
In all plant species investigated,  several copies of phenylalanine ammonia lyase gene are found, copies that probably respond differentially to internal and external stimuli. Indeed, gene transcription, and then enzyme activity, are under the control of both internal developmental and external environmental stimuli. Here are some examples that require increased enzyme activity.

  • The flowering.
  • The  production of lignin to strengthen the secondary wall of xylem cells.
  • The production of flower pigments that attract pollinators.
  • Pathogen infections, that require the production of phenylpropanoid phytoalexins, or exposure to UV rays.

⇑ Back to the top ⇑

Cinnamate 4-hydroxylase (C4H)

It belongs to the cytochrome P450 superfamily (EC 1.14.-.-), is a microsomal monooxygenase containing a heme cofactor, and dependent on both O2  and NADPH. It catalyzes the formation of p-coumaric acid  through the introduction of a hydroxyl group in 4-position of trans-cinnamic acid (this hydroxyl group is present in most flavonoids).

Flavonoid Biosynthesis
Fig. 4 – Cinnamate 4-Hydroxylase

This reaction is also part of the biosynthesis of hydroxycinnamic acids.
Increases in transcription rates and enzyme activity are observed in correlation with the synthesis of phytoalexins (in response to fungal infections), lignification as well as wounding.

⇑ Back to the top ⇑

4-Coumarate:CoA ligase (4CL)

With Mg2+ as a cofactor, it catalyzes the ATP-dependent activation of the carboxyl group of p-coumaric acid and other hydroxycinnamic acids, metabolically rather inert molecules, through the formation of the corresponding CoA-thioester.

Flavonoid Biosynthesis
Fig. 5 – 4-Coumarate:CoA Ligase

Generally, p-coumaric acid and caffeic acid are the preferred substrates, followed by ferulic acid and 5-hydroxyferulic acid, low activity against trans-cinnamic acid and none against sinapic acid. These CoA-thioesters are able to enter various reactions such as:

  • reduction to alcohol (monolignols) or aldehydes;
  • stilbene and flavonoid biosynthesis;
  • transfer to acceptor molecules.

It should finally be pointed out that the activation of the carboxyl group can also be obtained through an UDP-glucose-dependent transfer to glucose.

⇑ Back to the top ⇑

Biosynthesis of malonyl-CoA

Malonyl-CoA does not derived from the phenylpropanoid pathway, but from the reaction catalyzed by acetyl-CoA carboxylase (EC 6.4.1.2, the cytosolic form, see below). The enzyme, with biotin and Mg2+ as cofactors, catalyzes the ATP-dependent carboxylation of acetyl-CoA, using bicarbonate as a source of carbon dioxide (CO2).

Flavonoid Biosynthesis
Fig. 6 – Acetyl-CoA Carboxylase

It is found both in the plastids, where it participates in the synthesis of fatty acids, and the cytoplasm, and is the latter that catalyzes the formation of malonyl-CoA that is used in the biosynthesis of flavonoids and other compounds. Increases in the transcription rate of the gene and enzyme activity are induced in response to stimuli that increase the biosynthesis of these polyphenols, such as exposure to pathogenic fungi or UV-rays.
In turn, acetyl-CoA is produced in plastids, mitochondria, peroxisomes and cytosol through different metabolic pathways. The molecules used in the biosynthesis of malonyl-CoA, and therefore of the flavonoids, are  the cytosolic ones, produced in the reaction catalyzed by ATP-citrate lyase (EC 2.3.3.8) that cleaves citrate, in the presence of CoA and ATP, to form oxaloacetate and acetyl-CoA, plus ADP and inorganic phosphate.

⇑ Back to the top ⇑

The first steps in flavonoid biosynthesis

The first step in flavonoid biosynthesis is catalyzed by chalcone synthase (EC 2.3.1.74), an enzyme anchored to the endoplasmic reticulum and with no known cofactors.
From one p-coumaroyl-CoA and three malonyl-CoA, it catalyzes sequential condensation and decarboxylation reactions in the course of which a polyketide intermediate is formed. The polyketide undergoes cyclizations and aromatizations leading to the formation of the A ring. The product of the reactions is naringenin chalcone (2′,4,4′,6′-tetrahydroxychalcone), a 6′-hydroxychalcone and the first flavonoid to be synthesized by plants.

Flavonoid Biosynthesis
Fig. 7 – Naringenin Chalcone

The reaction, cytosolic, is irreversible due to the release of three CO2 and 4 CoA.
The B ring and the three-carbon bridge of the molecule originate from p-coumaroyl-CoA (and therefore from phenylalanine), the A ring from the three malonyl-CoA units.

Flavonoid Biosynthesis
Fig. 8 – Basic Flavonoid Skeleton

Also 6’-deoxychalcone can be produced; its synthesis is thought to involve an additional reduction step catalyzed by polyketide reductase (EC. 1.1.1.-).
Chalcone synthase from some plant species, such as barley (Hordeum vulgare), accepts as substrates also caffeoil-CoA, feruloil-CoA and cinnamoyl-CoA.
It is the most abundant enzyme of the phenylpropanoid pathway, probably because it has a low catalytic activity, and, in fact, is considered to be the rate-limiting enzyme in flavonoid biosynthesis.
As for phenylalanine ammonia lyase, chalcone synthase gene expression is under the control of both internal and external factors. In some plants, one or two isoenzymes are found, while in others up to 9.
Chalcone synthase belongs to polyketide synthase group, present in bacteria, fungi and plants. These enzymes are able to catalyze the production of polyketide chains through sequential condensations of acetate units provided by malonyl-CoA units. They also includes stilbene synthase (EC 2.3.1.146), which catalyzes the formation of resveratrol, a non flavonoid polyphenol compound that has attracted much interest because of its potential health benefits.
Generally, chalcones do not accumulate in plants because naringenin chalcone is converted to (2S)-naringenin, a flavanone, in the reaction catalyzed by chalcone isomerase (EC 5.5.1.6).

Flavonoid Biosynthesis
Fig. 9 – (2S)-Naringenin

The enzyme, the first of the flavonoid biosynthesis to be discovered, catalyzes a stereospecific isomerization and closes the C ring. Two types of chalcone isomerases are known, called type I and II. Type I enzymes use only 6′-hydroxychalcone substrates, like naringenin chalcone, while type II, prevalent in legumes, use both 6′-hydroxy- and 6′-deoxychalcone substrates.
It should be noted that with 6′-hydroxychalcones, isomerization can also occur nonenzymically to form a racemic mixture, both in vitro and in vivo, enough to allow a moderate synthesis of anthocyanins. On the contrary, under physiological conditions 6′-deoxychalcones are stable, and so the activity of type II chalcone isomerases is required to form flavanones.
The enzyme increases the rate of the reaction of 107 fold compared to the spontaneous reaction, but with a higher kinetics for the 6′-hydroxychalcones than 6′-deoxychalcones. Finally, it produces (2S)-flavanones, which are the biosynthetically required enantiomers.
As other enzymes in flavonoid biosynthesis, also chalcone isomerase gene expression is subject to strict control. And, as phenylalanine ammonia lyase and chalcone synthase, it is induced by elicitors.
In the reaction catalysed by flavanone-3β-hydroxylase (EC 1.14.11.9), (2S)-flavanones undergo a stereospecific isomerization that converts them into the respective (2R,3R)-dihydroflavonols. In particular, naringenin is converted into dihydrokaempferol.

Flavonoid Biosynthesis
Fig. 10 – Dihydrokaempferol

The enzyme is a cytosolic non-heme-dependent dioxygenase, dependent on Fe2+ and 2-oxoglutarate, and therefore belonging to the family of 2-oxoglutarate-dependent dioxygenase (which distinguishes them from the other hydroxylases of the flavonoid biosynthetic pathway which are cytochrome P450 enzymes).
Naringenin chalcone, (2S)-naringenin, and dihydrokaempferol are central intermediates in flavonoid biosynthesis, since they act as branch-point compounds from which the synthesis of distinct flavonoid subclasses can occur. For example, directly or indirectly:

Not all of these side metabolic pathways are present in every plant species, or are active within each tissue type of a given plant. Like enzymes previously seen, the activity of those involved in these “side-routes” is subject to strict control, resulting in a tissue-specific profile of flavonoid compounds. For example, grape seeds, flesh and skin have distinct anthocyanin, catechin, flavonol and condensed tannin profiles, whose synthesis and accumulation are strictly and temporally coordinated during the ripening process.

⇑ Back to the top ⇑

References

Andersen Ø.M., Markham K.R. Flavonoids: chemistry, biochemistry, and applications. CRC Press Taylor & Francis Group, 2006

de la Rosa L.A., Alvarez-Parrilla E., Gonzàlez-Aguilar G.A. Fruit and vegetable phytochemicals: chemistry, nutritional value, and stability. 1th Edition. Wiley J. & Sons, Inc., Publication, 2010

Heldt H-W. Plant biochemistry – 3th Edition. Elsevier Academic Press, 2005

Vogt T. Phenylpropanoid biosynthesis. Mol Plant 2010;3(1):2-20. doi:http://dx.doi.org/10.1093/mp/ssp106

Wink M. Biochemistry of plant secondary metabolism – 2nd Edition. Annual plant reviews (v. 40), Wiley J. & Sons, Inc., Publication, 2010

Lignans: definition, chemical structure, biosynthesis, metabolism, foods

Lignans: contents in brief

What are lignans?

Lignans are a subgroup of non-flavonoid polyphenols.
They are widely distributed in the plant kingdom, being present in more than 55 plant families, where they act as antioxidants and defense molecules against pathogenic fungi and bacteria.
In humans, epidemiological and physiological studies have shown that they can exert positive effects in the prevention of lifestyle-related diseases, such as type II diabetes and cancer. For example, an increased dietary intake of these polyphenols correlates with a reduction in the occurrence of certain types of estrogen-related tumors, such as breast cancer in postmenopausal women.
In addition, some lignans have also aroused pharmacological interest. Examples are:

  • podophyllotoxin, obtained from plants of the genus Podophyllum (Berberidaceae family); it is a mitotic toxin whose derivatives have been used as chemotherapeutic agents;
  • arctigenin and tracheologin, obtained from tropical climbing plants; they have antiviral properties and have been tested in the search for a drug to treat AIDS .

⇑ Back to the top ⇑

Chemical structure of lignans

Their basic chemical structure consists of two phenylpropane units linked by a C-C bond between the central atoms of the respective side chains (position 8 or β), also called β-β’ bond. 3-3′, 8-O-4′, or 8-3′ bonds are observed less frequently; in these cases the dimers are called neolignans. Hence, their chemical structure is referred to as (C6-C3)2, and they are included in the phenylpropanoid group, as well as their precursors: the hydroxycinnamic acids (see below).

Lignans
Fig. 1 – Phenylpropane unit

Based on their carbon skeleton, cyclization pattern, and the way in which oxygen is incorporated in the molecule skeleton, they can be divided into 8 subgroups: furans, furofurans, dibenzylbutanes, dibenzylbutyrolactones, dibenzocyclooctadienes, dibenzylbutyrolactols, aryltetralins and arylnaphthalenes. Furthermore, there is considerable variability regarding the oxidation level of both the propyl side chains and the aromatic rings.
They are not present in the free form in nature, but linked to other molecules, mainly as glycosylated derivatives.
Among the most common lignans, secoisolariciresinol (the most abundant one), lariciresinol, pinoresinol, matairesinol and 7-hydroxymatairesinol are found.

Note: They occur not only as dimers but also as more complex oligomers, such as dilignans and sesquilignans.

⇑ Back to the top ⇑

Biosynthesis of lignans

Lignans
Fig. 2 – Lignan Biosynthesis

In this section, we will examine the synthesis of some of the most common lignans.
The pathway starts from 3 of the 4 most common dietary hydroxycinnamic acids: p-coumaric acid, sinapic acid, and ferulic acid (caffeic acid is not a precursor of this subgroup of polyphenols). Therefore, they arise from the shikimic acid pathway, via phenylalanine.
The first three reactions reduce the carboxylic group of the hydroxycinnamates to alcohol group, with formation of the corresponding alcohols, called monolignols, that is, p-coumaric alcohol, sinapyl alcohol and coniferyl alcohol. These molecules also enter the pathway of lignin biosynthesis.

  • The first step, which leads to the activation of the hydroxycinnamic acids, is catalysed by hydroxycinnamate:CoA ligases, commonly called p-coumarate:CoA ligases (EC 6.2.1.12), with formation of the corresponding hydroxycinnamate-CoAs, namely, feruloil-CoA, p- coumaroyl-CoA and sinapil-CoA.
  • In the second step, a NADPH-dependent cinnamoyl-CoA: oxidoreductase, also called cinnamoyl-CoA reductase (EC1.2.1.44) catalyzes the formation of the corresponding aldehydes, and the release of coenzyme A.
  • In the last step, a NADPH-dependent cinnamyl alcohol dehydrogenase, also called monolignol dehydrogenase (EC 1.1.1.195), catalyzes the reduction of the aldehyde group to an alcohol group, with the formation of the aforementioned monolignols.

The next step, the dimerization of monolignols, involves the intervention of stereoselective mechanisms, or, more precisely, enantioselective mechanisms. In fact, most of the plant lignans exists as (+)- or (-)-enantiomers, whose relative amounts can vary from species to species, but also in different organs on the same plant, depending on the type of reactions involved.
The dimerization can occur through enzymatic reactions involving laccases (EC 1.10.3.2). These enzymes catalyze the formation of radicals that, dimerizing, form a racemic mixture. However, this does not explain how the enantiomeric mixtures found in plants are formed. The most accepted mechanism to explain the stereospecific synthesis involves the action of the laccase and of a protein able to direct the synthesis toward one or the other of the two enantiomeric forms: the dirigent protein. The reaction scheme might be: the enzyme catalyzes the synthesis of phenylpropanoid radicals that are orientated in such a way to obtain the desired stereospecific coupling by the dirigent protein.

Lignans
Fig. 3 – (-)-Matairesinol

For example, pinoresinol synthase, consisting of laccase and dirigent protein, catalyzes the stereospecific synthesis of (+)-pinoresinol from two units of coniferyl alcohol. (+)-Pinoresinol, in two consecutive stereospecific reactions catalyzed by NADPH-dependent pinoresinol/lariciresinol reductase (EC 1.23.1.2), is first reduced to (+)-lariciresinol, and then to (-)-secoisolariciresinol. (-)-Secoisolariciresinol, in the reaction catalyzed by NAD(P)-dependent secoisolariciresinol dehydrogenase (EC 1.1.1.331) is oxidized to (-)-matairesinol.

⇑ Back to the top ⇑

Metabolism of lignans by human gut microbiota

Their importance to human health is due largely to their metabolism by colonic microbiota, which carries out deglycosylations, para-dehydroxylations, and meta-demethylations without enantiomeric inversion. Indeed, this metabolization leads to the formation molecules with a modest estrogen-like activity (phytoestrogens), a situation similar to that observed with some isoflavones, such as those of soybean, some coumarins, and some stilbenes. These active metabolites are the so-called “mammalian lignans or enterolignans”, such as the aglycones of enterodiol and enterolactone, formed from secoisolariciresinol and matairesinol, respectively.
Studies conducted on animals fed diets rich in lignans have shown their presence as intact molecules, in low concentrations, in serum, suggesting that they may be absorbed as such from the intestine. These molecules exhibit estrogen-independent actions, both in vivo and in vitro, such as inhibition of angiogenesis, reduction of diabetes, and suppression of tumor growth.
Note: The term “phytoestrogen” refers to molecules with estrogenic or antiandrogenic activity, at least in vitro.

Once absorbed, they enter the enterohepatic circulation, and, in the liver, may undergo phase II reactions and be sulfated or glucuronidated, and finally excreted in the urine.

⇑ Back to the top ⇑

Food rich in lignans

The richest dietary source is flaxseed (linseed), that contains mainly secoisolariciresinol, but also lariciresinol, pinoresinol and matairesinol in good quantity (for a total amount of more than 3.7 mg/100 g dry weight). They are also found in sesame seeds.

Lignans
Fig. 4 – (-)-Secoisolariciresinol

Another important source is whole grains.
They are also present in other foods, but in concentrations from one hundred to one thousand times lower than those of flaxseed. Examples are:

  • beverages, generally more abundant in red wine, followed in descending order by black tea, soy milk and coffee;
  • fruits, such as apricots, pears, peaches, strawberries;
  • among vegetables, Brassicaceae, garlic, asparagus and carrots;
  • lentils and beans.

Their presence in grains and, to a lesser extent in red wine and fruit, makes them, at least in individuals who follow a Mediterranean-style eating pattern, the main source of phytoestrogens.

⇑ Back to the top ⇑

References

Andersen Ø.M., Markham K.R. Flavonoids: chemistry, biochemistry, and applications. CRC Press Taylor & Francis Group, 2006

de la Rosa L.A., Alvarez-Parrilla E., Gonzàlez-Aguilar G.A. Fruit and vegetable phytochemicals: chemistry, nutritional value, and stability. 1th Edition. Wiley J. & Sons, Inc., Publication, 2010

Heldt H-W. Plant biochemistry – 3th Edition. Elsevier Academic Press, 2005

Manach C., Scalbert A., Morand C., Rémésy C., and Jime´nez L. Polyphenols: food sources and bioavailability. Am J Clin Nutr 2004;79(5):727-47 [Abstract]

Satake H, Koyama T., Bahabadi S.E., Matsumoto E., Ono E. and Murata J. Essences in metabolic engineering of lignan biosynthesis. Metabolites 2015;5:270-90. doi:10.3390/metabo5020270

Tsao R. Chemistry and biochemistry of dietary polyphenols. Nutrients 2010;2:1231-46. doi:10.3390/nu2121231

van Duynhoven J., Vaughan E.E., Jacobs D.M., Kemperman R.A., van Velzen E.J.J, Gross G., Roger L.C., Possemiers S., Smilde A.K., Doré J., Westerhuis J.A.,and Van de Wiele T. Metabolic fate of polyphenols in the human superorganism. PNAS 2011;108(suppl. 1):4531-8. doi:10.1073/pnas.1000098107

Wink M. Biochemistry of plant secondary metabolism – 2nd Edition. Annual plant reviews (v. 40), Wiley J. & Sons, Inc., Publication, 2010