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Core
All tissues in the body can metabolise glucose (use glucose to produce the ATP they need for cellular processes).
However, some cells can only metabolise glucose. They have an absolute requirement for glucose because they cannot perform stage 3 or stage 4 of metabolism (stage 3 is the Krebs cycle and stage 4 is oxidative phosphorylation; the stages of metabolism are introduced here). The clearest example is the mature red blood cell, which has no mitochondria and so depends entirely on glycolysis for ATP. Without mitochondria, glycolysis is the only way for red blood cells to make ATP, so they depend on glucose from the blood. Neutrophils are a less strict example: they have a low density of mitochondria and rely predominantly on glycolysis for ATP production, although they can also use other fuels.
This also means that the body must be excellent at absorbing monosaccharides like glucose from the gut. Glucose and galactose cross the gut lining (the intestinal epithelium) mainly by active co-transport with sodium (the sugar and sodium cross together through one carrier, driven by the sodium gradient), via SGLT1 (sodium-glucose cotransporter 1). Fructose crosses by facilitated diffusion (passive movement through a carrier protein) via GLUT5, a member of the glucose transporter (GLUT) family. GLUT2 then carries these sugars out of the epithelial cells into the blood.
Glycolysis is the first stage in aerobic respiration and is the basis for anaerobic respiration. It is made up of ten progressive chemical reactions, split into two phases. It is done in many small steps for three reasons. It gives versatility (an intermediate, meaning a molecule made part-way through the pathway, can be taken out and used in another reaction). It gives fine control (with ten steps it is easy to slow down, speed up or stop the pathway at one point by inhibiting a single enzyme). And it conserves energy (the energy in glucose is released in small packages rather than in one destructive burst, so the cell can capture it in ATP and NADH).
Glycolysis acts as the central pathway of carbohydrate metabolism, and it occurs in all tissues, in the cytosol (the fluid inside the cell). It is exergonic (releases energy) and oxidative (glucose is oxidised, and NAD+, the oxidised form of NADH, is reduced to NADH). Glycolysis itself does not need oxygen. With the action of LDH (lactate dehydrogenase), which regenerates NAD+, it can continue anaerobically (without oxygen).
Functions of glycolysis include:
- Oxidation of glucose
- Production of NADH (2 per glucose)
- ATP synthesis (2 net per glucose)
- Production of three-carbon (3C) and six-carbon (6C) intermediates for other uses
Glycolysis has 10 reactions, 3 of which are irreversible (reactions 1, 3 and 10). These irreversible reactions are the main control points of glycolysis, so they are key to know. They are catalysed by the following enzymes:
Reaction 1 is catalysed by hexokinase (or glucokinase in the liver)
Reaction 3 is catalysed by Phosphofructokinase (PFK)
Reaction 10 is catalysed by Pyruvate Kinase
Diagram - Provides a summary of the reactions in glycolysis
SimpleMed original by Dr. Maddie Swannack
Phase 1 of Glycolysis Includes Reactions 1-3
This involves the phosphorylation of glucose (adding a phosphate group to it), making it negatively charged. This prevents passage of glucose back across the plasma membrane and increases the reactivity of the glucose. It uses up 2 moles of ATP. Delta G is the change in free energy, and a large negative value means the reaction releases a lot of energy. Reactions 1 and 3 have large, negative delta G values, so they are effectively irreversible.
Phase 2 of Glycolysis Includes Reactions 4-10
Reaction 4 splits the six-carbon (6C) sugar into two three-carbon (3C) molecules. Reaction 5 converts one of these, dihydroxyacetone phosphate (DHAP), into the other, glyceraldehyde-3-phosphate. This means reactions 6-10 happen twice per glucose molecule.
Reaction 6 captures one molecule of NADH each time, so 2 NADH per glucose.
Reactions 7 and 10 are the sites of ATP synthesis in glycolysis. Reaction 7 is catalysed by phosphoglycerate kinase and reaction 10 by pyruvate kinase. Each makes ATP by substrate-level phosphorylation, which means that a phosphate group is passed straight from an intermediate to ADP. Both reactions happen twice per glucose, so 2 ATP are made at each of them and 4 ATP in total per glucose. Reaction 10 also has a large negative delta G, so it is effectively irreversible. There is only a net gain of 2 ATP per glucose, as 2 ATP were used earlier in the pathway during the energy investment phase (phase 1).
Important Intermediates of Glycolysis
Intermediates of glycolysis can be removed from the pathway at many points to be used elsewhere. The intermediates listed below each have their own alternative uses.
- Glucose-6-Phosphate (G-6-P) - the phosphorylated intermediate of glucose that allows glycolysis to occur. Also, an important common intermediate (a molecule shared by two pathways) in the metabolism of galactose (see the galactose section below) - this means galactose can be used for glycolysis.
- Fructose-6-phosphate (Fructose-6-P) - even though it contains the word “fructose”, this is not a common intermediate in the metabolism of fructose.
- Glyceraldehyde-3-phosphate (Glyceraldehyde-3-P) - this is the common intermediate for glycolysis and fructose metabolism.
- 1,3-bisphosphoglycerate - in red blood cells, this is converted to 2,3-bisphosphoglycerate (2,3-BPG). This molecule lowers the affinity of haemoglobin for oxygen, so oxygen is released more readily to the tissues.
- 3-phosphoglycerate - an intermediate that can be diverted into other pathways, for example amino acid synthesis.
- Dihydroxyacetone phosphate (DHAP) - a 3C intermediate of glycolysis. Lipid synthesis uses glycerol-3-phosphate, which the liver can make from DHAP. Glycolysis can therefore supply the glycerol-3-phosphate needed for lipid synthesis in the liver. The liver can also make glycerol-3-phosphate from glycerol. The link between glycolysis and lipid synthesis is important in the metabolism of alcohol, covered in a later lesson.
- Pyruvate - the end point of the glycolysis reaction. It is the reactant for the Link Reaction, which converts pyruvate to acetyl-CoA (the two-carbon molecule that enters the Krebs cycle). This leads on to the Krebs cycle (see the next article on the oxygen-dependent pathway).
Diagram - The relationships between glycolysis and the metabolism of other sugars in the liver and how they link together
SimpleMed original by Dr. Maddie Swannack
Step 6 produces 2 NADH per glucose, so step 6 needs a supply of NAD+ to be converted into NADH.
Since levels of NAD+ in the cell are constant, the body needs a mechanism to regenerate NAD+ from NADH. In most cells in the body, this is the electron transport chain (the final stage of aerobic respiration that occurs in the mitochondria, part of oxidative phosphorylation).
However, some cells don’t have mitochondria, meaning they cannot perform the electron transport chain to regenerate their NAD+, so they need another method for NAD+ regeneration. This alternative method is also used in anaerobic respiration, such as during hard exercise. It is also used when the tissues are poorly perfused with blood, as in shock (circulatory failure, where the tissues do not get enough blood), including shock caused by severe heart failure. The electron transport chain needs oxygen to regenerate NAD+. Without enough oxygen, NADH builds up and NAD+ runs short.
This issue can be resolved by converting pyruvate to lactate with the enzyme lactate dehydrogenase. This process takes in NADH and converts it back to NAD+. The lactate is then carried in the blood to the liver, where it is converted back into pyruvate and used to make glucose (gluconeogenesis).
Diagram - The equation of pyruvate to lactate using lactate dehydrogenase
SimpleMed original by Dr. Maddie Swannack
If, however, anaerobic respiration happens for too long and lactate is allowed to build up, this can lead to two conditions:
- Hyperlactataemia - blood lactate above 2 mmol/L without acidosis. There is no change in blood pH at this point because of the buffering capacity of the blood (its ability to absorb extra acid).
- Lactic acidosis - blood lactate above 5 mmol/L (some sources use above 4 mmol/L), with a blood pH below 7.35. Blood pH is lowered at this point due to overcoming the blood’s buffering capacity. This is a marker of severe illness.
Allosteric Regulation of Glycolysis Enzymes
Allosteric regulation of enzymes occurs when an activator or inhibitor binds to the enzyme at a site that is not the active site (called the allosteric site). Enzymes can also be regulated by covalent modifications, such as phosphorylation (adding a phosphate group) or dephosphorylation (removing one). The irreversible reactions in glycolysis (1, 3 and 10) are the main sites of regulation. They operate far from equilibrium (they are strongly driven in one direction). Hexokinase is inhibited by its product, glucose-6-phosphate. PFK and pyruvate kinase are both controlled by activators and inhibitors.
For example, phosphofructokinase is controlled by both allosteric and hormonal regulation:
- Allosteric regulation - it is inhibited by high concentrations of ATP. High ATP is a signal that the cell has plenty of energy, so glycolysis can slow down. PFK is activated by AMP (adenosine monophosphate, a signal of low energy) and by fructose 2,6-bisphosphate (a signalling molecule made from fructose-6-phosphate).
- Hormonal regulation - in the liver, insulin increases PFK activity indirectly. It raises the level of fructose 2,6-bisphosphate, which activates PFK and so promotes glycolysis when glucose is plentiful. Glucagon has the opposite effect.
Diagram - The metabolism pathway for galactose, the monosaccharide which (when paired with glucose) makes the disaccharide lactose
SimpleMed original by Dr. Maddie Swannack
Deficiency in any of the three enzymes on the diagram (galactokinase, galactose-1-phosphate uridyltransferase (GALT) and UDP-galactose 4-epimerase, listed in blue) can lead to galactosaemia, a condition where galactose or its intermediates build up in the blood. Galactosaemia can lead to cataracts (clouding of the lens of the eye). Classic galactosaemia (a lack of GALT) can also cause liver damage in untreated newborns. In the lens, galactitol builds up and draws water in (osmotic swelling). A loss of active glutathione also adds oxidative damage from reactive oxygen species. Galactosaemia is inherited in an autosomal recessive pattern. Autosomal means that the gene is not on a sex chromosome. A child is affected only if they inherit a faulty copy of the gene from each parent. The faulty genes cause the deficiency in these enzymes.
If galactose is in excess, it enters a different pathway to normal and is converted to galactitol (a sugar alcohol) by the enzyme aldose reductase, which uses NADPH. This reduces the NADPH available. NADPH is needed for the biosynthesis of lipids and for glutathione (GSH) regeneration (covered in the article on oxidative stress). It is also needed to reduce inappropriate disulphide bonds (bonds between the sulphur atoms of two cysteines, a sulphur-containing amino acid). If these bonds are not reduced, proteins can become cross-linked, which may contribute to cataracts alongside the osmotic swelling described above. Glutathione is used in the clearance of reactive oxygen species (ROS), which are known to damage lipid bilayers and DNA.
The treatment for galactosaemia is a lifelong diet that restricts galactose. This means restricting milk and dairy products, which are the main source of galactose, because lactose is broken down into glucose and galactose. The diet reverses the early illness in newborns, but children remain at increased risk of long-term problems such as developmental delay and speech problems. Almost all girls and women with classic galactosaemia also develop premature ovarian insufficiency (loss of normal ovarian function earlier than expected).
Lactose is a disaccharide commonly found in dairy products in the diet. It is broken down into its monosaccharide components, galactose and glucose, by the enzyme lactase, before being absorbed into the body. Lactase sits on the brush border of the small intestine (the microvilli, tiny folds on the surface of the cells that line it).
Lactose intolerance means getting symptoms from the gut (such as tummy pain, bloating and diarrhoea) after eating lactose. They usually happen because the body makes too little of the enzyme lactase (lactase deficiency). If lactose cannot be digested and absorbed in the small intestine, it remains in the lumen. This makes the contents more concentrated (it lowers the water potential), so water is drawn in by osmosis. The extra water causes diarrhoea. Gut bacteria digest the unabsorbed lactose and produce gas, which causes flatulence.
Primary lactase deficiency is the commonest type, also called lactase non-persistence. It happens in people who do not have the lactase persistence allele (a gene variant that keeps lactase production going into adult life). These people produce lactase as babies (when their diet is almost exclusively milk). Lactase levels then fall at some point after weaning (when a baby moves from milk to solid food), even if they keep eating dairy products.
Secondary lactase deficiency is caused by damage to the small intestine, for example through gastroenteritis (such as rotavirus infection) or coeliac disease. Coeliac disease is an immune reaction to gluten (a protein found in wheat, barley and rye) that damages the lining of the small intestine. Secondary lactase deficiency can resolve once the cause has been treated or removed, but it can also persist for years.
Congenital lactase deficiency is a very rare, autosomal recessive disease where the patient has no lactase produced at all, leaving them unable to digest breast milk.
Diagram - The Pentose Phosphate pathway, involving the turning of glucose-6-Phosphate into 5 carbon sugars through the action of Glucose-6-Phosphate Dehydrogenase
SimpleMed original by Dr. Maddie Swannack
The Pentose Phosphate Pathway converts G-6-P (glucose-6-phosphate) from glycolysis to five-carbon (5C) sugar phosphates, providing NADPH (a coenzyme that donates electrons). NADPH is a vital molecule that helps the antioxidant molecule glutathione (GSH) to remain in its active state. The pathway requires Glucose-6-Phosphate dehydrogenase.
Glutathione can protect important cellular components from heavy metals and from damage caused by reactive oxygen species. These include free radicals (molecules with an unpaired electron), peroxides (molecules with an oxygen-oxygen bond) and lipid peroxides.
Glutathione reduces disulphide bonds formed within cytoplasmic proteins to cysteines (the amino acid that carries an SH group) by serving as an electron donor. In the process, glutathione is converted to its oxidised form, glutathione disulphide (GSSG). To be converted back to GSH, glutathione disulphide needs NADPH as an electron donor.
Glutathione helps to maintain proteins in their correct structure by keeping the SH groups in their reduced (hydrogenated) state.
If there is reduced NADPH production (such as in G-6-P dehydrogenase deficiency), inappropriate disulphide bonds can form within cells, causing proteins to aggregate. Red blood cells have no other source of NADPH, so reactive oxygen species also damage their haemoglobin. The denatured (unfolded) haemoglobin clumps together to form Heinz bodies. The damaged red blood cells become rigid and are removed by macrophages in the spleen. This destruction of red blood cells is called haemolysis. In G-6-P dehydrogenase deficiency, haemolysis usually happens only after an oxidative trigger, such as an infection, fava beans or certain drugs (for example primaquine, an antimalarial).
The 5C sugar phosphates produced in this pathway can be used in RNA and DNA synthesis (which require a 5C sugar backbone). They can also re-enter glycolysis after conversion to glyceraldehyde-3-phosphate (G-3-P).
Edited by: Dr. Ben Appleby and Dr. Marcus Judge
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