Reaction 1

The glucose is phosphorylated by ATP to give glucose 6-phosphate. This reaction, which is irreversible, is catalysed by hexokinase and requires Mg²⁺. As its name suggests hexokinase is not very specific and is able to phosphorylate a variety of hexose sugars.

ΔG0′=−nF Eo’

Reaction 2

The next step is a reversible isomerization reaction in which the six-membered pyranose ring of glucose is converted into the five-membered furanose ring of fructose under the influence of glucose 6-phosphate isomerase.

ΔG0′=−nF Eo’

Reaction 3

The fructose 6-phosphate is then converted into fructose 1,6-bisphosphate by a second phosphorylation with ATP which is catalysed by phosphofructokinase. Phosphofructokinase is an allosteric enzyme and the reaction, which is irreversible, is an important control point, i.e. rate-limiting step (page 337), for the glycolytic pathway.

ΔG0′=−nF Eo’

Reaction 4

At this stage fructose 1,6-bisphosphate is cleaved to give two triose phosphate molecules, namely glyceraldehyde 3-phosphate and dihydroxyacetone phosphate in a reversible reaction which is catalysed by aldolase.

ΔG0′=−nF Eo’

Because of the presence of triose phosphate isomerase the glyceraldehyde 3-phosphate and dihydroxyacetone phosphate are interconvertible. Glyceraldehyde 3-phosphate is on the direct pathway of glycolysis (Figure 17.1) and is continuously removed with the result that dihydroxyacetone phosphate is continuously converted into glyceraldehyde 3-phosphate. Not all the dihydroxyacetone phosphate is used in this way; some is converted to α-glycerophosphate which takes place in the α-glycerophosphate shuttle (page 230) and some may be used in the synthesis of triglycerides. However, when considering the oxidation of glucose it may be assumed that each molecule will give rise to two molecules of glyceraldehyde 3-phosphate which will both participate in the subsequent reactions.

Reaction 5

Up to this point two molecules of ATP have been used in the reaction sequence and no energy has been extracted from it. The next reaction, however, involves an oxidation in which the aldehyde group of the glyceraldehyde 3-phosphate is both dehydrogenated and phosphorylated and converted into the high-energy compound 1,3-diphosphoglycerate which is a mixed anhydride of a carboxylic acid and phosphoric acid. The energy required for this conversion comes from the oxidation of the aldehyde group and enables inorganic phosphate to act as the donor. The hydrogen atoms that are removed are transferred to the hydrogen-carrying

ΔG0′=−nF Eo’

coenzyme NAD⁺. The reaction is blocked by iodoacetate which combines with an −SH group of the enzyme glyceraldehyde 3-phosphate dehydrogenase and interferes with the substrate binding.

Reaction 6

This is the first reaction in which ATP is produced. It is an example of substrate level phosphorylation in which the energy required to convert ADP to ATP is obtained from an energy-rich substrate in a reaction catalysed by a specific kinase, in this case phosphoglycerate kinase.

ΔG0′=−nF Eo’

The last phase of glycolysis consists of three steps in which the 3-phosphoglycerate produced in reaction 6 is converted to pyruvate and another molecule of ATP is formed.

Reaction 7

By an intramolecular rearrangement, the phosphate group of 3-phosphoglycerate is transferred to the 2-position in a phosphate transfer reaction catalysed by phosphoglyceromutase.

ΔG0′=−nF Eo’

Reaction 8

The next step is a dehydration reaction whereby 2-phosphoglycerate is converted to phosphoenolpyruvate (PEP). The dehydration raises the group transfer potential of the phosphate group so that phosphoenolpyruvate is a high-energy compound. The reaction which is catalysed by enolase is dependent on Mg²⁺ and is inhibited by fluoride.

ΔG0′=−nF Eo’

Reaction 9

The high-energy phosphoenolpyruvate is used in a second substrate level phosphorylation catalysed by pyruvate kinase in which ADP is converted to ATP and pyruvate is produced. The reaction is irreversible under normal physiological conditions.

ΔG0′=−nF Eo’

Energy production and the importance of glycolysis

During the conversion of one molecule of glucose to two molecules of pyruvate, two molecules of ATP are used to prime reactions 1 and 3 but, since reactions 5−9 occur twice over, for every molecule of glucose metabolized, a total of four molecules of ATP are produced, two each in reactions 6 and 9, giving a net synthesis of two molecules of ATP. Thus the overall reaction is

Glucose+2Pi+2ADP+2NAD+→2pyruvate+2ATP+2NADH+2H+

Pyruvate is an important compound. After entering the mitochondria it may be converted to acetyl-CoA (page 232) which is then oxidized to carbon dioxide and water by the joint activities of the citrate cycle and electron-transport chain with the production of much greater quantities of ATP than are produced by glycolysis alone. It also has a role in amino acid synthesis as it may be transaminated to form alanine (page 281). Pyruvate is also an important intermediate in gluconeogenesis the process whereby glucose is synthesized from non-carbohydrate sources (page 237). In bacteria a variety of end products may be formed from pyruvate depending both on the nature of the bacterium and the environmental conditions. When oxygen is in short supply, muscle tissue converts pyruvate to lactate while yeasts ferment it to ethanol.

The three-dimensional structures of seven glycolytic enzymes, three dehydrogenases, three kinases and a mutase have been worked out. All of them, in spite of the diversity of the reactions they catalyse, contain a region which has a similar structural pattern in which 150 amino acids take part. This suggests that all these enzymes are derived from a common primeval gene and that the glycolytic pathway must have developed at a very early stage in evolution.

Anaerobic glycolysis in muscle

When glycolysis is occurring under aerobic conditions the hydrogen atoms released in the oxidative reaction take the pathway shown above and are oxidized to water via the reactions of the electron-transport chain. However, when, as in a muscle that is being strenuously exercised, there is a shortage of oxygen, the mitochondrial system, which is essentially aerobic, cannot operate effectively and an alternative method must be found to oxidize the NADH produced during glycolysis. Under these conditions, instead of the NADH being used to convert dihydroxyacetone phosphate into glycerophosphate, it is used to convert pyruvate into lactate by the following reaction which is catalysed by lactate dehydrogenase:

CH3·CO·COOH+NADH+H+⇌Lactate dehydrogenaseCH3·CHOH·COOH+NAD+

In this way NAD⁺ is regenerated and the muscle cells can continue to produce small amounts of ATP glycolytically. ATP may also be derived from phosphocreatine and from the myokinase reaction.

Quite large amounts of lactate may be produced in muscle as a result of anaerobic glycolysis and its fate is considered later (page 239). It is mainly derived from muscle glycogen since, although when resting the muscles obtain their energy by metabolizing glucose and fatty acids obtained directly from the blood, during exercise the stored muscle glycogen is called upon. This has the advantage that, at a time when ATP is in short supply, one molecule is ‘saved’ for every glucosyl residue of glycogen used instead of glucose itself. This is because the glucosyl residues are in a higher energy state than free glucose and are phosphorylated by a low-energy phosphoryl donor, i.e. inorganic phosphate (page 234) rather than ATP, which is needed for the phosphorylation of glucose.

Anaerobic glycolysis in yeast

In yeast the enzyme pyruvate decarboxylase catalyses the decarboxylation of pyruvate to CO₂ and acetaldehyde. Pyruvate decarboxylase requires Mg²⁺ and thiamine pyrophosphate as a coenzyme. The acetaldehyde is then reduced to ethanol in a reaction catalysed by alcohol dehydrogenase in which the NADH produced during glycolysis is oxidized and NAD⁺ is regenerated

CH3·CO·COOH→Mg2+,TPPPyruvatedecarboxylaseCH3·CHO+CO2CH3·CHOAcetaldehyde+NADH+H+→AlcoholdehydrogenaseCH3·CH2OHEthanol+NAD+                                                             

The net reaction for the fermentation of glucose is

Gulcose+2Pi+2ADP→2ethanol+2CO2+2ATP

Other microorganisms have different ways of metabolizing pyruvate. End products include lactic acid, butyric acid and propionic acid as well as various alcohols. Some of the reactions are considered in more detail in connection with the metabolism of plaque.