The word ‘Glycolysis’ has been derived from a Greek word glykys, meaning “sweet” and lysis, meaning “breaking down”. The pathway is also known as the Embden-Meyerhof-Parnas (EMP) pathway. All organisms perform glycolysis to extract energy by breaking down one mole of glucose (6-Carbon sugar) into 2 moles of pyruvate (3-Carbon compound) to give net 2 ATP and 2 NADH. The glycolytic pathway is series of 10 sequential reactions occurring in the cytosol of all the cells. This pathway is carried out in two phases – the first five steps constitute the preparatory phase and the next five as the payoff phase. It is the only pathway that can occur in both aerobic and anaerobic condition.

The preparatory phase

Step I: In this step, a glucose (Glc) molecule is phosphorylated to convert it into glucose-6-phosphate (Glc-6-P) at an expense of one ATP molecule. This reaction is catalysed by an enzyme that involves the transfer of a phosphoryl group from an ATP molecule to a glucose molecule. It is an irreversible reaction, catalysed by the enzyme called ‘hexokinase’. The hexokinase, like all other kinases, requires divalent metal ion such as Mn2+ or Mg2+ for its enzymatic activity.

What is need of phosphorylation of glucose in the first step? – This process has two notable reasons (a) Transfer of the phosphoryl group from ATP to the carbon 6 of glucose makes glucose-6-phosphate negatively charged, thereby not allowing the diffusion of glucose through the membrane. (b) Addition of the phosphoryl group destabilises the glucose molecule.

Step II: An isomerisation step, where, in a reversible reaction, phosphoglucoisomerase converts glucose-6-phosphate into its isomer fructose-6-phosphate by rearranging the chemical structure of the aldose sugar to ketose sugar.

Step III: This step is catalysed by an enzyme called ‘phosphofructokinase’ (PFK). It is an allosteric enzyme that catalyses the phosphorylation of the hydroxyl group on the carbon-1 of fructose-6-phosphate to give fructose 1,6-bisphosphate. Here a second ATP molecule is used as a high-energy phosphate donor. The formation of the fructose 1,6-bisphosphate after the phosphorylation of fructose-6-phosphate in this irreversible reaction step makes it a committed step to glycolysis, as fructose 1,6-bisphosphate cannot participate in any other pathway other than glycolysis.

Step IV: Aldolase is an enzyme that catalyses a reversible reaction involved in the cleaving of the six-carbon sugar, that is, fructose 1,6-bisphosphate into two three-carbon isomers, that is, glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP).

Step V: An isomerisation step, where, in a reversible reaction, an enzyme called triosephosphate isomerase (TIM) catalyses the reversible interconversion of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Thus the glycolytic pathway continues with two molecules of the one isomer.

Till now on the glycolytic pathway, we have found that a single molecule of glucose (6-carbon sugar) has been converted into two molecules of glyceraldehyde-3-phosphate (3-carbon sugar) at the expense of two ATP molecules. This phase is called preparatory-phase.

From now on both the 3-carbon sugar (glyceraldehyde-3-phosphate) molecule will proceed into the payoff phase. Let’s further look into the payoff phase.

The glycolytic pathway
Image credit: Nelson, D. L, Cox, M. M., courtesy Wikimedia Commons, from Nelson, D. L, Cox, M. M 2004. Lehninger Principles of Biochemistry, 4th edition. W. H. Freeman.

The payoff phase

Step VI: This step is the first energy extraction step, where, two molecules of glyceraldehyde 3-phosphate or 3-phosphoglyceraldehyde is catalysed by an enzyme ‘glyceraldehyde 3-phosphate dehydrogenase’ (GAPDH) to give two molecules of 1, 3-bisphosphoglycerate (1,3-BPG). The reaction happens in the two-step – With the oxidation of the carbon-1 aldehyde group on glyceraldehyde 3-phosphate to give NADH along with proton and followed by phosphorylation of the carboxylic acid at the carbon-1 position on glyceraldehyde 3-phosphate. Here, nicotinamide adenine dinucleotide (NAD+) is a cofactor of the Glyceraldehyde-3-phosphate dehydrogenase (GADPH).

Step VII: 1, 3-bisphosphoglycerate is catalysed by phosphoglycerate kinase, leading to the donation of a high energy phosphate to ADP to give ATP and 3-phosphoglycerate (3PG) is produced. This reaction is an example of substrate-level phosphorylation.

Step VIII: This step is an isomerisation reaction catalysed by phosphoglycerate mutase, where the phosphate group from the carbon-3 of 3-phosphoglycerate is rearranged to carbon-2 position to give 2-phosphoglycerate (2PG).

Step IX: This step is a dehydration reaction catalysed by enolase, involved in the release of the water molecule from the 2-phosphoglycerate to form phosphoenolpyruvate (PEP) with a high energy enol-phosphate bond. Fluoride is a competitive inhibitor of enolase.

Step X: This is a substrate level phosphorylation reaction catalysed by an enzyme pyruvate kinase. In this step, phosphoenolpyruvate (PEP) is catalysed to give pyruvic acid (or pyruvate) with the transfer of a high energy phosphate group to an ADP molecule to give an ATP molecule.


Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002. Section 16.1, Glycolysis Is an Energy-Conversion Pathway in Many Organisms. Available from:

Qin J, Chai G, Brewer JM, Lovelace LL, Lebioda L. Fluoride Inhibition of Enolase: Crystal Structure and Thermodynamics. Biochemistry. 2006;45(3):793-800. doi:10.1021/bi051558s.

Stelios Kolomvounis, Biology 1. OpenStax CNX. Sep 30, 2015

Chaudhry R, Bhimji SS. Biochemistry, Carbohydrate, Glycolysis. [Updated 2018 Feb 6]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2018 Jan-. Available from:

External links:

HyperPhysics – Glycolysis

Biology LibreTexts – Glycolysis

Lumen Learning – Glycolysis

News-Medical.Net – What is Glycolysis?

Last Updated: Jan 9, 2019



  1. How much energy is produced by glycolysis of one mole of glucose under an anaerobic condition?
    (a) 7 ATP molecules
    (b) 2 ATP molecules
    (c) 5 ATP molecules
    (d) 10 ATP molecules
  2. Where does glycolysis occur?
    (a) mitochondria
    (b) cytosol
    (c) smooth endoplasmic reticulum
    (d) endoplasmic reticulum
  3. What glucose is phosphorylated in the first step of glycolysis?
  4. Why the step catalysed by ‘phosphofructokinase’ (PFK) is considered as the committed reaction step in the glycolytic pathway?
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