Glycolysis Pathway Steps

Glycolytic pathway steps

Glycolysis is multi-step enzymatic process for the conversion of glucose into two molecules of Pyruvic acid (Pyruvate) with the release of energy in the form of ATP and NADH. Altogether there are 10 steps in the glycolytic process.

Glycolysis is the sequence of reactions that metabolize one molecule of glucose to two molecules of pyruvate with the concomitant net production of two molecules of ATP.

Steps of glycolysis are grouped on two phases, Preparatory phase and Pay-off phase.


Steps in Preparatory phase:

1. Phosphorylation of Glucose: It is first step of the glycolytic pathway in which phosphorylation of glucose occurs at C-6 to yield glucose 6-phosphate, with ATP as the phosphoryl donor.

  • This reaction is irreversible under intracellular conditions, catalyzed by hexokinase which requires Mg2+ for its activity.

This step is notable for two reasons:

  • Glucose 6-phosphate cannot diffuse through the membrane, because of its negative charge.
  • Addition of the phosphoryl group begins to destabilize glucose, thus facilitating its further metabolism.


2.Isomerisation of Glucose 6-Phosphate to Fructose 6-Phosphate: The enzyme phosphohexose isomerase (phosphoglucose isomerase) catalyzes the reversible isomerization of glucose 6-phosphate, an aldose sugar molecule to fructose 6-phosphate, a ketose.

This reaction includes additional steps because both glucose 6-phosphate and fructose 6-phosphate are present primarily in the cyclic forms. The enzyme must first open the six membered ring of glucose 6-phosphate, catalyze the isomerization, and then promote the formation of the five-membered ring of fructose 6- phosphate.

This step has critical role in overall chemistry of glycolysis. The rearrangement of carbonyl and hydroxyl groups at C1 and C2 is necessary prelude to following two steps.

  • The phosphorylation that occurs in the next reaction (step 3) requires that the group at C-1 first be converted from a carbonyl to an alcohol, and
  • Subsequent reaction (step 4) cleavage of the bond between C-3 and C-4 requires a carbonyl group at C-2.



3.Phosphorylation of Fructose 6-Phosphate to Fructose 1,6- Bisphosphate: An enzyme phosphofructokinase-1 (PFK-1) catalyzes the transfer of a phosphoryl group from ATP to fructose 6-phosphate to yield fructose 1,6-bisphosphate.

  • This is irreversible and one of regulatory step of glycolysis.

The activity of PFK-1 is increased whenever the cell’s ATP supply is depleted or when the ATP breakdown products, ADP and AMP (particularly the latter), are in excess, leading to regulation of glycolysis.


4.Cleavage of Fructose 1,6-Bisphosphate: The enzyme fructose 1,6-bisphosphate aldolase, often called simply aldolase, catalyzes a reversible aldol condensation. Fructose 1,6-bisphosphate is cleaved to yield two different triose phosphates, glyceraldehyde-3-phosphate, an aldose, and dihydroxyacetone phosphate, a ketose C-2.

  • At equilibrium, 96% of the triose phosphate is dihydroxyacetone phosphate. However, the reaction proceeds readily from dihydroxyacetone phosphate to glyceraldehyde 3-phosphate because the subsequent reactions of glycolysis remove this product.


5.Interconversion of the Triose Phosphates: Only one of the two triose phosphates formed by aldolase, glyceraldehyde 3-phosphate, can be directly degraded in the subsequent steps of glycolysis. The other product, dihydroxyacetone phosphate, is rapidly and reversibly converted to glyceraldehyde 3-phosphate by the enzyme, triose phosphate isomerase.

 This reaction completes the preparatory phase of glycolysis in which, glucose molecule is phosphorylated at C-1 and C-6 and then cleaved to form two molecules of glyceraldehyde 3-phosphate.


Steps in Pay-off phase:

6.Oxidation of Glyceraldehyde 3-Phosphate to 1,3-Bisphosphoglycerate: The first step in the payoff phase is the oxidation of glyceraldehyde 3-phosphate to 1,3 bisphosphoglycerate, catalyzed by glyceraldehyde 3- phosphate dehydrogenase. The aldehyde group of glyceraldehyde 3-phosphate is oxidized, not to a free carboxyl group but to a carboxylic acid anhydride with phosphoric acid.

1,3-Bisphosphoglycerate is an acyl phosphate. Such compounds have a high phosphoryl transfer potential; one of its phosphoryl groups is transferred to ADP in the next step in glycolysis.

The reaction catalyzed by glyceraldehyde 3- phosphate dehydrogenase is the sum of two processes:

  • Oxidation of the aldehyde to a carboxylic acid by NAD+
  • Joining of the carboxylic acid and orthophosphate to form the acyl-phosphate product.


 7.Phosphoryl Transfer from 1,3-Bisphosphoglycerate to ADP: The enzyme phosphoglycerate kinase transfers the high-energy phosphoryl group from the carboxyl group of 1,3-bisphosphoglycerate to ADP, forming ATP and 3- phosphoglycerate.

Steps 6 and 7 of glycolysis together constitute an energy-coupling process in which 1,3-bisphosphoglycerate is the common intermediate; it is formed in the first reaction (which is endergonic), and its acyl phosphate group is transferred to ADP in the second reaction (which is strongly exergonic).



8.Conversion of 3-Phosphoglycerate to 2-Phosphoglycerate: This step requires enzyme phosphoglycerate mutase which catalyzes a reversible shift of the phosphoryl group between C-2 and C-3 of glycerate; Mg2+ is essential for this reaction.

 The reaction occurs in two steps:

  • A phosphoryl group attached to a His residue of the mutase is transferred to the hydroxyl group at C-2 of 3- phosphoglycerate, forming 2,3 bisphosphoglycerate.
  • The phosphoryl group at C-3 of 2,3-BPG is then transferred to the same His residue, producing 2- phosphoglycerate and regenerating the phosphorylated enzyme.

Enzyme mutase functions as a phosphatase. It converts 2,3-bisphosphoglycerate into 2-phosphoglycerate. However, the phosphoryl group remains linked to the enzyme and this phosphoryl group is transferred to 3-phosphoglycerate to reform 2,3 bisphosphoglycerate.


9.Dehydration of 2-Phosphoglycerate to Phosphoenolpyruvate: In the second glycolytic reaction that generates a compound with high phosphoryl group transfer potential, enolase promotes reversible removal of a molecule of water from 2-phosphoglycerate to yield phosphoenolpyruvate (PEP).


 10.Transfer of the Phosphoryl Group from Phosphoenolpyruvate to ADP: The last step in glycolysis is the transfer of the phosphoryl group from phosphoenolpyruvate to ADP, catalyzed by pyruvate kinase, which requires K and either Mg2+ or Mn2+.

Formation of pyruvate ends the pay off phase of glycolysis.

In this substrate-level phosphorylation, the product pyruvate first appears in its enol form, then tautomerizes rapidly and nonenzymatically to its keto form, which predominates at pH 7.

Phosphoglycerate kinase catalyzes the transfer of the phosphoryl group from the acyl phosphate of 1,3- bisphosphoglycerate to ADP and results in formation of ATPFormation of ATP in this manner is referred to as substrate-level phosphorylation because the phosphate donor, 1,3- BPG, is a substrate.

Thus, the outcomes of the reactions catalyzed by glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase are:

  • Glyceraldehyde 3-phosphate, an aldehyde, is oxidized to 3-phosphoglycerate, a carboxylic acid.
  • NAD+ is concomitantly reduced to NADH.
  • ATP is formed from Pi and ADP at the expense of carbon oxidation energy.