10 Steps of Glycolysis Process

step by step process of glycolytic pathway

The breakdown of the six-carbon glucose into two molecules of the three-carbon pyruvate occurs in 10 steps, the first 5 of which constitute the preparatory phase and the last 5 0f these steps are called payoff phase or energy-conserving stage.


steps of glycolysis pathway


10 steps of glycolysis process:

Steps of Glycolysis process 01: Phosphorylation of glucose :

In the first step of the glycolysis process, D-glucose is turned into glucose-6-phosphate using ATP as a phosphate donor in the reaction. Here, glucose is phosphorylated in the process of glycolysis. Phosphorylation is the process through which a phosphate group is added to a molecule which is derived from ATP. Hexokinase requires Mg or any other metal ions from its activity, just like all other kinases. Atoms of Mg^2+ helps to shield the negative charges produced from the phosphate groups from the ATP molecule.


steps of glycolysis process


In the reaction of phosphorylation, ATP turns into ADP with the help of an enzyme which is known as “hexokinase”. It works in many eukaryotic cells and prokaryotic cells, and an additional enzyme “glucokinase” found in the liver is also involved in this reaction. The function of glucokinase is to remove glucose from the blood, to store glucose as glycogen in the liver for further purposes to be used in the process.


“Hexokinase” is an enzyme that catalyzes different ring structures like glucose 6 carbon. In the presence of this particular enzyme, the reaction is proceeded and facilitated in the required way. In the reaction, energy loss is happening in the form of heat.

Thus, glucose-6-phosphate is formed in the process. Glucose-6-phosphate is an important component of the junction of many metabolic pathways.


SEE MORE:  Summary of Glycolysis


Steps of Glycolysis process 02: Isomerization/Conversion of Glucose-6-phosphate :

From the first process, we got the produced  Glucose-6-phosphate. Now in this second step, the conversion of Glucose-6-phosphate to fructose-6-phosphate occurs. It is termed as a reversible isomerization reaction due to its flow in both ways.

steps of glycolysis process


For it’s a conversion from glucose-6-phosphate to fructose-6-phosphate, an enzyme is involved, named “phosphohexos isomerase”. Just like the name of both the enzyme and title states, it helps the produced glucose in the Isomerization reaction or to rearrange itself. Phosphohexos isomerase helps to accelerate the reaction. In this reaction, there involves a change of carbon-oxygen bond. The glucose is a six-membered ring structure. Due to the change, it turns into a five-membered ring structure. This reaction has a mentionable role in the glycolytic pathway, which is a very critical contribution in the overall biochemistry.


Steps of Glycolysis process 03: Phosphorylation of fructose 6-phosphate :

This step is considered as the committed step in the process of glycolysis due to its reaction and it’s a contribution to metabolism rather than storing glucose and then transforming it into any other compound. The produced product in the above step, fructose-6-phosphate, again goes through reaction to transform itself into fructose 1,6-biphosphate. Here, phosphofructokinase (one of the complex known enzyme) helps to change it with co-factor magnesium. This reaction is irreversible in various cellular conditions.

steps of glycolysis process


This step is very much similar to the first step where ATP is turned into ADP and Phospholyration is involved too. Here, a phosphate group is added to the carbon. Like, in step 1, magnesium is involved to shield negative charges. That, helps the compound to change from fructose-6-phosphate to fructose 1,6-biphosphate. An enzyme, named “photofructokinases” catalyzes the reaction to facilitate its activities. The reaction that the enzyme facilitates is irreversible. The mechanism of the enzyme photofructokinases accelerates when the cell lacks ATP. And, the mechanism decelerates when there is excess/enough ATP. Thus the affinity of the enzyme is lowered for the process. This is a more important reaction since it helps in metabolism rather than storing or converting glucose into another form.


SEE MORE: Does glycolysis require oxygen


Steps of Glycolysis 04: Cleavage of fructose 1, 6-biphosphate:

The C-C bond scission reaction is what makes this step very unique. Here, fructose 1,6 biphosphate do different products. Since fructose has two phosphates on its two ends, it splits in the middle with two phosphates which are named: dihydroxyacetone phosphate and glyceraldehyde 3-phosphate or, Fructose 1,6-biphosphate are cleaved to get two different types of phosphates which are known as dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The enzyme that catalyzes the reaction is known as fructose diphosphate aldolase, often called as simple aldolase.

steps of glycolysis process


In this reaction, fructose diphosphate aldolase is catalyzed and utilized. It cleaves fructose 1,6-biphosphate to get a 3-carbon molecule. There are two classes of aldolase and are found in the body of different organisms. Mg is not needed in animal tissues but comes in need of many microbes when Zn does its activities for the fulfillment. The metal ion polarizes the carbonyl group. The Cleavage between C-3 and C-4 depends on the presence of the Carbonyl group in the reaction of the process of glycolysis, in this step. The reaction flows in either direction, making the condition reversible. Thus, a reversible aldol condensation type reaction is noticed here.


SEE MORE: Reactants Products and Equation of Glycolysis Process


Steps of Glycolysis process 05: Triosephosphate isomerase :

Only one of the produced product from the above step remains constant in this step. That is, glyceraldehyde 3-phosphate. The other produced product, which is known as Dihydroxyacetone phosphate further transforms itself into glyceraldehyde 3-phosphate by triosephosphate isomerase as it is a catalyst. This isomerization reaction works reversibly. Just like a similar reaction occurs in step 2 in this process of glycolysis reactions.

steps of glycolysis process

The reaction mechanism is very similar to the reaction mechanism of step 2. Here, in this further reaction, glyceraldehyde 3-phosphate is turned into 1,3-bisphosphoglycerate after reacting properly. The enzyme that catalyzes or speeds the reaction is named ‘glyceraldehyde phosphate dehydrogenase’. Now, in the glycolytic pathway, a 3-carbon molecule is ensured. But glucose hasn’t completely turned into pyruvate yet. Thus, the preparatory phase of glycolysis came to an end due to this reaction and this very step.


Steps of Glycolysis process 06: Oxidative Phosphorylation of Glyceraldehyde 3-phosphate :

There are two energy-conserving reactions of the process of glycolysis where step 06 is the first of them and step 09 is the second of them. In step 06, 1,3-bisphosphoglycerate is formed from Glyceraldehyde 3-phosphate.

It is the only one reaction in the total glycolytic pathway where NAD+ is converted to NADH. The reaction is facilitated by an enzyme, phosphoglycerate kinase.


steps of glycolysis process



Glyceraldehyde 3-phosphate dehydrogenase is a tetramer of four identical subunits. Here, each of the moles of the enzyme consists of 4 moles of NAD+. The aldehyde group of Glyceraldehyde 3-phosphate is dehydrogenated in this complex reversible reaction. Glyceraldehyde 3-phosphate’aldehyde group is oxidized to a carboxylic acid anhydride with phosphoric acid. Much energy of Glyceraldehyde 3-phosphate’s aldehyde group is conserved in the reaction. The action mechanism of Glyceraldehyde 3-phosphate dehydrogenase is very complex and also involves 3 complex steps for further reactions; Covalent binding of substrate and SH group, Oxidation of thiohemiacetal and reduction of NAD+, Phosphorolysis of thioester. All these reactions in this step at last result in the production of 3-phosphoglycerate.


Steps of Glycolysis process 07: Transfer from 1,3-bisphosphoglycerate to ADP:


Here, in this reaction ATP is generated, which has been produced in the process of glycolysis for the first time. Here, 2 moles of triosephosphate are produced for one mole of glucose, so 2 moles of ATP are generated when one molecule of glucose is oxidized. The transfer of phosphate group from carboxylic group 3-phosphoglyceroyl phosphate to ADP. These reactions are accelerated by the enzyme phosphoglycerate kinase and thus producing ATP.

steps of glycolysis process


Phosphoglycerate kinase requires a divalent metal ion like Magnesium, Zinc, etc, acting as co-factor to conduct any reaction.

In the reaction, 1,3-bisphosphoglycerate and ADP in the presence of phosphoglycerate kinase as the required enzyme and with co-factor Magnesium produces 3-phosphoglycerate and ATP.

So, from the step 06 and step 07, the net results are, NAD+ is converted into NADH, ATP is produced from ADP and Glyceraldehyde 3-phosphate is oxidized and is converted or changed into 3-phosphoglycerate and a carboxylic acid.


In other words, the energy which is released through oxidation is conserved by the coupled formation of ATP, ADP, and Pi. These types of reactions can also be called as substrate-level phosphorylations. In this reaction, oxidative phosphorylation is joined to electron transport, so it is known as respiratory-chain phosphorylation.


This step is associated with the transfer of a high-energy phosphate group of 3-phospoglycerol phosphate’s carboxylic group. The metal ion used in the process of reactions of this step interacts with ADP or ATP to form the reactive complex for the reaction of the process of glycolysis.


Steps of Glycolysis process 08: Isomerization of 3-phosphoglycerate :


Now, in the main reaction, 3-phosphoglycerate is transformed into 2-phosphoglycerate. The enzyme that facilitates or catalyzes the reaction is known as phosphoglycerate mutase.

In the reaction of this step, in the reaction, the inter-molecular shift of 3-phosphoglycerate happens.  So, it transfers the phosphate group from carbon-3 to carbon-2 of the molecule. The enzyme phosphoglycerate mutase which is a dimer of identical subunits helps to accelerate this reaction with the help of Magnesium as co-factor to shield the negative charges as Mg^2+ is a metal ion.

steps of glycolysis process


The rearrangement of 3-phosphoglycerate happens in this reaction. The reaction as it goes, it occurs in two different steps. So, firstly, the reaction of 3-phosphoglycerate and phosphoenzyme results in the production of free enzyme and 2,3-diphosphoglycerate which at last produces at 2-phosphoglycerate.  It requires a catalyst and it’s co-factor to facilitate the reaction. Therefore, 3-phosphoglycerate is rearranged in the presence of phosphoglycerate mutase and it’s metal ion co-factor, Magnesium, which leads to yield the product

‘2-phosphoglycerate’. The enzyme, phosphoglycerate mutase is a dimer of identical subunits and is also phosphorylated at the start which is continuously regenerated by the reaction cycle.


Steps of Glycolysis process 09: Dehydration of 2-phosphoglycerate to Phosphoenolpyruvate :


In this reaction, the produced 2-diphosphoglycerate from the above step is dehydrated to Phosphoenolpyruvate with the help of the action of enolase (a dimer with identical sub-units) and requires metal ions, Magnesium or Zinc ions, that act as the enzyme’s co-factor. The simultaneous presence of Fluoride and phosphate inhibits enolase in its activities and actions.

steps of glycolysis process

Here, the reaction is catalyzed by an enzyme which is known as enolase, this enzyme helps to accelerate the reaction. This enzyme helps to dehydrate the produced product of the above step, 2-phosphoglycerate. The enzyme, enolase, promotes the removal of a water molecule in a reaction that was reversible to yield the required product. It dehydrates 2-phosphoglycerate by removing a water group of the existing product and thus reduces it to Phosphoenolpyruvate. This is a reversible reaction and has a small free energy change value for its further activities. Moreover, the reaction is stabilized with the help of Mg which is a metal ion, an essential ion for the reaction to proceed. A compound with relatively low Phosphoryl group transfer potential is converted into relatively high Phosphoryl group transfer.


Steps of Glycolysis process 10: Transfer of phosphate from PEP to ADP :


This is the last step of glycolysis. In this process, ATP is regenerated, just like step 7. In this step and the reaction, Phosphoenolpyruvate is turned or converted into pyruvate, the required product. The reaction is facilitated with the help of an enzyme which is known as pyruvate kinase. This is one of the physiologically irreversible reactions in the process of glycolysis.

In the first step of this reaction, Phosphoenolpyruvate reacts with ADP in presence of catalysts like pyruvate kinase and it’s co-factor Magnesium (a metal ion) and thus the reaction produces the final product known as pyruvate and ATP.

steps of glycolysis process

The reaction proceeds when enol pyruvate changes its arrangement at a quick pace and thus takes a new form. The new form is known as ketopyruvate. The pyruvate at first comes in the enol form then, after the reaction, it transforms itself into keto form.


The reaction stands, Phosphoenolpyruvate in addition to ADP and also hydrogen, in presence of pyruvate kinase and metal ions like Mg, Zn, etc produces the required product ketopyruvate. Here, ADP is transformed and converted into ATP. The reaction that is facilitated above is just another example of the substrate-level phosphorylation of glycolysis. This is the transfer of Phosphoryl group from Phosphoenolpyruvate to ADP.


Pyruvate kinase, the enzyme, can be of three major different forms; M, L and A type which can be found on different parts of the body of different organisms. These come in a lot of help in different ways in the process of glycolysis. The reaction produces a lot of free energy which is released as heat later.


Now in aerobic glycolysis, Oxidation happens when pyruvate goes to the citric acid cycle.

Whereas, when in anaerobic glycolysis, lactate dehydrogenase converts pyruvate to lactate. Thus, the process of glycolysis ends here, in the way explained above.


The net reaction to the steps of glycolysis process:



Much of this energy is conserved by the coupled phosphorylation of four molecules of ADP to ATP. The net yield is two molecules of ATP per molecule of glucose used because two molecules of ATP were invested in the preparatory phase. Energy is also conserved in the payoff phase in the formation of two molecules of the electron carrier NADH per molecule of glucose.

steps of glycolysis process

In the sequential reactions of glycolysis, three types of chemical transformations are particularly noteworthy:

(1) degradation of the carbon skeleton of glucose to yield pyruvate;

(2) phosphorylation of ADP to ATP by compounds with high phosphoryl group transfer potential, formed during glycolysis; and

(3) transfer of a hydride ion to NAD, forming NADH.

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