• Introduction
  • The basics of glycolysis
  • Energy-requiring phase
  • Step 1
  • Step 2
  • Step 3
  • Step 4
  • Step 5
  • Energy-releasing phase
  • Step 6
  • Step 7
  • Step 8
  • Step 9
  • Step 10
  • What happens to pyruvate?
  • Final thoughts
  • Electron carrier: Small organic molecules that switch between oxidized and reduced forms and transport electrons during metabolic reactions.
  • NADH: A reduced form of Nicotinamide adenine dinucleotide that carries electrons from one reaction to another. An important cofactor in metabolism consisting of two nucleotides and their phosphate groups. 
  • NAD+: An oxidized form of Nicotinamide adenine dinucleotide that accepts electrons from other reactions and becomes reduced. 
  • Pyruvate: The end product of glycolysis, and an important molecule in multiple biological pathways, such as the citric acid cycle.
  • Substrate phosphorylation: Formation of ATP from ADP and a phosphorylated intermediate, rather than inorganic phosphate. 


The name glycolysis stems from the Greek words of glyk (sweet) and lysis (dissolution) which accurately describes the breakdown of glucose to generate energy through cellular respiration. With the presence of oxygen, this process consists of glycolysis, the citric acid cycle, and oxidative phosphorylation.

These pathways are interconnected and each of them has its own specific function in breaking down glucose to generate ATP. Once fully oxidized, a single glucose molecule produces 30-32 molecules of ATP. This energy is used in exercise as well as maintaining regular bodily functions (breathing, heartbeat, hormonal activity, cell repair, etc.).

Glycolysis is the first phase of cellular respiration, as it breaks down glucose into 2 pyruvates and 2 NADH. In aerobic conditions, these molecules can be used in the citric acid cycle and oxidative phosphorylation. Interestingly, glycolysis is also crucial for anaerobic respiration because it does not require oxygen to function. In this case, pyruvate can be reduced to lactate through fermentation.

This post explains the basic mechanisms involved in glycolysis, and why it is one of the main building blocks of life.

The basics of glycolysis

Glycolysis refers to a series of reactions where a six-carbon glucose molecule is split into two three-carbon molecules called pyruvate. This process is also anaerobic (does not require oxygen) and takes place in the cytoplasm of the cell. 

This entire process consists of ten different reactions divided into two distinct phases. The first half is called the energy-requiring phase, where the cell uses two ATPs to split a six-carbon glucose molecule into two three-carbon molecules. The second half of glycolysis is called the energy-releasing phase. Here, the energy is extracted from the molecules and stored as four molecules of ATP, two molecules of NADH, and two molecules of pyruvate.

However, since the first phase requires two ATPs to work, glycolysis has a net gain of two ATP molecules for every molecule of glucose.

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The first step of cellular respirationSplits glucose into pyruvateAnaerobic processTakes place in the cytoplasmProduces 2 ATP, 2 NADH, & 2 pyruvates per glucose molecule

The energy-requiring phase

The energy-requiring phase, also known as the preparatory phase, describes the process of using two ATP molecules to form an unstable sugar with two phosphate groups. This is then split into two smaller three-carbon sugar molecules that are isomers of each other. 

The energy-requiring phase of glycolysis consists of five distinct steps, each of them catalyzed by a specific enzyme. 

Step 1

The first step of glycolysis is catalyzed by an enzyme called hexokinase. This process begins by adding two phosphate groups from two ATP to a glucose molecule (C6H12O6). As the glucose molecule is phosphorylated it produces glucose-6-phosphate, which is a more reactive form of glucose. This also keeps glucose inside the cell because glucose with a phosphate cannot cross the plasma membrane. 

Step 2

The second phase of glycolysis is catalyzed by isomerase. In this step, a glucose-6-phosphate (C6H13O9P) is converted into its isomer, fructose-6-phosphate. 

Step 3

The third step utilizes an enzyme called phosphofructokinase that phosphorylates (adds a phosphate) fructose-6-phosphate (C6H13O9P) to produce fructose-1,6-biphosphate.

Not only does phosphofructokinase drive this pathway, but it also regulates the speed at which this reaction occurs. The enzyme is more active when ADP levels are elevated and slows down with sufficient concentration of ATP. 

Step 4

The fourth step of glycolysis is catalyzed by an enzyme called aldolase which splits fructose-1,6-biphosphate (C6H14O12P2) into two three-carbon molecules; dihydroxyacetone-phosphate (DHAP) and glyceraldehyde-3-phosphate. From these two, only glyceraldehyde-3-phosphate is able to directly continue to the next phase of glycolysis. 

Step 5

The fifth and last step of the energy-requiring phase is catalyzed by triosephosphate isomerase. This enzyme transforms DHAP into its isomer, glyceraldehyde-3-phosphate. This effectively continues the pathway with two molecules of the same isomer. 

At this point, two ATP molecules are used to break down a single molecule of glucose. 

Glycolysis breaks down glucose to form 2 ATP, 2 pyruvate molecules, and 2 NADH.

The energy-releasing phase

The energy-releasing phase, also called the payoff phase, describes the series of reactions that the three-carbon sugars formed in the first half undergo in order to produce pyruvate. This entire process produces four molecules of ATP as well as two molecules of NADH. 

Also note that since a glucose molecule is split into two smaller sugar molecules, each reaction occurs twice before moving onto the next reaction. 

Step 6

The sixth step of glycolysis oxidizes the sugar (glyceraldehyde-3-phosphate) with the help of an enzyme called glyceraldehyde-3-phosphate dehydrogenase. This process also releases high-energy electrons that are picked up by the electron carrier NAD+, reducing it to NADH and H+. Then, a second phosphate is added to the sugar, which phosphorylates it into 1,3-biphosphoglycerate. 

Note that the continuation of this reaction depends on the availability of the oxidized form of the electron carrier (NAD+). Therefore, NADH must constantly be oxidized back to NAD+ to maintain this process.

In aerobic conditions, NADH can be oxidized and the electrons from the released hydrogen can be used to generate ATP. In anaerobic conditions (without oxygen) the pathway continues by fermentation. This also produces NAD+ from NADH. 

Step 7

The seventh step is catalyzed by phosphoglycerate kinase, which causes 1,3-bisphosphoglycerate (C3H8O10P2) to donate one of its phosphate groups to ADP. This forms a single molecule of ATP as well as 3-phosphoglycerate. 

Step 8

Step eight of glycolysis is catalyzed by a mutase enzyme. Here, the remaining phosphate group of 3-phosphoglycerate (C3H7O7P) is transferred from the third carbon to the second carbon. Thus, converting 3-phosphoglycerate into its isomer, 2-phosphoglycerate. 

Step 9

The ninth step is catalyzed by enolase, which causes 2-phosphoglycerate (C3H7O7P) to lose its water molecule. This dehydration reaction produces phosphoenolpyruvate (PEP).

Step 10

The final step of glycolysis is catalyzed by the enzyme pyruvate kinase. In this stage, phosphoenolpyruvate (C3H5O6P) donates its phosphate group to ADP, creating a second molecule of ATP. As PEP loses its phosphate, it is converted into pyruvate which is the end product of glycolysis. 

Because pyruvate kinase is a rate-limiting enzyme that catalyzes the production of pyruvate, this last step will not occur if it is not available in sufficient quantities. While this still allows glycolysis to continue, it only produces two ATP molecules during the energy-releasing phase. 

What happens to pyruvate?

In aerobic conditions, pyruvate can be oxidized in the mitochondria to produce a total of two ATP. This process also produces Acetyl-CoA, which enters the citric acid cycle, further continuing the breakdown of glucose. Keep in mind that to fully oxidize a glucose molecule, the muscles must have a sufficient supply of oxygen. In practice, this means that ATP can be produced with oxygen as long as the intensity of the exercise remains low enough.

In anaerobic conditions, ATP must be produced through fermentation. Here, pyruvate molecules are reduced to lactate by NADH, leaving NAD+ as a byproduct. Through a process known as the Cori cycle, the lactate produced by the muscles is transported to the liver and converted back to glucose (gluconeogenesis), and sent back to the muscles. Anaerobic respiration takes over during shorter and more intense exercises when the muscle’s oxygen need surpasses the oxygen supply. 

Glycolysis is the first phase of cellular respiration.

Final thoughts

Glycolysis is one of the earliest metabolic pathways to evolve among living organisms. It is also the first pathway used in the breakdown of glucose to generate energy. During the first stage of glycolysis, two ATPs are used to cleave a six-carbon glucose molecule into two three-carbon sugars. In the second half of this process, ATP is extracted and the high-energy electrons from hydrogen are attached to NAD+.

During substrate phosphorylation (formation of ATP by the transfer of a phosphate group), four ATP molecules are formed, producing a net gain of two NADH and two ATP molecules. 

Once all ten reactions have taken place, a glucose molecule is broken down into two molecules of pyruvate, two molecules of NADH, and two ATP molecules. Pyruvate can then be shuttled into the mitochondria to go through a series of reactions called the citric acid cycle. This allows cellular respiration to continue to produce even more ATP. 

What else would you like to know about glycolysis? Let us know in the comments. 


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