Glycolysis Pathway: Definition, Steps

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Glycolysis is the metabolic pathway that breaks down glucose to produce energy in the form of ATP (adenosine triphosphate) and other by-products such as pyruvate and NADH. It is a key process that occurs in the cytoplasm of cells and is anaerobic, meaning it does not require oxygen.

  • Glucose is the primary component in the metabolism of plants, animals, and many microorganisms due to the rich potential energy and good fuel.
  • Glucose stores as a starch or glycogen, when energy demands these are released as glucose to produce ATP either aerobically or anaerobically.
  • In animal and plants, there are four major pathway of glucose utilization such as; the synthesis of complex polysaccharides destined for the extracellular space; stored in cells (as a polysaccharide or as sucrose); oxidized to a three-carbon compound (pyruvate) via glycolysis to provide ATP and metabolic intermediates, or oxidized via the pentose phosphate (phosphogluconate) pathway to yield ribose 5-phosphate for nucleic acid synthesis and NADPH for reductive biosynthetic processes.
  • The photosynthetic organisms get their glucose by reducing atmospheric CO2 to triose and then triose to glucose.
  • The non-photosynthetic organisms get their glucose from simpler three or four carbon precursors via gluconeogenesis. It is reverse glycolysis with the helps of different glycolytic enzymes.

What is glycolysis?

  • Glycolysis is a type of metabolic pathway where one molecule of glucose degraded into 2 molecules of 3 carbon-containing pyruvate molecules through a  series of enzyme-catalyzed reactions.
  • Glycolysis pathway also known as Embden-Meyerhof-Parnas Pathway.
  • Glycolysis is a Greek Word where Glykys means Sweet and Lysis means Splitting.
  • The glycolysis is a 10 step process, where the first 5 steps is known as Preparatory phase and the last 5 steps known as payoff phase.
  • In Preparatory phase Glucose is converted into Glyceraldehyde 3 phosphate and Dihydroxyacetone phosphate. In the payoff phase, the Glyceraldehyde 3 phosphate is converted into 3 carbon-containing pyruvate.
  • In Preparatory phase 2 molecules of ATP are used up while in payoff phase 4 molecules of ATP are generated.

Salient features of glycolysis

  • Glycolysis is a process that occurs in all cells. This pathway’s enzymes are found in the cytosomal portion of cells.
  • Glycolysis can occur in two conditions: anaerobic (without oxygen) and aerobic (with oxygen). Under anaerobic conditions, lactate is the end product. Under aerobic conditions, pyruvate forms, which is then oxidized into CO2 and H2O.
  • Glycolysis is a key pathway to ATP synthesis in tissues without mitochondria. erythrocytes, cornea, lens etc.
  • Glycolysis is essential for the brain, which is dependent upon glucose for energy. Before glucose can be oxidized to H2O and CO2, it must undergo glycolysis.
  • Glycolysis (anaerobic), may be summarized as the net reaction Glucose + 2ADP + 2Pi o 2Lactate + 2ATP
  • Glycolysis is a central pathway in metabolism, with many of its intermediates serving as branch points to other pathways. The intermediates of glycolysis can be used to synthesize amino acids and fat.
  • The synthesis of glucose will be possible by reversing glycolysis and making alternate arrangements at irreversible steps.

Glycolysis Steps/10 steps of glycolysis

Glycolysis Pathway: Definition, Steps
Glycolysis Pathway: Definition, Steps | Image Source:*R6_3FLZHGlAaLXJA.png

Preparatory Phase

  1. Phosphorylation of Glucose: In this step, D-Glucose is phosphorylated with the help of the enzyme hexokinase. The hexokinase enzyme is a class of transferase enzyme. This enzyme transfers the phosphoryl group from ATP to the OH group of C6 carbon in Glucose and form Glucose 6 phosphate. In this step, Mg2+ is required.
  2. Conversation of Glucose 6 Phosphate to Fructose 6 Phosphate: In this step, the enzyme Phosphohexoisomerase catalyze the conversation reaction of Glucose 6 Phosphate to Fructose 6 Phosphate. This enzyme triggers the isomerization of Glucose 6 phosphate. In this step, Mg2+ is required.
  3. Phosphorylation of Fructose 6 Phosphate: In this step, enzyme phosphofructokinase-1 (PFK-1) catalyzes the phosphorylation reaction of Fructose 6 Phosphate and forms Fructose 1,6 Bisphosphate.
  4. Cleavage of Fructose 1,6 Bisphosphate: Enzyme aldolase or fructose 1,6-bisphosphate aldolase cleave the Fructose 1,6 Bisphosphate into two triose phosphates such as Glyceraldehyde 3 phosphate (aldose) and Dihydroxyacetone phosphate (Ketose). In this step, Mg2+ is required.
  5. Interconversion of triose phosphates: The enzyme triose phosphate isomerase converts the Dihydroxyacetone phosphate into Glyceraldehyde 3 phosphate.

Payoff Phase

  1. Oxidation of Glyceraldehyde 3 phosphate: The enzyme Glyceraldehyde 3 phosphate dehydrogenase converts the Glyceraldehyde 3 phosphate into 1,3-Bisphosphoglycerate with the help of 2 molecule of Pi and 2 molecules of NAD+.
  2. Conversation of 1,3-Bisphosphoglycerate to 3 phosphoglycerate: The enzyme phosphoglycerate kinase transfer one molecule of a phosphoryl group from 1,3-Bisphosphoglycerate to ADP and forms 3 phosphoglycerate and ATP. In this step, Mg2+ is required.
  3. Conversation of 3 phosphoglycerate to 2 phosphoglycerate: The enzyme phosphoglycerate mutase catalyzes the reversible shift of phosphoryl group between C-2 and C-3 of glycerate and forms 2 phosphoglycerate from  3 phosphoglycerate. In this step, Mg2+ is required.
  4. Dehydration of 2-Phosphoglycerate to Phosphoenolpyruvate: The enzyme enolase removes one molecule of H2O from 2-Phosphoglycerate and forms Phosphoenolpyruvate.
  5. Transfer of the Phosphoryl Group from Phosphoenolpyruvate to ADP: The enzyme pyruvate kinase transfers the phosphoryl group from Phosphoenolpyruvate to ADP to yield pyruvate and ATP. In this step K+ and either Mg2+ or Mn2+ si required.
Glycolysis Pathway: Definition, Steps
Glycolysis Pathway Steps

The overall equation of glycolysis is;

Glucose + 2NADP+ + 2Pi = 2 Pyruvate + 2ATP + 2NADPH + 2H+ + 2H2O


Glycolysis equation

A summary of the process of glycolysis cab be written as follows:

C6H12O6 + 2ADP + 2Pi + 2NAD+   →   2C3H4O3 + 2H2O + 2ATP + 2NADH + 2H+

In words, the equation is written as:

Glucose + Adenosine diphosphate + Phosphate  +  Nicotinamide adenine dinucleotide

Pyruvate + Water + Adenosine triphosphate + Nicotinamide adenine dinucleotide + Hydrogen ions

Production of ATP in glycolysis

Table 13.1 shows the details of ATP production in glycolysis (from glucose). Under anaerobic conditions, 2 ATP are synthesized while, under aerobic conditions, 8 or 6 ATP are synthesized–depending on the shuttle pathway that operates. One more ATP is produced when glycolysis takes place from glycogen. Because glucose 1-phosphate is directly produced by glycogen, which then forms glucose 6-phosphate, no ATP is required to activate glucose. Anaerobic glycolysis produces 3 ATP from glycogen.

Glycolysis and shuttle pathways

The NADH that is produced during glycolysis can participate, in the presence of oxygen and mitochondria, in the shuttle pathways to the synthesis ATP. Three ATP molecules can be generated from each NADH molecule if the cytosolic NADH uses the malate-aspartate shuttle. This contrasts with the glycerolphosphate-sea shuttle, which produces only 2 ATP.

Irreversible steps in glycolysis

The majority of reactions that occur during glycolysis can be reversed. The three steps that are catalysed in glycolysis by the enzymes, hexokinase or glucokinase as well as phosphofructokinase, pyruvate and glucokinase are irreversible. These three stages are responsible for controlling glycolysis. With alternate arrangements at the three irreversible steps, the reversal in glycolysis leads to the synthesis from pyruvate of glucose (gluconeogenesis).

Enzymes of Glycolysis

The enzymes that catalyze glycolytic reaction in most cells are found in the extra-mitochondrial portion of the cell’s cytosol. Nearly all enzymes involved with glycolysis require Mg2+. This is a common trait. Here are some enzymes that can catalyze different steps in glycolysis.

  • Hexokinase
  • Phosphoglucoisomerase
  • Phosphofructokinase
  • Aldolase
  • Phosphotriose isomerase
  • Glyceraldehyde 3-phosphate dehydrogenase
  • Phosphoglycerate kinase
  • Phosphoglycerate mutase
  • Enolase
  • Pyruvate kinase
  • When more than one enzyme catalyzes the same reaction but is encoded by different genes is known as isozymes.
  • In glycolysis, 2 molecules of ATP and 2 Molecules of NADPH is generated.

Fates of Pyruvate

Depending on the metabolism and organism, the pyruvate may follow one of three routes.

1. Oxidation of pyruvate

Aerobic organisms move the pyruvate to the mitochondria, where it is oxidized (acetyl Co A) into the acetyl groups of acetylcoenzyme. This involves the release one mole CO2. The acetyl CoA is then completely oxidized to CO2 and H2O through the citric acid cycle. This is the same pathway as glycolysis in plants and aerobic organisms.

2. Lactic acid fermentation

The pyruvate can’t be oxidized in conditions that lack oxygen, such as in the skeletal muscles cells. Anaerobic glycolysis reduces the pyruvate to lactate in such situations. Other anaerobic organisms also produce lactate from glucose through the process of lactic acids fermentation.

3. Alcoholic Fermentation

Some microbes, such as brewer’s yeast convert the pyruvate from glucose anaerobically into ethanol or CO2. This is the oldest form of glucose metabolism, and it is only possible in low oxygen conditions.

Glycolysis products/end product of glycolysis

After both the energy-requiring phase and the energy-releasing phase have occurred in glycolysis, the final reaction produces two pyruvate molecules and two ATP molecules.

The presence of oxygen allows the pyruvate molecules to be broken down by oxidation in cellular metabolism. This will give rise to more energy in form of ATP or carbon dioxide. The NADH molecules move back and forth between the reduced and oxidized states (NAD+, NADH).

Substrates Entry Points

Three ways substrates can enter glycolysis pathways are known as “entry points”. These are:

  1. Dietary glucose – glucose is directly absorbed from the gastrointestinal tract into the bloodstream and enters the pathway.
  2. Glycogenolysis – Glycogenolysis is when glucose is released from the liver and enters the pathway.
  3. Other monosaccharides – Galactose, fructose, and other monosaccharides enter the glycolysis pathway at different levels through common intermediates.

Glycogen stored in skeletal muscles cannot be completely broken down into glucose. It cannot be completely broken down into glucose and can therefore only be used to fuel glycolysis in the skeletal muscle cells where it is stored.

Transport into Cells

To allow cells to use circulating glucose, it must move from the bloodstream into the intracellular space. Various transporters (GLUT 1-4) transport glucose into cells. They all have different kinetics, and regulation methods depending on the function of glycolysis.

N.B. For an explanation of the term Km, please see our article on enzyme kinetics.

GLUT transporterKey featureLocationReason
GLUT-1Low Km, so highly activeAll cellsRegulates basal uptake even at low glucose levels, ensures a constant supply of energy for survival
GLUT-2Concentration-dependent, due to high KmLiver, pancreatic isletsActs as a glucose sensor – increases uptake in high glucose levels for storage. Also regulates insulin release from the pancreas
GLUT-4Insulin-dependentMuscle, adipose, heartIncreases uptake in presence of insulin (i.e. after meals) for storage

Phases of Glycolysis

Glycolysis is a two-part process. First, energy is used to produce high-energy intermediates. These intermediates then release their energy in the second phase.

  • Energy investment phase –  two ATP molecules are required to make high-energy intermediates.
  • Energy pay out phase – The intermediate is metabolized, producing four ATP molecules as well as two NADH molecules.

Importance of Glycolysis

Glycolysis is the initial step in the process of reducing glucose to produce energy for the cellular metabolism.

The energy found in the sugar glucose bonds makes up nearly all of the energy that living cells use. Two ways glucose can enter heterotrophic cells are available. Secondary active transport is one method. This transport occurs against the glucose concentration gradient. Another mechanism is a group known as GLUT proteins. These integral proteins are also known to be glucose transporter proteins. These transporters facilitate the diffusion of glucose. Glycolysis is the first step in the process of converting glucose into energy. Both prokaryotic as well as eukaryotic cells have cytoplasmic glycolysis. Because it is used by almost all living organisms, it was likely one of the first metabolic pathways to evolve. This process doesn’t require oxygen, and it is therefore anaerobic.

Glycolysis, which is the first step in cellular respiration’s main metabolic pathways to generate ATP, is the first. Two distinct phases are required to convert the six-carbon glucose ring into two three-carbon sugars, pyruvate, through a series enzymatic reactions. The first phase of glycolysis is energy-intensive. The second phase converts to pyruvate into ATP and NADH, which are then used by the cells to produce energy. The net result of glycolysis is two pyruvate molecules and two ATP molecules. Two NADH molecules are also produced for the cell’s energy. The glycolytic pathway links to the Krebs Cycle where additional ATP is produced to meet the cells’ energy requirements.

Structure of glycolysis components in Fischer projections and polygonal model

Fischer projections depict the chemical changes that occur in glycolysis intermediates. This image can be used to compare with the polygonal model representation. A video shows another comparison of Fischer projections with Poligonal Models in glycolysis. You can also see video animations on YouTube for another metabolic pathway (Krebs Cycle), and for the representation and application of Polygonal Models in Organic Chemistry.

Structure of glycolysis components in Fischer projections and polygonal model
Structure of glycolysis components in Fischer projections and polygonal model | Image Source:

Interactive pathway map

Interactive pathway map
Interactive pathway map | Image Source:

Glycolysis in disease

1. Diabetes

Insulin signals trigger cellular glucose uptake. The glucose is then broken down by glycolysis, which lowers blood sugar levels. Diabetes can lead to hyperglycemia due to low insulin levels. This is when glucose levels rise and cells don’t properly absorb it. Through gluconeogenesis, hepatocytes also contribute to hyperglycemia. Glycolysis occurs in the hepatocytes and controls the hepatic glucose generation. Hyperglycemia is when the liver produces too much glucose without having the ability to break it down.

2. Genetic diseases

Glycolytic mutations, which are rare due to the importance of the metabolic pathway, cause inability to breathe and eventually death. Some mutations can be seen, one example being Pyruvate-kinase deficiency which causes chronic hemolytic anemia.

3. Cancer

Malignant tumor cells can perform glycolysis at a rate ten times faster that their non-cancerous tissue counterparts. Hypoxia, which is a decrease in O2 supply within tumor cells, can often be caused by a lack of capillary support during their genesis. These cells are dependent on anaerobic metabolic processes, such as glycolysis to produce ATP (adenosine Triphosphate). Certain glycolytic enzymes are overexpressed in tumor cells, which can lead to higher rates of glycolysis. 

These enzymes can be Isoenzymes of traditional glycolysis enzymes that are more susceptible to feedback inhibition. By generating enough ATP from the anaerobic pathway, the increase in glycolytic activity counteracts hypoxia. Otto Warburg first identified this phenomenon in 1930. It is known as the Warburg Effect. According to the Warburg hypothesis, cancer is more likely due to dysfunctional mitochondrial metabolism than uncontrolled cell growth. There are many theories that explain the Warburg effect. One theory is that increased glycolysis may be a natural protective process and that malignant changes could be caused by energy metabolism.

This high glycolysis rate is important for medical purposes. High aerobic glycolysis by malignant tumours can be used clinically to diagnose and monitor the treatment response to cancers.

Research continues to improve mitochondrial metabolism and treatment of cancer by reducing glycolysis, starving cancerous cells with various new methods, including a ketogenic diet.

Intermediates for other pathways

These metabolic pathways all heavily depend on glycolysis for metabolites.

  • The pentose-phosphate pathway begins with dehydrogenation glucose-6-phosphate. This is the first intermediate produced by glycolysis and produces various pentose sugars and NADPH to synthesize fatty acids, cholesterol, and other nutrients.
  • Glycogen synthesis begins with glucose-6-phosphate at beginning of glycolytic pathway.
  • Glycerol, for the formation of triglycerides and phospholipids, is produced from the glycolytic intermediate glyceraldehyde-3-phosphate.
  • There are many post-glycolytic routes:
    • Synthesis of fatty acids
    • Cholesterol synthesis
    • Citric acid cycles lead to: Amino acid synthesis, Nucleotide and Tetrapyrrole syntheses.

Glycolysis and gluconeogenesis share many intermediates, but they are not functionally related. Both pathways have two regulatory steps that, when they are active in one, are inactive in another. Both processes cannot be active simultaneously. If both sets of reactions were simultaneously active, the net result would be hydrolysis of four high-energy phosphate bonds per reaction cycle (two ATP, two GTP).

Change in free energy for each step of glycolysis

StepReactionΔG°’ / (kJ/mol)ΔG / (kJ/mol)
1Glucose + ATP4− → Glucose-6-phosphate2− + ADP3− + H+−16.7−34
2Glucose-6-phosphate2− → Fructose-6-phosphate2−1.67−2.9
3Fructose-6-phosphate2− + ATP4− → Fructose-1,6-bisphosphate4− + ADP3− + H+−14.2−19
4Fructose-1,6-bisphosphate4− → Dihydroxyacetone phosphate2− + Glyceraldehyde-3-phosphate2−23.9−0.23
5Dihydroxyacetone phosphate2− → Glyceraldehyde-3-phosphate2−7.562.4
6Glyceraldehyde-3-phosphate2− + Pi2− + NAD+ → 1,3-Bisphosphoglycerate4− + NADH + H+6.30−1.29
71,3-Bisphosphoglycerate4− + ADP3− → 3-Phosphoglycerate3− + ATP4−−18.90.09
83-Phosphoglycerate3− → 2-Phosphoglycerate3−4.40.83
92-Phosphoglycerate3− → Phosphoenolpyruvate3− + H2O1.81.1
10Phosphoenolpyruvate3− + ADP3− + H+ → Pyruvate + ATP4−−31.7−23.0

Concentrations of metabolites in erythrocytes

CompoundConcentration / mM
Dihydroxyacetone phosphate0.14

Frequently Asked Questions (FAQs) of Glycolysis 

Where does glycolysis take place?

Glycolysis takes place in the cytoplasm of cells.

What is aerobic glycolysis?

Aerobic glycolysis is the process of oxidation of glucose into pyruvate followed by the oxidation of pyruvate into CO2 and H2O in the presence of a sufficient amount of oxygen.

What is anaerobic glycolysis?

Anaerobic glycolysis is the process that takes place in the absence of enough oxygen resulting in the reduction of pyruvate into lactate and reoxidation of NADH into NAD+.

does glycolysis require oxygen?

Glycolysis requires no oxygen. It is an anaerobic type of respiration performed by all cells, including anaerobic cells that are killed by oxygen.

is glycolysis aerobic or anaerobic?

Glycolysis is an anaerobic process. None of its nine steps involve the use of oxygen.

how many nadh are produced by glycolysis?

Two NADH molecules are produced by glycolysis.

what does glycolysis produce?

Glucose is the source of almost all energy used by cells. Overall, glycolysis produces two pyruvate molecules, a net gain of two ATP molecules, and two NADH molecules.

in glycolysis, atp molecules are produced by _.
a. photophosphorylation
b. oxidative phosphorylation
c. photosynthesis
d. cellular respiration
e. substrate-level phosphorylation

Ans: e. substrate-level phosphorylation.

For each glucose that enters glycolysis, _____ acetyl coa enter the citric acid cycle.

For each glucose that enters glycolysis, 2 acetyl CoA enter the citric acid cycle.

why is atp required for glycolysis?

Energy is needed at the start of glycolysis to split the glucose molecule into two pyruvate molecules. These two molecules go on to stage II of cellular respiration. The energy to split glucose is provided by two molecules of ATP.

in addition to atp, what are the end products of glycolysis?

The end products of glycolysis are: pyruvic acid (pyruvate), adenosine triphosphate (ATP), reduced nicotinamide adenine dinucleotide (NADH), protons (hydrogen ions (H2+)), and water (H2O). Glycolysis is the first step of cellular respiration, the process by which a cell converts nutrients into energy.

how many atp are produced in glycolysis?

Glycolysis produces 2 ATP

which kind of metabolic poison would most directly interfere with glycolysis?

A poison that closely mimics the structure of glucose but is not metabolized. If the poison cannot be metabolized, then no NADH, pyruvate or ATP will be produced in glycolysis.

where does glycolysis take place in eukaryotic cells?

Glycolysis takes place in the cytoplasm. Within the mitochondrion, the citric acid cycle occurs in the mitochondrial matrix, and oxidative metabolism occurs at the internal folded mitochondrial membranes (cristae).

why is glycolysis considered to be one of the first metabolic pathways to have evolved?

Photosynthesis is one of the earliest reactions where carbon dioxide and water come together to form glucose. In glucose the energy of the sun is trapped. Glycolysis breaks down glucose molecules in carbon dioxide and water. Breaking down glucose releases energy.
Most of the cells respire anaerobically. All these cells have glycolysis in their metabolic pathway. Therefore it is one of the earliest metabolic pathways



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