5.3 ATP and Glycolysis

Learning Objectives

By the end of this section, you will be able to:

  • Explain how ATP is used by the cell as an energy source
  • Describe glycolysis and list the products and their quantities from the breakdown of one glucose molecule

Adenosine Triphosphate, an Energy Carrier

A living cell cannot store significant amounts of free energy. Excess free energy would result in an increase of heat in the cell, which would result in excessive thermal motion that could damage and then destroy the cell. Rather, a cell must be able to handle that energy in a way that enables the cell to store the energy safely and release it for use only as needed. Living cells accomplish this by using the compound adenosine triphosphate (ATP). ATP is often called the “energy currency” of the cell, and, like currency, this versatile compound can be used to fill any energy need of the cell. How? It functions similarly to a rechargeable battery.

ATP has the adenine base attached to a ribose attached to a chain of three phosphate groups (Figure 5.10). Much energy is released when the phosphodiester bond between the beta and gamma phosphate groups is hydrolyzed. Sequential hydrolysis of ATP produces adenosine diphosphate (ADP) and adenosine monophosphate (AMP). The addition of a phosphate group to a molecule requires energy. Phosphate groups are negatively charged and thus repel one another when they are arranged in series, as they are in ADP and ATP. This repulsion makes the ADP and ATP molecules inherently unstable. The release of one or two phosphate groups from ATP, a process called dephosphorylation, releases energy.

Figure 5.10 The molecular structure of ATP. The negative charges on the phosphate group naturally repel each other, requiring energy to bond them together and releasing energy when these bonds are broken.

When ATP is broken down, usually by the removal of its terminal phosphate group, energy is released. The cell uses the energy to do work, usually by the released phosphate binding to another molecule, activating it. For example, in the mechanical work of muscle contraction, ATP supplies the energy to move the contractile muscle proteins. Consider the active transport work of the sodium-potassium pump in cell membranes. ATP alters the structure of the integral protein that functions as the pump, changing its affinity for sodium and potassium. In this way, the cell performs work, pumping ions against their electrochemical gradients. ATP is a small, relatively simple molecule, but within its bonds contains the potential for a quick burst of energy that can be harnessed to perform cellular work. This molecule can be thought of as the primary energy currency of cells in the same way that money is the currency that people exchange for things they need. ATP is used to power the majority of energy-requiring cellular reactions.

Overview of Glycolysis

You have read that nearly all of the energy used by living things comes to them in the bonds of the sugar, glucose. Glycolysis is the first step in the breakdown of glucose to extract energy for cell metabolism. Many living organisms carry out glycolysis as part of their metabolism. Glycolysis takes place in the cytoplasm of most prokaryotic and all eukaryotic cells. The process does not use oxygen directly and therefore is termed anaerobic.

Glucose enters heterotrophic cells in two ways. One method is through secondary active transport in which the transport takes place against the glucose concentration gradient. The other mechanism uses a group of integral proteins called the glucose transporter (GLUT) proteins. These transporters assist in the facilitated diffusion of glucose.

Glycolysis begins with the six-carbon, ring-shaped structure of a single glucose molecule and ends with two molecules of a three-carbon sugar called pyruvate, also called pyruvic acid. Glycolysis consists of two distinct phases. In the first part of the glycolysis pathway, energy is used to make adjustments so that the six-carbon sugar molecule can be split evenly into two three-carbon pyruvate molecules. In the second part of glycolysis, ATP and nicotinamide adenine dinucleotide (NADH) are produced (Figure 5.11). The illustration shows a simplified process of glucose moving through the stages of glycolysis.  First, two ATP are used to activate glucose. Second, the glucose is split into two branches, with one NADH and two ATP produced from each branch. The net products are two pyruvate molecules, two NADH, and two ATP.

If the cell cannot catabolize the pyruvate molecules further, there is only a net production of two ATP molecules from one molecule of glucose. For example, mature mammalian red blood cells are only capable of glycolysis, which is their sole source of ATP. If glycolysis is interrupted, these cells would eventually die.

A graphic shows glucose at the top with an arrow pointing down to fructose diphosphate, which then splits into two glyceraldehyde 3-phosphate molecules. Each of these forms one NADH and two ATP molecules in the process of each becoming a pyruvate molecule.
Figure 5.11 Glycolysis begins with an energy investment phase which requires two ATP to phosphorylate the starting glucose molecule. The 6-carbon intermediate is then split into two 3-carbon sugar molecules. In the energy recovery phase, each 3-carbon sugar is then oxidized to pyruvate with the energy transferred to form two NADH and two ATP. (Credit: Rao, A. and Ryan, K. Department of Biology, Texas A&M University)

First Half of Glycolysis (Energy-Requiring Steps)

The reactions of the first half of glycolysis are shown in Figure 5.12.

Step 1. The first step in glycolysis is catalyzed by hexokinase, an enzyme with broad specificity that catalyzes the phosphorylation of six-carbon sugars. Hexokinase phosphorylates glucose using ATP as the source of the phosphate, producing glucose-6-phosphate, a more reactive form of glucose. This reaction prevents the phosphorylated glucose molecule from continuing to interact with the GLUT proteins, and it can no longer leave the cell because the negatively charged phosphate will not allow it to cross the hydrophobic interior of the plasma membrane.

Step 2. In the second step of glycolysis, an isomerase converts glucose-6-phosphate into one of its isomers, fructose-6-phosphate (this isomer has a phosphate attached at the location of the sixth carbon of the ring). An isomerase is an enzyme that catalyzes the conversion of a molecule into one of its isomers. (This change from phosphoglucose to phosphofructose allows the eventual split of the sugar into two three-carbon molecules.)

Step 3. The third step is the phosphorylation of fructose-6-phosphate, catalyzed by the enzyme phosphofructokinase. A second ATP molecule donates a high-energy phosphate to fructose-6-phosphate, producing fructose-1,6-bisphosphate.

Step 4. The newly added high-energy phosphates further destabilize fructose-1,6-bisphosphate. The fourth step in glycolysis employs an enzyme, aldolase, to cleave fructose-1,6-bisphosphate into two three-carbon isomers: dihydroxyacetone phosphate and glyceraldehyde-3-phosphate.

Step 5. In the fifth step, an isomerase transforms the dihydroxyacetone-phosphate into its isomer, glyceraldehyde-3-phosphate. Thus, the pathway will continue with two molecules of a glyceraldehyde-3-phosphate. At this point in the pathway, there is a net investment of energy from two ATP molecules in the breakdown of one glucose molecule.

Figure 5.12 The first half of glycolysis uses two ATP molecules in the phosphorylation of glucose, which is then split into two three-carbon molecules.

Second Half of Glycolysis (Energy-Releasing Steps)

The reactions of the second half of glycolysis are shown in Figure 5.13. So far, glycolysis has cost the cell two ATP molecules and produced two small, three-carbon sugar molecules. Both of these molecules will proceed through the second half of the pathway, and sufficient energy will be extracted to pay back the two ATP molecules used as an initial investment and produce a profit for the cell of two additional ATP molecules and two NADH molecules.

Step 6. The sixth step in glycolysis oxidizes the sugar (glyceraldehyde-3-phosphate), extracting high-energy electrons, which are picked up by the electron carrier NAD+, producing NADH. The sugar is then phosphorylated by the addition of a second phosphate group, producing 1,3-bisphosphoglycerate. Note that the second phosphate group does not require another ATP molecule.

Here again is a potential limiting factor for this pathway. The continuation of the reaction depends upon the availability of the oxidized form of the electron carrier, NAD+. Thus, NADH must be continuously oxidized back into NAD+ in order to keep this step going. If NAD+ is not available, the second half of glycolysis slows down or stops. If oxygen is available in the system, the NADH will be oxidized readily, though indirectly, and the high-energy electrons from the hydrogen released in this process will be used to produce ATP. In an environment without oxygen, an alternate pathway (fermentation) can provide the oxidation of NADH to NAD+.

Step 7. In the seventh step, catalyzed by phosphoglycerate kinase (an enzyme named for the reverse reaction), 1,3-bisphosphoglycerate donates a high-energy phosphate to ADP, forming one molecule of ATP. (This is an example of substrate-level phosphorylation.) A carbonyl group on the 1,3-bisphosphoglycerate is oxidized to a carboxyl group, and 3-phosphoglycerate is formed.

Step 8. In the eighth step, the remaining phosphate group in 3-phosphoglycerate moves from the third carbon to the second carbon, producing 2-phosphoglycerate (an isomer of 3-phosphoglycerate). The enzyme catalyzing this step is a mutase (isomerase).

Step 9. Enolase catalyzes the ninth step. This enzyme causes 2-phosphoglycerate to lose water from its structure; this is a dehydration reaction, resulting in the formation of a double bond that increases the potential energy in the remaining phosphate bond and produces phosphoenolpyruvate (PEP).

Step 10. The last step in glycolysis is catalyzed by the enzyme pyruvate kinase (the enzyme in this case is named for the reverse reaction of pyruvate’s conversion into PEP) and results in the production of a second ATP molecule by substrate-level phosphorylation and the compound pyruvic acid (or its salt form, pyruvate). Many enzymes in enzymatic pathways are named for the reverse reactions, since the enzyme can catalyze both forward and reverse reactions (these may have been described initially by the reverse reaction that takes place in vitro, under nonphysiological conditions).

Figure 5.13 The second half of glycolysis involves phosphorylation without ATP investment (step 6) and produces two NADH and four ATP molecules per glucose.

Outcomes of Glycolysis

Glycolysis begins with glucose and produces two pyruvate molecules, four new ATP molecules, and two molecules of NADH. (Note: two ATP molecules are used in the first half of the pathway to prepare the six-carbon ring for cleavage, so the cell has a net gain of two ATP molecules and two NADH molecules for its use). If the cell cannot catabolize the pyruvate molecules further, it will harvest only two ATP molecules from one molecule of glucose.

The last step in glycolysis will not occur if pyruvate kinase, the enzyme that catalyzes the formation of pyruvate, is not available in sufficient quantities. In this situation, the entire glycolysis pathway will proceed, but only two ATP molecules will be made in the second half. Thus, pyruvate kinase is a rate-limiting enzyme for glycolysis.


Gain a better understanding of the breakdown of glucose by glycolysis by visiting this site to see the process in action.

Section Summary

Glycolysis is the first pathway used in the breakdown of glucose to extract energy. Because it is used by nearly all organisms on earth, it must have evolved early in the history of life. Glycolysis consists of two parts: The first part prepares the six-carbon ring of glucose for separation into two three-carbon sugars. Energy from ATP is invested into the molecule during this step to energize the separation. The second half of glycolysis extracts ATP and high-energy electrons from hydrogen atoms and attaches them to NAD+. Two ATP molecules are invested in the first half and four ATP molecules are formed during the second half. This produces a net gain of two ATP molecules per molecule of glucose for the cell.



anaerobic: does not require oxygen

adenosine triphosphate (ATP): the cell’s energy currency

glycolysis: the process of breaking glucose into two three-carbon molecules with the production of ATP and NADH

Media Attribution

  • Figure 5.11 by Rao, A. and Ryan, K. Department of Biology, Texas A&M University


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Introduction to Biology Copyright © 2023 by Natasha Ramroop Singh, PhD is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

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