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7.2C: The Energy-Releasing Steps of Glycolysis

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  • In the second half of glycolysis, energy is released in the form of 4 ATP molecules and 2 NADH molecules.


    Outline the energy-releasing steps of glycolysis


    Key Points

    • The net energy release in glycolysis is a result of two molecules of glyceraldehyde-3- phosphate entering the second half of glycolysis where they are converted to pyruvic acid.
    • Substrate -level phosphorylation, where a substrate of glycolysis donates a phosphate to ADP, occurs in two steps of the second-half of glycolysis to produce ATP.
    • The availability of NAD+ is a limiting factor for the steps of glycolysis; when it is unavailable, the second half of glycolysis slows or shuts down.

    Key Terms

    • NADH: nicotinamide adenine dinucleotide (NAD) carrying two electrons and bonded with a hydrogen (H) ion; the reduced form of NAD

    Second Half of Glycolysis (Energy-Releasing Steps)

    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 where sufficient energy will be extracted to pay back the two ATP molecules used as an initial investment while also producing a profit for the cell of two additional ATP molecules and two even higher-energy NADH molecules.


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

    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, there 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 non-physiological conditions).