Second Half of Glycolysis (Energy-Releasing Steps)


This illustration shows the steps in the second half of glycolysis. In step six, the enzyme glyceraldehydes dash 3 dash phosphate dehydrogenase produces one N A D H molecule and forms 1 3 dash bisphosphoglycerate. In step seven, the enzyme phosphoglycerate kinase removes a phosphate group from the substrate, forming one A T P molecule and 3 dash phosphoglycerate. In step eight, the enzyme phosphoglycerate mutase rearranges the substrate to form 2 dash phosphoglycerate. In step nine, the enzyme enolase rearranges the substrate to form phosphoenolpyruvate. In step ten, a phosphate group is removed from the substrate, forming one A T P molecule and pyruvate.
The second half of glycolysis involves phosphorylation without ATP investment (step 6) and produces two NADH and four ATP molecules per glucose.

Source: OpenStax Biology 2e

OpenStax Biology 2e

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 even higher-energy NADH molecules.

Step 6 (see image). 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.

– What is an important pyridine nucleotide that functions as an oxidative cofactor in eukaryotic cells, plays a key role in the production of energy through redox reactions, and serves as a cofactor for dehydrogenases, reductases and hydroxylases, making it a major carrier of H+ and e- in major metabolic pathways such as glycolysis, the triacarboxylic acid cycle, and fatty acid synthesis?

Here 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.

– What do you call the production of ATP by way of an electron transport chain and using oxygen as the final electron acceptor. ?

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).

– What is an oxidation reaction in which a substance loses both a proton and an electron?

Substrate-level phosphorylation (SLP) is directly phosphorylating ADP with a phosphate and energy provided from a coupled reaction. SLP will only occur if there is a reaction that releases sufficient energy to allow the direct phosphorylation of ADP. Oxidative phosphorylation is when ATP is generated from the oxidation of NADH and FADH2 and the subsequent transfer of electrons and pumping of protons. That process generates an electrochemical gradient, which is required to power the ATP synthase.


Clark, M., Douglas, M., Choi, J. Biology 2e. Houston, Texas: OpenStax. Access for free at:


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