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To identify the reactions that involve substrate-level phosphorylation, first, write down the 10 reactions in the glycolytic pathway. These reactions are: 1. Glucose → Glucose-6-phosphate 2. Glucose-6-phosphate → Fructose-6-phosphate 3. Fructose-6-phosphate → Fructose-1,6-bisphosphate 4. Fructose-1,6-bisphosphate → Dihydroxyacetone phosphate + Glyceraldehyde-3-phosphate 5. Dihydroxyacetone phosphate ↔ Glyceraldehyde-3-phosphate 6. Glyceraldehyde-3-phosphate → 1,3-bisphosphoglycerate 7. 1,3-bisphosphoglycerate → 3-phosphoglycerate 8. 3-phosphoglycerate → 2-phosphoglycerate 9. 2-phosphoglycerate → Phosphoenolpyruvate 10. Phosphoenolpyruvate → Pyruvate

Now, look at each reaction and determine which ones involve the direct transfer of a phosphate group to ADP or GDP to form ATP or GTP. These are the reactions involving substrate-level phosphorylation. There are two reactions in glycolysis that involve substrate-level phosphorylation: 1. Reaction 6: Glyceraldehyde-3-phosphate → 1,3-bisphosphoglycerate In this step, an inorganic phosphate group (Pi) is added to glyceraldehyde-3-phosphate (G3P), which results in 1,3-bisphosphoglycerate (1,3-BPG). Concurrently, a molecule of NAD+ is reduced to form NADH. 2. Reaction 7: 1,3-bisphosphoglycerate → 3-phosphoglycerate In this step, the high-energy phosphate group is transferred from 1,3-bisphosphoglycerate to ADP to form ATP and 3-phosphoglycerate (3-PG). This reaction is an example of substrate-level phosphorylation because the phosphate group is transferred directly to ADP, generating ATP. Thus, the substrate-level phosphorylations in the glycolytic pathway occur in reactions 6 and 7.