Final answer:
ATP is first made in glycolysis during the conversion of phosphoenolpyruvate to pyruvate by pyruvate kinase, resulting in the production of 2 molecules of ATP.
Step-by-step explanation:
The step in which ATP is first made in glycolysis is the conversion of phosphoenolpyruvate (substrate) to pyruvate (product) by the enzyme pyruvate kinase.
This step occurs in the second phase of glycolysis and is responsible for generating 2 molecules of ATP through substrate-level phosphorylation.
The reaction is as follows: phosphoenolpyruvate + ADP -> pyruvate + ATP.