Question: how many times does substrate level phosphorylation occur in glycolysis?
Answer: 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.