What is the rate-limiting step of glycolysis and which enzyme catalyzes it?

• A. Glucose-6-phosphate to fructose-6-phosphate by phosphoglucose isomerase.
• B. Glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate by GAPDH.
• C. Fructose-6-phosphate to fructose-1,6-bisphosphate by PFK-1.
• D. Pyruvate to acetyl-CoA by PDH.

Answer: (C)

The rate at which glycolysis proceeds is governed by a single control point: the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate by phosphofructokinase-1. This step commits the carbon skeleton to glycolysis and has a strongly negative ΔG under cellular conditions, making it effectively irreversible and the gatekeeper for flux through the pathway. Because of its pivotal role, the enzyme is tightly regulated by the cell’s energy status: ATP inhibits PFK-1 when energy is plentiful, while AMP and ADP activate it when energy is needed; citrate and low pH also modulate its activity. Once this committed step takes place, downstream reactions proceed more readily, so they do not usually bottleneck the overall rate of glycolysis. The other steps listed are either near equilibrium or occur after the commitment point, or in the case of the pyruvate dehydrogenase step, belong to the link between glycolysis and the mitochondria rather than glycolysis itself.