The payoff phase oxidizes each 3-carbon glyceraldehyde-3-phosphate to pyruvate, harvesting energy as NADH and ATP. Because every glucose produces 2 G3P, each payoff step runs twice per glucose.
The Five Payoff Steps
Each G3P runs through these five steps. Because one glucose produces two G3P, multiply every yield by 2 for the whole-glucose total.
Per G3P: 2 ATP + 1 NADH + 1 pyruvate Per glucose (×2): 4 ATP + 2 NADH + 2 pyruvate
Net Yield
Per molecule of glucose:
4 ATP produced (2 per G3P × 2 G3P) in the payoff
2 ATP consumed in the investment phase
Net: 2 ATP
Plus: 2 NADH and 2 pyruvate
Substrate-Level Phosphorylation
The two ATPs in the payoff phase are made by substrate-level phosphorylation - a high-energy phosphate group is transferred directly from a phosphorylated intermediate to ADP. This is different from oxidative phosphorylation (ETC + ATP synthase), which uses a proton gradient.
Arsenic Poisoning
Arsenite (As3+) inhibits the sulfhydryl groups needed by GAPDH. Arsenate (As5+) mimics phosphate, becomes incorporated at step 6 but then spontaneously hydrolyzes instead of phosphorylating ADP in step 7 - effectively uncoupling glycolysis without producing ATP. Both are lethal because glycolysis is required in all cells.
What is the overall net yield from glycolysis per molecule of glucose?
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Net 2 ATP, 2 NADH, and 2 pyruvate. Investment phase consumes 2 ATP; payoff phase produces 4 ATP and 2 NADH per glucose. Subtracting investment from payoff gives the net of 2 ATP. Pyruvate then enters either aerobic pathways (TCA cycle) or fermentation.
What is substrate-level phosphorylation, and where does it happen in glycolysis?
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Substrate-level phosphorylation is direct transfer of a phosphate from a high-energy substrate to ADP, producing ATP. It happens twice in glycolysis: at step 7 (phosphoglycerate kinase transfers from 1,3-BPG) and step 10 (pyruvate kinase transfers from PEP). It is independent of the electron transport chain.
Why is GAPDH step 6 a common target of poisoning (e.g., arsenic)?
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GAPDH couples the oxidation of G3P (producing NADH) to the formation of a high-energy acyl-phosphate (1,3-BPG), which feeds step 7's ATP production. Blocking GAPDH halts glycolysis' energy output. Arsenite inhibits the enzyme directly; arsenate mimics phosphate, enters the reaction, but spontaneously hydrolyzes instead of forming a useful product, robbing the cell of ATP.
