In 1906 Harden and Young, in a series of classic studies on the fermentation of glucose to ethanol and CO2 by extracts of brewer's yeast, made the following observations.

(A) Inorganic phosphate was essential to fermentation; when the supply of phosphate was exhausted, fermentation ceased before all the glucose was used.
(B) During fermentation under these conditions, ethanol, CO2, and a sugar phosphate accumulated.
(C) When arsenate was substituted for phosphate, no sugar phosphate acumulated, but the fermentation proceeded until all the glucose was converted to ethanol and CO2.

Answer the following questions.

1. Which enzyme of glycolysis requires inorganic phosphate and therefore stops when no phosphate is available?
(a) glyceraldehyde 3-phosphate dehydrogenase
(b) phosphoglycerate mutase
(c) phosphofructokinase-1
(d) phosphoglycerate kinase

2. What sugar phosphate accumulates under these conditions?
(a) glucose 1,6-biphosphate
(b) glucose 1-phosphate
(c) fructose 1-phosphate
(d) fructose 1,6-biphosphate

3. Arsenate substitution for phosphate generated an acyl arsenate compound that immediately degraded. What glycolysis intermediate was a product of the spontaneous degradation of this acyl arsenate?
(a) glycerol 3-phosphate
(b) 3-phosphoglycerate
(c) dihydroxyacetone phosphate
(d) glyceraldehyde 3-phosphate

During this process, the G-3-P is hereby changed or converted into 1,3-bisphosphoglycerate in presence of NAD+ and Pi. Note that the exhaustion of Pi signal the end of the glycolysis process.

Because there's is glucose in abundance (excess), the glucose is thus, phosphorylated to ATP but no Pi gets released.

b.

During fermentation, CO₂ and Ethanol are produced. But if there's no fermentation of ethanol in the absence of oxygen (anaerobic condition) , the NADP+ will piled up.. So, no new NAD+ would be available for further glycolysis.

Pyruvate is converted to ethanol and CO2 to replenish NAD+ for glycolysis to proceed.

c.

The hexose phosphate is piled up or accumulated to fructose 1, 6 bisphosphate.

The point between the energy input reaction preceding it and the energy forming reaction after it serves as the intermediary of the reaction.

Because of unavailability of P, G-3-P is not broken down. Hence, the reaction will backflow to fructose 1, 6 bisphosphate, which is more stable.