Question

8. Requirement for Phosphate in Ethanol Fermentation 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: 1) Inorganic phosphate was essential to fermentation; when the supply of phosphate was exhausted, fermentation ceased before all the glucose was used. 2) During fermentation under these conditions, ethanol, CO2, and a hexose bisphosphate accumulated. Questions: a) Why did fermentation cease when the supply of phosphate (Pi) was exhausted? b) Why did ethanol and CO2 accumulate? Was the conversion of pyruvate to ethanol and CO2 essential? Why? Identify the hexose bisphosphate that accumulated. Why did it accumulate?

Answer 1

1) a. Phosphate is required in the glyceraldehyde 3-phosphate (G-3-P) dehydrogenase reaction. G-3-P is converted to 1,3-bisphosphoglycerate in presence of NAD+ and Pi. Glycolysis ceases if Pi is exhausted. As there is excess of glucose, it is phosphorylated to ATP. However, no Pi is release.

b. Yeast produces ethanol and CO2 by fermentation. NADP+ will accumulate if no ethanol fermentation occurs in anaerobic conditions. Hence, 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 accumulated is fructose 1, 6 bisphosphate. This intermediate is the point between the energy input reaction preceding it and the energy forming reaction after it. Due to Pi unavailability, G-3-P is not broken down. Hence, the reaction will backflow to fructose 1, 6 bisphosphate, which is more stable.

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