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Out of 10 steps in glycolysis, only one reaction- Glyceraldehyde 3-phosphate (G3P) to 1,3-bisphosphoglycerate (PGP), uses NAD+ and thereby producing NADH. Furthermore, this very step is solely responsible for net gain of 2ATP in glycolysis since here phosphorylation occurs without the expenditure of ATP.

Why is NAD+ used only in this step? Is it possible to predict by other information that this is the only step to require NAD+? Is it possible to make this reaction happen without the involvement of NAD(P)+? Is it possible to skip this step and directly produce 3-phosphoglycerate (Lets not worry even if we cannot produce ATP in this process). I am interested in this because, this step necessitates the ferementation for regeneration of NAD+ in anaerobic condition. E. coli produced acetate as a side effect, as expected during anaerobic situations, but also rather unexpectedly during aerobic and high-growth rate situation (acetate-swtich, overflow problem). Production of acetate is unwanted in many industrial applications.

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NAD+ is important in this step, since it is co-factor for the glyceraldehyde-3-phosphate dehydrogenase (G3PDH), which acts as a acceptor for the hydrogen atom from the C1 (see below).

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If you look at the reaction, the aldehyde from the C1 is oxidized to a carboxylic acid which in a second step is turned into a phosphoester. To do so, a cysteine from the active center of the G3PDH attacks the G3P and forms a hemiacetal. This is oxidized into a thioester, the hydride which is split off in this step is transfered to the NAD+ bound by the G3PDH. In the second step the thioester formed between the enzyme and the G3P is attacked by a molecule of inorganic phosphate to form 1,3-BPG. This step is only made possible by splitting the very energy rich thioester bond. So for this reaction to happen you need both steps, as the formation of an energy rich phosphate ester bond would otherwise not happen. See here for more details.

To go directly from G3P to 3-PG you would need an enzyme which oxidizes the aldehyde group in the G3P. And then you need again an acceptor for the hydrogen which is split off here.

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