In the first stage of glycolysis, the two molecules of ATP are broken down into 2 ADPs + 2 Pi through hydrolysis, then in the second stage of glycolysis they are phosphorilazed to obtain 2 ATPs. How can this process happen twice to yield 4 molecules of ATP if we only have 2 ADPs to begin with?

Note: I'm not asking why the process happens twice, since it's easy to see that the Fructose-1,6-biphosphate is being broken down into two Glyceraldehyde phosphate molecules. I'm asking how can the process of producing two ATPs happen twice if we only broke down two ATP molecules into ADP.


There are many other sources of ADP in the cell: various proteins that use ATP as an energy source hydrolyze it to form ADP + phosphate, thereby extracting energy. This ADP can then be re-phosphorylated by glycolysis to form ATP again. If glycolysis did not produce a net yield of ATP it would be pointless, so it really should be unbalanced in this sense.

  • $\begingroup$ This idea was my best guess, however most of the sources I checked out on glycolysis omitted this information. So, to be clear, what you're saying is that ADP doesn't have to come directly from the previous steps of glycolysis, but it usually comes from other sources in the cell? $\endgroup$ – BrozzSama Apr 3 '16 at 22:16
  • $\begingroup$ Yes. The whole point of producing ATP is that it serves as a common source of energy for various energy-demanding reactions in the cell. And those reactions turn ATP back into ADP. $\endgroup$ – Roland Apr 3 '16 at 22:19

I think I understand your question, and stumbled upon it because i was wondering the same thing while studying for my Microbio test. I know its a year late, but someday someone else might need this info...so here it is.

To put it simply: there is a second pair of phosphates added to the two 3-carbon molecules cleaved from the preparatory stage before the 4 ATP are finally formed. These get added by an enzyme while reducing the NAD+ molecules.

In depth: The first pair of phosphates the cell uses to synthesize ATP come from the initial 2 ATP invested during the prep stage, but an additional 2 phosphates are added when the cell oxidizes the two Glyceraldehyde, 3 Phosphate (G3P) molecules to form NADH. In this step, two electrons are taken away from the G3Ps and an available inorganic phosphate is added to the "1" position, forming "1,3 Biphosphoglyceric Acid " (1,3BPG). Afterwards, the molecules each have 2 phosphates for a total of 4 phosphates available to get oxidized and used to synthesize 4 ATPs.

This step, (the addition of an inorganic phosphate as an intermediary step) is SO overlooked by regular textbooks that its not even funny. mine doesnt even list the enzyme; I had to look it up. but yeah, that's the source of the additional phosphates available for forming the 4 ATP molecules of glycolysis, and possibly why you didnt see it from the diagram in your textbook (you're not alone!).

Hope that helps!

Sources, as of 4/18/17:


It may help if I suggest two ways of looking at the situation.

  1. The first way, which tries to reflect your line of thinking (as I understand it), looks at the situation as two reactions being needed to maintain ADP in the cell. The first reaction produces ATP:

Glycolysis: ADP + Pi → ATP

Whereas the second reaction regenerates ADP by using the ATP

Biosynthesis / Movement / Electrochemical work etc: ATP → ADP + Pi

If the second reaction doesn’t occur, the first one will stop too, but that is OK as the cell has no need to produce ATP if there is plenty.

  1. A second (perhaps more sophisticated) way is to regard the situation as an equilibrium:

ADP + Pi ⇋ ATP

The position of this equilibrium (and hence the relative concentrations of ADP and ATP) will vary depending on the situation in the cell. The concentrations of ADP and ATP will effect glycolysis (and other aspects of metabolism) both by simple mass action, and also be something you may not have yet met, the regulation of certain key enzymes (allosteric regulation). (An equilibrium between ATP, ADP and AMP also exists.)

Finally, there are both open systems and (semi-) closed systems in biological chemistry. The conversion of foodstuffs into chemical energy and the structural components of a cell is obviously an open system — the substrates for glycolysis ultimately come from outside the organism. The interconversion of ATP and ADP (and NAD+/NADH) can be regarded as (semi-) closed, as the main part of the chemical moiety is being conserved. However one should not forget that, initially, when the organism is growing and cells are dividing, the adenosine backbone has to be synthesized, and it requires energy to do this and more energy to add phosphates (also from the diet) to the ribose.


i know that is simple because if you can just look at the first step there were 2ATP only and the energy in this molecule is used to phosphorylate the glucose molecule to give fructose biphosphate and 2ADP and 2pi is left this to molecule may reorganize by the substrate level phosphoryalation to give 4ATP molecules as you know the enery is come from the chamical energy from the GP(glyceraldehyde 3 phosphate ) as initially we have 2ADP and 2Pi but finally we have 4ATP because the energy is enough to reconstract additional 2ADP and 2Pi which comes from other non glycolisis reactions


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