Re: During glycolysis, NAD+ turns into NADH + H+. Where do the 2 Hs come from?

I'm learning glycolysis. Every source I can find shows the glyceraldehyde 3-phosphate (PGAL) -> 1,3- bisphosphoglycerate (PGAP) step of glycolysis converting NAD+ into NADH + H+. When I count the total hydrogen atoms present, I find there are five before but six afterwards. Where's the extra hydrogen coming from?

Hi Zak,

Thanks for submitting your question to the MadSci Network. Glycolysis is a process that is easy to summarize, but for which the details can be elusive and complex. For example, here is an answer that I wrote summarizing glycolysis (1173974025.Bc) in which I explicitly avoided the discussion of the conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate (or as I was taught, 1,3-diphosphoglycerate).

So, lets focus on that reaction. I note that you said that every source you had consulted had summarized the reaction as G3P + NAD⁺ + Pi --> 1,3-BPG + NADH + H⁺. While that is a correct summary of the reaction, the specifics of the reaction are more complicated, and because you want to know where the hydrogens come from, we need to look at the activity of the glyceraldehyde 3-phosphate dehydrogenase enzyme. If you don't want to go into the gritty details right off the bat, you can jump to the end of this answer, where I cut to the chase and explain the summary of the reaction with which you are familiar.

I don't know what sources you have been using, but I usually recommend a college-level biochemistry textbook for questions like this. Biochemistry, by L. Stryer, is available online, from the National Center for Biotechnology Information (NCBI) Bookshelf database of bioscience reference texts, which makes it a very useful resource for in-depth biochemistry questions.

Stryer Chapter 16 discusses glycolysis and gluconeogenesis, and it is section 16.1.5 and Figure 16.8 (below) that hold the answers to your questions.

So, the yellow curve in that figure represents the glycerladehyde 3-phosphate dehydrogenase enzyme, and in particular, the active site. The active site coordinates a cysteine residue, a histidine residue, and an NAD⁺ molecule around the G3P molecule. The cysteine residue contains a sulfhydryl (-SH) group; this is the source of one of the hydrogen ions (H⁺ or proton) you are asking about, as you can see in the top left image of the figure. The proton in the sulfhydryl group is transferred to the oxygen in the keto group, while the sulfur attacks the carbon.

In the image to the right, the histidine is protonated by the proton that originally came from the sulfhydryl group, and the proton from the glyceraldehyde 3-phosphate is transferred to the NAD⁺, forming NADH in the image below.

That NADH then leaves the active site, and a new NAD⁺ molecule moves into the active site. In the image in the middle on the left, the glyceraldehyde 3-phosphate has formed a thioester (thio means sulfur) with the cysteine residue of the active site. Now, in order for an enzyme to function as a catalyst, it has to be unchanged by the reaction; this means that the cysteine and the protonated histidine have to be returned to their former (intact) states. It looks like we have enough protons (the sulfhydryl group could be reconstituted with the proton on the histidine), but the carbon in the thioester would be short an electron. So, we need a molecule that is willing to share an electron.

Thats where the phosphate (abbreviated as Pi) comes in. When I was teaching biochemistry, I found that students were often confused by the structure of phosphate. Recall that Pi is generated by the hydrolysis of the gamma phosphate in a nucleotide triphosphate, so the free phosphate molecule (orthophosphate) is described as HPO₄^-2, and not as PO₄^-3. I suspect that this might be the source of your confusion, as the proton that recharges the sulfhydryl group comes from the phosphate group!

So, in the bottom left image of the figure, Pi (HPO₄^-2) is used to phosphorylate the thioester, transferring its proton to the cysteine, and resulting in the generation of 1,3-BPB. The protonated histidine loses its proton, which is released into solution (H⁺).

When you take the enzyme out of the equation (because it is unchanged when the reaction is complete), you have what appears to be a proton transferred from G3P to NAD⁺, forming NADH, and a proton (H⁺) being released from Pi when it is incorporated to the (apparently) deprotonated G3P to form 1,3-BPG. Here's a diagram, with the two protons in question shown in bold (the R in G3P and 1,3-BPG represents the remainder of the molecule that is not involved in the reaction).

     H       HO
     |        |
   O=C   +  O=P-O- + NAD+
     |        |
     R        O-

    G3P  orthophosphate

         |
         V

         O    
        / \
     O=C O=P-O- + NADH + H+
       |   |
       R   O-

      1,3-BPG

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