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Stage 2: Conversion to Pyruvate
In this section, we will look at the reactions that convert our two 3-carbon molecules of glyceraldehyde-3-phosphate (GAP) into pyruvate, the product of glycolysis. This conversion occurs in five steps that we will review below. At this point, we will also see where oxygen comes into play in glycolysis so that in the next section, we can look at the differences between aerobic and anaerobic glycolysis. Keep in mind in this section that since we have split our 6-carbon molecule into two 3-carbon molecules, each of these reactions is occurring in both of the 3-carbon molecules.
In this step, two main events take place: 1) glyceraldehyde-3-phosphate is oxidized by the coenzyme nicotinamide adenine dinucleotide (NAD); 2) the molecule is phosphorylated by the addition of a free phosphate group. The enzyme that catalyzes this reaction is glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

The chemistry that takes place in this reaction is more complex than that of the previous reactions we've discussed. Knowledge of organic chemistry is needed to understand the specific mechanisms of the conversion. Generally, the enzyme GAPDH contains appropriate structures and holds the molecule in a conformation such that it allows the NAD molecule to pull a hydrogen off the GAP, converting the NAD to NADH. The phosphate group then attacks the GAP molecule and releases it from the enzyme to yield 1,3 bisphoglycerate, NADH, and a hydrogen atom. We will come back to the role of this NAD/NADH molecule in the next section.
In this step, 1,3 bisphoglycerate is converted to 3-phosphoglycerate by the enzyme phosphoglycerate kinase (PGK). This reaction involves the loss of a phosphate group from the starting material. The phosphate is transferred to a molecule of ADP that yields our first molecule of ATP. Since we actually have two molecules of 1,3 bisphoglycerate (because there were two 3-carbon products from stage 1 of glycolysis), we actually synthesize two molecules of ATP at this step. With this synthesis of ATP, we have cancelled the first two molecules of ATP that we used, leaving us with a net of 0 ATP molecules up to this stage of glycolysis.

Again, we see that an atom of magnesium is involved to shield the negative charges on the phosphate groups of the ATP molecule.
This step involves a simple rearrangement of the position of the phosphate group on the 3 phosphoglycerate molecule, making it 2 phosphoglycerate. The molecule responsible for catalyzing this reaction is called phosphoglycerate mutase (PGM). A mutase is an enzyme that catalyzes the transfer of a functional group from one position on a molecule to another.
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