Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyses the conversion of glyceraldehyde 3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3BPG) during glycolysis, as well as the reverse of this reaction during gluconeogenesis. This reaction requires the cofactor NAD⁺, which functions as an electron acceptor, and an inorganic phosphate. This is the sixth reaction in glycolysis, and it is very important because it yields NADH which is used later in the cycle to produce ATP.

This reaction couples an oxidation step with a phosphorylation step. An interesting part of the overall reaction is that the unfavorable formation of an acylphosphate from a carboxylate is driven by the favorable oxidation of the aldehyde.¹¹ The two reactions are coupled by a thioester intermediate in order to preserve the free energy released by the oxidation step. At physiological temperatures the DG of the reaction equals -0.4 kcals/mol and is therefore reversible in the body.¹²

The reaction begins when a hydride ion is removed from the aldehyde group of G3P. This is not a favorable reaction due to the dipolar nature of the carbonyl group it was originally attached to.¹² The sulfhydryl group of the Cys-149 acts as a nucleophile to allow the hydride ion to leave.⁷ The aldehyde substrate then reacts with the Cys-149 residue in the active site of the enzyme to form a tetrahedral hemithioacetal intermediate. The hydride ion is then transferred to the NAD which is also tightly bound to the active site. This results in the formation of NADH and an energy rich thioester intermediate. The NADH then dissociates from the active site and another NAD molecule binds in its place. This is the limiting step of the reaction. The thioester is then attacked by the inorganic phosphate to form 1,3BPG.¹