Glyceraldehyde 3-phosphate dehydrogenase (abbreviated GAPDH) () is an enzyme of about 37kDa that catalyzes the sixth step of glycolysis and thus serves to break down glucose for energy and carbon molecules. In addition to this long established metabolic function, GAPDH has recently been implicated in several non-metabolic processes, including transcription activation, initiation of apoptosis, ER to Golgi vesicle shuttling, and fast axonal, or axoplasmic transport. In sperm, a testis-specific isoenzyme GAPDHS is expressed. Structure Under normal cellular conditions, cytoplasmic GAPDH exists primarily as a tetramer. This form is composed of four identical 37-kDa subunits containing a single catalytic thiol group each and critical to the enzyme's catalytic function. Nuclear GAPDH has increased isoelectric point (pI) of pH 8.3–8.7. Of note, the cysteine residue C152 in the enzyme's active site is required for the induction of apoptosis by oxidative stress. Notably, post-translational modifications of cytoplasmic GAPDH contribute to its functions outside of glycolysis. GAPDH is encoded by a single gene that produces a single mRNA transcript with 8 splice variants, though an isoform does exist as a separate gene that is expressed only in spermatozoa. Reaction Two-step conversion of G3P The first reaction is the oxidation of glyceraldehyde 3-phosphate (G3P) at the position-1 (in the diagram it is shown as the 4th carbon from glycolysis), in which an aldehyde is converted into a carboxylic acid (ΔG°'=-50 kJ/mol (−12kcal/mol)) and NAD+ is simultaneously reduced endergonically to NADH. The energy released by this highly exergonic oxidation reaction drives the endergonic second reaction (ΔG°'=+50 kJ/mol (+12kcal/mol)), in which a molecule of inorganic phosphate is transferred to the GAP intermediate to form a product with high phosphoryl-transfer potential: 1,3-bisphosphoglycerate (1,3-BPG). This is an example of phosphorylation coupled to oxidation, and the overall reaction is somewhat endergonic (ΔG°'=+6.3 kJ/mol (+1.5)). Energy coupling here is made possible by GAPDH. Mechanism GAPDH uses covalent catalysis and general base catalysis to decrease the very large activation energy of the second step (phosphorylation) of this reaction. 1: Oxidation First, a cysteine residue in the active site of GAPDH attacks the carbonyl group of GAP, creating a hemithioacetal intermediate (covalent catalysis). The hemithioacetal is deprotonated by a histidine residue in the enzyme's active site (general base catalysis). Deprotonation encourages the reformation of the carbonyl group in the subsequent thioester intermediate and ejection of a hydride ion. Next, an adjacent, tightly bound molecule of NAD+ accepts the hydride ion, forming NADH while