Glyceraldehyde 3-phosphate dehydrogenase

Glyceraldehyde 3-phosphate dehydrogenase (abbreviated as GAPDH or less commonly as G3PDH) is an is_associated_with::enzyme of ~37kDa that catalyzes the sixth step of is_associated_with::glycolysis and thus serves to break down is_associated_with::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 is_associated_with::apoptosis, ER to Golgi vesicle shuttling, and fast axonal, or is_associated_with::axoplasmic transport. In sperm, a testis-specific is_associated_with::isoenzyme is_associated_with::GAPDHS takes its role.

Metabolic function
As its name indicates, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyses the conversion of is_associated_with::glyceraldehyde 3-phosphate to D -is_associated_with::glycerate 1,3-bisphosphate. This is the 6th step in the glycolytic breakdown of glucose, an important pathway of energy and carbon molecule supply which takes place in the is_associated_with::cytosol of eukaryotic cells. The conversion occurs in two coupled steps. The first is favourable and allows the second unfavourable step to occur.

Two-step conversion of glyceraldehyde-3-phosphate
The first reaction is the oxidation of is_associated_with::glyceraldehyde 3-phosphate at the carbon 1 position (in the diagram it is shown as the 4th carbon from glycolysis), in which an is_associated_with::aldehyde is converted into a is_associated_with::carboxylic acid (ΔG°'=-50 kJ/mol (-12kcal/mol)) and NAD+ is simultaneously reduced endergonically to NADH.

The energy released by this highly is_associated_with::exergonic oxidation reaction drives the is_associated_with::endergonic second reaction (ΔG°'=+50 kJ/mol (+12kcal/mol)), in which a molecule of inorganic is_associated_with::phosphate is transferred to the GAP intermediate to form a product with high phosphoryl-transfer potential: is_associated_with::1,3-bisphosphoglycerate (1,3-BPG).

This is an example of is_associated_with::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 of catalysis
GAPDH uses covalent catalysis and general base catalysis to decrease the very large and positive activation energy of the second step of this reaction. First, a is_associated_with::cysteine residue in the active site of GAPDH attacks the carbonyl group of GAP, creating a is_associated_with::hemithioacetal intermediate (covalent catalysis). Next, an adjacent, tightly bound molecule of NAD+ accepts a is_associated_with::hydride ion from GAP, forming is_associated_with::NADH; GAP is concomitantly oxidized to a is_associated_with::thioester intermediate using a molecule of water. This thioester species is much higher in energy than the is_associated_with::carboxylic acid species that would result in the absence of GAPDH (the carboxylic acid species is so low in energy that the energy barrier for the second step of the reaction (phosphorylation) would be too high, and the reaction, therefore, too slow and equilibrium too unfavorable for a living organism). Donation of the hydride ion by the hemithioacetal is facilitated by its deprotonation by a is_associated_with::histidine residue in the enzyme's active site (general base catalysis). Deprotonation encourages the reformation of the carbonyl group in the thioester intermediate and ejection of the hydride ion. NADH leaves the active site and is replaced by another molecule of NAD+, the positive charge of which stabilizes the negatively charged carbonyl oxygen in the transition state of the next and ultimate step. Finally, a molecule of is_associated_with::inorganic phosphate attacks the thioester and forms a tetrahedral intermediate, which then collapses to release 1,3-bisphosphoglycerate, and the is_associated_with::thiol group of the enzyme's cysteine residue.

Enzyme regulation
This protein may use the is_associated_with::morpheein model of is_associated_with::allosteric regulation.

Additional functions
GAPDH, like many other enzymes, has multiple functions. In addition to catalysing the 6th step of is_associated_with::glycolysis, recent evidence implicates GAPDH in other cellular processes.GAPDH has been described to exhibit higher order multifunctionality in the context of maintaining cellular iron homeostasis. This came as a surprise to researchers but it makes evolutionary sense to re-use and adapt existing proteins instead of evolving a novel protein from scratch.

GAPDH can also be inhibited by arsenate, inhibiting glycolysis in red blood cells and causing hemolytic anemia.

Transcription and apoptosis
GAPDH can itself activate transcription. The OCA-S transcriptional coactivator complex contains GAPDH and is_associated_with::lactate dehydrogenase, two proteins previously only thought to be involved in is_associated_with::metabolism. GAPDH moves between the is_associated_with::cytosol and the nucleus and may thus link the metabolic state to gene transcription.

In 2005, Hara et al. showed that GAPDH initiates is_associated_with::apoptosis. This is not a third function, but can be seen as an activity mediated by GAPDH binding to is_associated_with::DNA like in transcription activation, discussed above. The study demonstrated that GAPDH is S-nitrosylated by NO in response to cell stress, which causes it to bind to the protein is_associated_with::SIAH1, a is_associated_with::ubiquitin ligase. The complex moves into the nucleus where Siah1 targets nuclear proteins for degradation, thus initiating controlled cell shutdown. In subsequent study the group demonstrated that is_associated_with::deprenyl, which has been used clinically to treat is_associated_with::Parkinson's disease, strongly reduces the apoptotic action of GAPDH by preventing its S-nitrosylation and might thus be used as a drug.

Metabolic switch
GAPDH acts as reversible metabolic switch under oxidative stress. When cells are exposed to is_associated_with::oxidants, they need excessive amounts of the antioxidant cofactor is_associated_with::NADPH. In the cytosol, NADPH is reduced from NADP+ by several enzymes, three of them catalyze the first steps of the is_associated_with::Pentose phosphate pathway. Oxidant-treatments cause an inactivation of GAPDH. This inactivation re-routes temporally the metabolic flux from glycolysis to the Pentose Phosphate Pathway, allowing the cell to generate more NADPH. Under stress conditions, NADPH is needed by some antioxidant-systems including is_associated_with::glutaredoxin and is_associated_with::thioredoxin as well as being essential for the recycling of is_associated_with::gluthathione.

ER to Golgi transport
GAPDH also appears to be involved in the vesicle transport from the is_associated_with::endoplasmic reticulum (ER) to the is_associated_with::Golgi apparatus which is part of shipping route for secreted proteins. It was found that GAPDH is recruited by rab2 to the is_associated_with::vesicular-tubular clusters of the ER where it helps to form COP 1 vesicles. GAPDH is activated via is_associated_with::tyrosine is_associated_with::phosphorylation by Src.

Cellular location
All steps of glycolysis take place in the is_associated_with::cytosol and so does the reaction catalysed by GAPDH. Research in is_associated_with::red blood cells indicates that GAPDH and several other glycolytic enzymes assemble in complexes on the inside of the is_associated_with::cell membrane. The process appears to be regulated by phosphorylation and oxygenation. Bringing several glycolytic enzymes close to each other is expected to greatly increase the overall speed of glucose breakdown.

Usage of GAPDH as loading control
Because the GAPDH gene is often stably and constitutively expressed at high levels in most tissues and cells, it is considered a is_associated_with::housekeeping gene. For this reason, GAPDH is commonly used by biological researchers as a loading control for is_associated_with::western blot and as a control for is_associated_with::qPCR. However, researchers have reported different regulation of GAPDH under specific conditions. For example, the transcription factor MZF-1 has been shown to regulate the GAPDH gene. Therefore, the use of GAPDH as loading control has to be considered carefully.