Glyceraldehyde 3-phosphate dehydrogenase

Glyceraldehyde 3-phosphate dehydrogenase (abbreviated as GAPDH or less commonly as G3PDH) (EC number|1.2.1.12) is an enzyme 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 cite journal |author= A. Tarze, A. Deniaud, M. Le Bras, E. Maillier, D. Molle, N. Larochette, N. Zamzami, G. Jan, G. Kroemer, and C. Brenner |title= GAPDH, a novel regulator of the pro-apoptotic mitochondrial membrane permeabilization |journal=Oncogene |volume=26 |issue=18 |pages=2606–2620 |year=2007 |pmid= 17072346 |doi= 10.1038/sj.onc.1210074] , and ER to Golgi vesicle shuttling.

Metabolic function

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyses the conversion of glyceraldehyde 3-phosphate as the name indicates. This is the 6th step of the breakdown of glucose (glycolysis), an important pathway of energy and carbon molecule supply located in the cytosol of eukaryotic cells. Glyceraldehyde 3-phosphate is converted to D-glycerate 1,3-bisphosphate in two coupled steps. The first is favourable and allows the second unfavourable step to occur.

Overall reaction catalysed

Enzymatic Reaction
foward_enzyme=glyceraldehyde phosphate dehydrogenase
reverse_enzyme=
substrate=glyceraldehyde 3-phosphate
product=D-glycerate 1,3-bisphosphate
reaction_direction_(forward/reversible/reverse)=reversible
minor_foward_substrate(s)=NAD⁺ + P_i
minor_foward_product(s)=NADH + H⁺
minor_reverse_substrate(s)=NADH + H⁺
minor_reverse_product(s)=NAD⁺ + P_i
substrate_

product_

Two-step conversion of glyceraldehyde 3-phosphate

The first reaction is the oxidiation of glyceraldehyde 3-phosphate at the carbon 1 position (the 4th carbon from glycolysis which is shown in the diagram), 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-Biphosphoglycerate (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 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 cysteine residue in the active site of GAPDH attacks the carbonyl group of GAP, creating a hemithioacetal intermediate (covalent catalysis). Next, an adjacent, tightly bound molecule of NAD⁺ accepts a hydride ion from GAP, forming NADH; GAP is concomitantly oxidized to a thioester intermediate using a molecule of water. This thioester species is much higher in energy than the 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 great, and the reaction therefore too slow, for a living organism). Donation of the hydride ion by the hemithioacetal is facilitated by its deprotonation by a 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 inorganic phosphate attacks the thioester and forms a tetrahedral intermediate, which then collapses to release 1,3-bisphosphoglycerate, and the thiol group of the enzyme's cysteine residue.

Additional functions

GAPDH is multifunctional like an increasing number of enzymes. In addition to catalysing the 6th step of glycolysis, recent evidence implicates GAPDH in other cellular processes. This came as a surprise to researchers but it makes evolutionary sense to re-use and adapt an existing proteins instead of evolving a novel protein from scratch.

Transcription and apoptosis

Zheng et al. discovered in 2003 that GAPDH can itself activate transcription. The "OCA-S" transcriptional coactivator complex contains GAPDH and lactate dehydrogenase, two protein previously only thought to be involved in metabolism. GAPDH moves between the cytosol and the nucleus and may thus link the metabolic state to gene transcription.cite journal |author=Zheng L, Roeder RG, Luo Y |title=S phase activation of the histone H2B promoter by OCA-S, a coactivator complex that contains GAPDH as a key component |journal=Cell |volume=114 |issue=2 |pages=255–66 |year=2003 |pmid=12887926 |doi=]

In 2005, Hara et al. showed that GAPDH initiates apoptosis. This is not a third function, but can be seen as an activity mediated by GAPDH binding to 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 "Siah1", a ubiquitin ligase. The complex moves into the nucleus where Siah1 targets nuclear proteins for degradation, thus initiating controlled cell shutdown.cite journal |author=Hara MR, Agrawal N, Kim SF, "et al" |title=S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding |journal=Nat. Cell Biol. |volume=7 |issue=7 |pages=665–74 |year=2005 |pmid=15951807 |doi=10.1038/ncb1268] In subsequent study the group demonstrated that deprenyl, which has been used clinically to treat Parkinson's disease, strongly reduces the apoptotic action of GAPDH by preventing its S-nitrosylation and might thus be used as a drug.cite journal |author=Hara MR, Thomas B, Cascio MB, "et al" |title=Neuroprotection by pharmacologic blockade of the GAPDH death cascade |journal=Proc. Natl. Acad. Sci. U.S.A. |volume=103 |issue=10 |pages=3887–9 |year=2006 |pmid=16505364 |doi=10.1073/pnas.0511321103]

Metabolic Switch

GAPDH acts as reversible metabolic swich under oxidative stress. When cells are exposed to Oxidants, they need excessive amounts of the antioxidant cofactor NADPH. In the cytosol, NADPH is reduced from NADP+ by several enzymes, three of them catalyze the first steps of the 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. cite journal |author=Ralser M et al |title=Dynamic rerouting of the carbohydrate flux is key to counteracting oxidative stress. |journal=J Biol |volume=6 |issue=11 |year=2007 |pmid=18154684] . Under stress conditions, NADPH is needed by some antioxidnt-systems including glutaredoxin and thioredoxin as well as it is essential for the recycling of gluthathione.

ER to Golgi transport

GAPDH also appears to be involved in the vesicle transport from the endoplasmic reticulum (ER) to the Golgi apparatus which is part of shipping route for secreted proteins. It was found that GAPDH is recruited by rab2 to the vesicular-tubular clusters of the ER where it helps to form COP 1 vesicles. GAPDH is activated via tyrosine phosphorylation by Src.cite journal |author=Tisdale EJ, Artalejo CR |title=A GAPDH mutant defective in Src-dependent tyrosine phosphorylation impedes Rab2-mediated events |journal=Traffic |volume=8 |issue=6 |pages=733–41 |year=2007 |pmid=17488287 |doi=10.1111/j.1600-0854.2007.00569.x]

Cellular location

All steps of glycolysis take place in the cytosol and so does the reaction catalysed by GAPDH. Research in red blood cells indicates that GAPDH and several other glycolytic enzymes assemble in complexes on the inside of the cell membrane. The process appears to be regulated by phosphorylation and oxygenation. cite journal |author=Campanella ME, Chu H, Low PS |title=Assembly and regulation of a glycolytic enzyme complex on the human erythrocyte membrane |journal=Proc. Natl. Acad. Sci. U.S.A. |volume=102 |issue=7 |pages=2402–7 |year=2005 |pmid=15701694 |doi=10.1073/pnas.0409741102] Bringing several glycolytic enzymes close to each other is expected to greatly increased the overall speed of glucose breakdown.

Miscellaneous

Because the GAPDH gene is often stably and constitutively expressed at high levels in most tissues and cells, it is considered a housekeeping gene. For this reason, GAPDH is commonly used by biological researchers as a loading control for western blot and as a control for RT-PCR. However, many researchers report different regulation of GAPDH under specific conditions. Therefore, the use of GAPDH as loading control has to be controlled carefully.

Sources

Glycolysis text book references

*Voet, D. and Voet, J. G. (2004) "Biochemistry", Third Edition. J. Wiley & Sons, Hoboken, NJ.
*Berg, Jeremy M., Tymoczko, John L., & Stryer, Lubert (2007) "Biochemistry", Sixth Edition. W. H. Freeman and Co., NY.
* [http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=glyceraldehyde+3+phosphate+dehydrogenase&rid=mcb.figgrp.4342&WebEnv=0bpB8XePphZ8qSS3b9o1BB3FMZtXPr7yFc3MxfLR12WUi7sKapf987mBijj9A0v-LwF_W_lLjUKNwY%40D45D6EC76612AEB0_0018SID&WebEnvRq=1 diagram of the GAPDH reaction mechanism] from Lodish MCB at NCBI bookshelf
* [http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=glyceraldehyde+3+phosphate+dehydrogenase&rid=mboc4.figgrp.297&WebEnv=0qL7ctlqrxJxTMzSHUlui3y2aeU6B8K6Tblugar02bi5Eetekc7g1j_m9gRDhWr1NM3L7U4G-5GFjf%40D45D6EC76612AEB0_0018SID&WebEnvRq=1 similar diagram] from Alberts The Cell at NCBI bookshelf

Cited research

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