- Glyceraldehyde 3-phosphate dehydrogenase
Glyceraldehyde 3-phosphate dehydrogenase (abbreviated as GAPDH or less commonly as G3PDH) (EC number|188.8.131.52) is an
enzymethat catalyzes the sixth step of glycolysisand thus serves to break down glucosefor 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 apoptosiscite 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.
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyses the conversion of
glyceraldehyde 3-phosphateas 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 cytosolof eukaryotic cells. Glyceraldehyde 3-phosphate is converted to D- glycerate 1,3-bisphosphatein two coupled steps. The first is favourable and allows the second unfavourable step to occur.
Overall reaction catalysed
glyceraldehyde phosphate dehydrogenase
minor_foward_substrate(s)=NAD+ + Pi
minor_foward_product(s)=NADH + H+
minor_reverse_substrate(s)=NADH + H+
minor_reverse_product(s)=NAD+ + Pi
Two-step conversion of glyceraldehyde 3-phosphate
The first reaction is the oxidiation of
glyceraldehyde 3-phosphateat the carbon 1 position (the 4th carbon from glycolysis which is shown in the diagram), in which an aldehydeis 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 exergonicoxidation reaction drives the endergonicsecond reaction (ΔG°'=+50 kJ/mol (+12kcal/mol)), in which a molecule of inorganic phosphateis 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 phosphorylationcoupled 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
cysteineresidue in the active site of GAPDH attacks the carbonyl group of GAP, creating a hemithioacetalintermediate (covalent catalysis). Next, an adjacent, tightly bound molecule of NAD+accepts a hydride ionfrom GAP, forming NADH; GAP is concomitantly oxidized to a thioesterintermediate using a molecule of water. This thioester species is much higher in energy than the carboxylic acidspecies 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 histidineresidue 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 phosphateattacks the thioester and forms a tetrahedral intermediate, which then collapses to release 1,3-bisphosphoglycerate, and the thiolgroup of the enzyme's cysteine residue.
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
2003that 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 cytosoland 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=]
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 DNAlike 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]
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 glutaredoxinand thioredoxinas 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 apparatuswhich is part of shipping route for secreted proteins. It was found that GAPDH is recruited by rab2 to the vesicular-tubular clustersof the ER where it helps to form COP 1 vesicles. GAPDH is activated via tyrosine phosphorylationby 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]
All steps of glycolysis take place in the
cytosoland so does the reaction catalysed by GAPDH. Research in red blood cellsindicates 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.
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 blotand 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.
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
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