Glyceraldehyde 3-phosphate dehydrogenase
|Symbols||; G3PD; GAPD; HEL-S-162eP|
|External IDs||ChEMBL: GeneCards:|
|RNA expression pattern|
|File:PBB GE GAPDH 212581 x at tn.png|
|File:PBB GE GAPDH 213453 x at tn.png|
|File:PBB GE GAPDH 217398 x at tn.png|
|Glyceraldehyde 3-phosphate dehydrogenase, NAD binding domain|
File:PDB 1cer EBI.jpg|
determinants of enzyme thermostability observed in the molecular structure of thermus aquaticus d-glyceraldehyde-3-phosphate dehydrogenase at 2.5 angstroms resolution
|Glyceraldehyde 3-phosphate dehydrogenase, C-terminal domain|
File:PDB 2czc EBI.jpg|
crystal structure of glyceraldehyde-3-phosphate dehydrogenase from pyrococcus horikoshii ot3
Glyceraldehyde 3-phosphate dehydrogenase (abbreviated as GAPDH or less commonly as G3PDH) (EC 22.214.171.124) is an enzyme of ~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 takes its role.
- 1 Metabolic function
- 2 Additional functions
- 3 Metabolic switch
- 4 Cellular location
- 5 Usage of GAPDH as loading control
- 6 References
- 7 Further reading
As its name indicates, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyses the conversion of glyceraldehyde 3-phosphate to D-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 cytosol of eukaryotic cells. The conversion occurs in two coupled steps. The first is favourable and allows the second unfavourable step to occur.
Overall reaction catalyzed
|glyceraldehyde 3-phosphate||glyceraldehyde phosphate dehydrogenase||D-glycerate 1,3-bisphosphate|
|File:D-glyceraldehyde-3-phosphate wpmp.png||File:D-glycerate 1,3-bisphosphate.svg|
|NAD+ +Pi||NADH + H+|
|NAD+ +Pi||NADH + H+|
Two-step conversion of glyceraldehyde-3-phosphate
The first reaction is the oxidation of glyceraldehyde 3-phosphate at the carbon 1 position (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 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 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 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.
Interactive pathway map
GAPDH, like many other enzymes, has multiple functions. In addition to catalysing the 6th step of 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 lactate dehydrogenase, two proteins 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.
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. 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.
GAPDH acts as reversible metabolic switch 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. Under stress conditions, NADPH is needed by some antioxidant-systems including glutaredoxin and thioredoxin as well as being 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.
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. 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 housekeeping gene. For this reason, GAPDH is commonly used by biological researchers as a loading control for western blot and as a control for 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.
- Tarze A, Deniaud A, Le Bras M, Maillier E, Molle D, Larochette N et al. (Apr 2007). "GAPDH, a novel regulator of the pro-apoptotic mitochondrial membrane permeabilization". Oncogene 26 (18): 2606–20. PMID 17072346. doi:10.1038/sj.onc.1210074.
- Zala D, Hinckelmann MV, Yu H, Lyra da Cunha MM, Liot G, Cordelières FP et al. (Jan 2013). "Vesicular glycolysis provides on-board energy for fast axonal transport". Cell 152 (3): 479–91. PMID 23374344. doi:10.1016/j.cell.2012.12.029.
- Selwood T, Jaffe EK (Mar 2012). "Dynamic dissociating homo-oligomers and the control of protein function". Archives of Biochemistry and Biophysics 519 (2): 131–43. PMC 3298769. PMID 22182754. doi:10.1016/j.abb.2011.11.020.
- Boradia VM, Raje M, Raje CI (Dec 2014). "Protein moonlighting in iron metabolism: glyceraldehyde-3-phosphate dehydrogenase (GAPDH)". Biochemical Society Transactions 42 (6): 1796–801. PMID 25399609. doi:10.1042/BST20140220.
- Zheng L, Roeder RG, Luo Y (Jul 2003). "S phase activation of the histone H2B promoter by OCA-S, a coactivator complex that contains GAPDH as a key component". Cell 114 (2): 255–66. PMID 12887926. doi:10.1016/S0092-8674(03)00552-X.
- Hara MR, Agrawal N, Kim SF, Cascio MB, Fujimuro M, Ozeki Y et al. (Jul 2005). "S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding". Nature Cell Biology 7 (7): 665–74. PMID 15951807. doi:10.1038/ncb1268.
- Hara MR, Thomas B, Cascio MB, Bae BI, Hester LD, Dawson VL et al. (Mar 2006). "Neuroprotection by pharmacologic blockade of the GAPDH death cascade". Proceedings of the National Academy of Sciences of the United States of America 103 (10): 3887–9. PMC 1450161. PMID 16505364. doi:10.1073/pnas.0511321103.
- Agarwal AR, Zhao L, Sancheti H, Sundar IK, Rahman I, Cadenas E (Nov 2012). "Short-term cigarette smoke exposure induces reversible changes in energy metabolism and cellular redox status independent of inflammatory responses in mouse lungs". American Journal of Physiology. Lung Cellular and Molecular Physiology 303 (10): L889–98. PMID 23064950. doi:10.1152/ajplung.
- Ralser M, Wamelink MM, Kowald A, Gerisch B, Heeren G, Struys EA et al. (2007). "Dynamic rerouting of the carbohydrate flux is key to counteracting oxidative stress". Journal of Biology 6 (4): 10. PMC 2373902. PMID 18154684. doi:10.1186/jbiol61.
- Tisdale EJ, Artalejo CR (Jun 2007). "A GAPDH mutant defective in Src-dependent tyrosine phosphorylation impedes Rab2-mediated events". Traffic 8 (6): 733–41. PMID 17488287. doi:10.1111/j.1600-0854.2007.00569.x.
- Campanella ME, Chu H, Low PS (Feb 2005). "Assembly and regulation of a glycolytic enzyme complex on the human erythrocyte membrane". Proceedings of the National Academy of Sciences of the United States of America 102 (7): 2402–7. PMC 549020. PMID 15701694. doi:10.1073/pnas.0409741102.
- Barber RD, Harmer DW, Coleman RA, Clark BJ (May 2005). "GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues". Physiological Genomics 21 (3): 389–95. PMID 15769908. doi:10.1152/physiolgenomics.00025.2005.
- Piszczatowski RT, Rafferty BJ, Rozado A, Tobak S, Lents NH (Aug 2014). "The glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) is regulated by myeloid zinc finger 1 (MZF-1) and is induced by calcitriol". Biochemical and Biophysical Research Communications 451 (1): 137–41. PMID 25065746. doi:10.1016/j.bbrc.2014.07.082.
- Voet D, Voet JG (2010). Biochemistry. New York: Wiley. ISBN 0-470-57095-4.
- Stryer, Lubert; Berg, Jeremy Mark; Tymoczko, John L. (2002). Biochemistry, Fifth Edition & Lecture Notebook. San Francisco: W. H. Freeman. ISBN 0-7167-9804-2.
- diagram of the GAPDH reaction mechanism from Lodish MCB at NCBI bookshelf
- similar diagram from Alberts The Cell at NCBI bookshelf