Glyceraldehyde 3-phosphate dehydrogenase, NAD binding domain
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 (abbreviatedGAPDH) (EC1.2.1.12) is anenzyme of about 37kDa that catalyzes the sixth step ofglycolysis and thus serves to break downglucose for energy and carbon molecules. In addition to this long established metabolic function, GAPDH has recently been implicated in several non-metabolic processes, includingtranscription activation, initiation ofapoptosis,[4]ER-to-Golgi vesicle shuttling, and fast axonal, oraxoplasmic transport.[5] In sperm, a testis-specificisoenzymeGAPDHS is expressed.
Under normal cellular conditions,cytoplasmic GAPDH exists primarily as atetramer. This form is composed of four identical 37-kDa subunits containing a single catalyticthiol group each and critical to the enzyme's catalytic function.[6][7] Nuclear GAPDH has increasedisoelectric point (pI) of pH 8.3–8.7.[7] Of note, thecysteineresidue C152 in the enzyme'sactive site is required for the induction of apoptosis byoxidative stress.[7] Notably,post-translational modifications of cytoplasmic GAPDH contribute to its functions outside of glycolysis.[6]
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 inspermatozoa.[7]
The first reaction is the oxidation ofglyceraldehyde 3-phosphate (G3P) at the position-1 (in the diagram it is shown as the 4th carbon from glycolysis), in which analdehyde is converted into acarboxylic acid (ΔG°'=-50 kJ/mol (−12kcal/mol)) and NAD+ is simultaneously reduced endergonically to NADH.
The energy released by this highlyexergonic oxidation reaction drives theendergonic second reaction (ΔG°'=+50 kJ/mol (+12kcal/mol)), in which a molecule of inorganicphosphate 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 ofphosphorylation 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.
GAPDH uses covalent catalysis and general base catalysis to decrease the very large activation energy of the second step (phosphorylation) of this reaction.
First, acysteine residue in the active site of GAPDH attacks the carbonyl group of G3P, creating ahemithioacetal intermediate (covalent catalysis).
The hemithioacetal is deprotonated by ahistidine 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 ahydride ion.
Next, an adjacent, tightly bound molecule ofNAD+ accepts thehydride ion, formingNADH while the hemithioacetal is oxidized to athioester.
This thioester species is much higher in energy (less stable) than thecarboxylic acid species that would result if G3P were oxidized 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 unfavorable for a living organism).
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 ofinorganic phosphate attacks the thioester and forms a tetrahedral intermediate, which then collapses to release 1,3-bisphosphoglycerate, and thethiol group of the enzyme's cysteine residue.
As its name indicates, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyses the conversion ofglyceraldehyde 3-phosphate toD-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 thecytosol of eukaryotic cells. The conversion occurs in two coupled steps. The first is favourable and allows the second unfavourable step to occur.
One of the GAPDHmoonlighting functions is its role in adhesion and binding to other partners. Bacterial GAPDH fromMycoplasma andStreptococcus and fungal GAPDH fromParacoccidioides brasiliensis are known to bind with the human extracellular matrix component and act in adhesion.[9][10][11] GAPDH is found to be surface bound contributing in adhesion and also in competitive exclusion of harmful pathogens.[12] GAPDH fromCandida albicans is found to cell-wall associated and binds toFibronectin andLaminin.[13] GAPDH fromprobiotics species are known to bind human colonicmucin and ECM, resulting in enhanced colonization of probiotics in the human gut.[14][15][16] Patel D. et al., showed thatLactobacillus acidophilus GAPDH binds with mucin, acting in adhesion.[17]
GAPDH can itself activatetranscription. TheOCA-S transcriptional coactivator complex contains GAPDH andlactate dehydrogenase, two proteins previously only thought to be involved inmetabolism. GAPDH moves between thecytosol and thenucleus and may thus link the metabolic state to gene transcription.[18]
In 2005, Hara et al. showed that GAPDH initiatesapoptosis. This is not a third function, but can be seen as an activity mediated by GAPDH binding toDNA like in transcription activation, discussed above. The study demonstrated that GAPDH isS-nitrosylated by NO in response to cell stress, which causes it to bind to the proteinSIAH1, aubiquitin ligase. The complex moves into the nucleus where Siah1 targets nuclear proteins fordegradation, thus initiating controlled cell shutdown.[19] In subsequent study the group demonstrated thatdeprenyl, which has been used clinically to treatParkinson's disease, strongly reduces the apoptotic action of GAPDH by preventing its S-nitrosylation and might thus be used as a drug.[20]
GAPDH acts as a reversible metabolic switch under oxidative stress.[21] When cells are exposed tooxidants, they need excessive amounts of the antioxidant cofactorNADPH. In the cytosol, NADPH is reduced from NADP+ by several enzymes, three of them catalyze the first steps of thepentose 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.[22] Under stress conditions, NADPH is needed by some antioxidant-systems includingglutaredoxin andthioredoxin as well as being essential for the recycling ofgluthathione.
GAPDH, like many other enzymes, has multiple functions. In addition to catalysing the 6th step ofglycolysis, 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,[24] specifically as achaperone protein for labile heme within cells.[25] 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.
Because the GAPDH gene is often stably and constitutively expressed at high levels in most tissues and cells, it is considered ahousekeeping gene. For this reason, GAPDH is commonly used by biological researchers as aloading control forwestern blot and as a control forqPCR. However, researchers have reported different regulation of GAPDH under specific conditions.[26] For example, the transcription factorMZF-1 has been shown to regulate the GAPDH gene.[27] Hypoxia also strongly upregulates GAPDH.[28] Therefore, the use of GAPDH as loading control has to be considered carefully.
All steps of glycolysis take place in thecytosol and so does the reaction catalysed by GAPDH. Inred blood cells, GAPDH and several other glycolytic enzymes assemble in complexes on the inside of thecell membrane. The process appears to be regulated by phosphorylation and oxygenation.[29] Bringing several glycolytic enzymes close to each other is expected to greatly increase the overall speed of glucose breakdown. Recent studies have also revealed that GAPDH is expressed in an iron dependent fashion on the exterior of the cell membrane a where it plays a role in maintenance of cellular iron homeostasis.[30][31]
GAPDH is overexpressed in multiple human cancers, such as cutaneousmelanoma, and its expression is positively correlated with tumor progression.[32][33] Its glycolytic and antiapoptotic functions contribute to proliferation and protection of tumor cells, promotingtumorigenesis. Notably, GAPDH protects againsttelomere shortening induced bychemotherapeutic drugs that stimulate thesphingolipidceramide. Meanwhile, conditions likeoxidative stress impair GAPDH function, leading to cellular aging and death.[7] Moreover, depletion of GAPDH has managed to inducesenescence in tumor cells, thus presenting a novel therapeutic strategy for controlling tumor growth.[34]
GAPDH has been implicated in several neurodegenerative diseases and disorders, largely through interactions with other proteins specific to that disease or disorder. These interactions may affect not only energy metabolism but also other GAPDH functions.[6] For example, GAPDH interactions withbeta-amyloid precursor protein (betaAPP) could interfere with its function regarding thecytoskeleton or membrane transport, while interactions withhuntingtin could interfere with its function regarding apoptosis, nucleartRNA transport,DNA replication, andDNA repair. In addition, nuclear translocation of GAPDH has been reported inParkinson's disease (PD), and several anti-apoptotic PD drugs, such asrasagiline, function by preventing the nuclear translocation of GAPDH. It is proposed that hypometabolism may be one contributor to PD, but the exact mechanisms underlying GAPDH involvement in neurodegenerative disease remains to be clarified.[35] TheSNP rs3741916 in the5'UTR of theGAPDH gene may be associated with late onsetAlzheimer's disease.[36]
Rheb to sequester theGTPase during low glucose conditions;[7]
Siah1 to form a complex that translocates to the nucleus, where itubiquitinates and degrades nuclear proteins during nitrosative stress conditions;[7]
GAPDH's competitor of Siah protein enhances life (GOSPEL) to block GAPDH interaction with Siah1 and, thus, cell death in response to oxidative stress;[7]
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^Tarze A, Deniaud A, Le Bras M, Maillier E, Molle D, Larochette N, Zamzami N, Jan G, Kroemer G, Brenner C (April 2007). "GAPDH, a novel regulator of the pro-apoptotic mitochondrial membrane permeabilization".Oncogene.26 (18):2606–2620.doi:10.1038/sj.onc.1210074.PMID17072346.S2CID20291542.
^Brassard J, Gottschalk M, Quessy S (August 2004). "Cloning and purification of the Streptococcus suis serotype 2 glyceraldehyde-3-phosphate dehydrogenase and its involvement as an adhesin".Veterinary Microbiology.102 (1–2):87–94.doi:10.1016/j.vetmic.2004.05.008.PMID15288930.
^Kinoshita H, Uchida H, Kawai Y, Kawasaki T, Wakahara N, Matsuo H, Watanabe M, Kitazawa H, Ohnuma S, Miura K, Horii A, Saito T (June 2008). "Cell surface Lactobacillus plantarum LA 318 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) adheres to human colonic mucin".Journal of Applied Microbiology.104 (6):1667–1674.doi:10.1111/j.1365-2672.2007.03679.x.PMID18194256.S2CID22346488.
^Patel DK, Shah KR, Pappachan A, Gupta S, Singh DD (October 2016). "Cloning, expression and characterization of a mucin-binding GAPDH from Lactobacillus acidophilus".International Journal of Biological Macromolecules.91:338–346.doi:10.1016/j.ijbiomac.2016.04.041.PMID27180300.
^Hara MR, Agrawal N, Kim SF, Cascio MB, Fujimuro M, Ozeki Y, Takahashi M, Cheah JH, Tankou SK, Hester LD, Ferris CD, Hayward SD, Snyder SH, Sawa A (July 2005). "S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding".Nature Cell Biology.7 (7):665–674.doi:10.1038/ncb1268.PMID15951807.S2CID1922911.
^Agarwal AR, Zhao L, Sancheti H, Sundar IK, Rahman I, Cadenas E (November 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 –L898.doi:10.1152/ajplung.00219.2012.PMID23064950.
^Boradia VM, Raje M, Raje CI (December 2014). "Protein moonlighting in iron metabolism: glyceraldehyde-3-phosphate dehydrogenase (GAPDH)".Biochemical Society Transactions.42 (6):1796–1801.doi:10.1042/BST20140220.PMID25399609.
^Piszczatowski RT, Rafferty BJ, Rozado A, Tobak S, Lents NH (August 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):137–141.Bibcode:2014BBRC..451..137P.doi:10.1016/j.bbrc.2014.07.082.PMID25065746.
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^Ramos D, Pellín-Carcelén A, Agustí J, Murgui A, Jordá E, Pellín A, Monteagudo C (January 2015). "Deregulation of glyceraldehyde-3-phosphate dehydrogenase expression during tumor progression of human cutaneous melanoma".Anticancer Research.35 (1):439–444.PMID25550585.
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