Glyceraldehyde 3-phosphate dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase
GAPDH with NAD+ and Pi bound to the active site. PDB rendering based on 2CZC.
Available structures PDB Ortholog search: PDBe, RCSB List of PDB id codes 1U8F, 1ZNQ, 3GPD
Identifiers
Symbols	GAPDH; G3PD; GAPD
External IDs	OMIM: 138400 MGI: 3646088 HomoloGene: 107053 ChEMBL: 2284 GeneCards: GAPDH Gene
EC number	1.2.1.12
Gene Ontology Molecular function • glyceraldehyde-3-phosphate dehydrogenase (NAD+) (phosphorylating) activity • protein binding • microtubule binding • peptidyl-cysteine S-nitrosylase activity • NADP binding • NAD binding Cellular component • nucleus • cytoplasm • lipid particle • cytosol • plasma membrane • microtubule cytoskeleton • ribonucleoprotein complex • perinuclear region of cytoplasm • extracellular vesicular exosome Biological process • microtubule cytoskeleton organization • carbohydrate metabolic process • glucose metabolic process • gluconeogenesis • glycolysis • negative regulation of translation • peptidyl-cysteine S-trans-nitrosylation • small molecule metabolic process • protein stabilization • neuron apoptotic process • cellular response to interferon-gamma Sources: Amigo / QuickGO
RNA expression pattern
More reference expression data
Orthologs
Species	Human	Mouse
Entrez	2597	14433
Ensembl	ENSG00000111640	ENSMUSG00000057666
UniProt	P04406	P16858
RefSeq (mRNA)	NM_001256799	NM_008084
RefSeq (protein)	NP_001243728	NP_032110
Location (UCSC)	Chr 12: 6.64 – 6.65 Mb	Chr 6: 125.16 – 125.17 Mb
PubMed search
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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
Identifiers
Symbol	Gp_dh_N
Pfam	PF00044
Pfam clan	CL0063
InterPro	IPR020828
PROSITE	PDOC00069
SCOP	1gd1
SUPERFAMILY	1gd1
Available protein structures: Pfam structures PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

Glyceraldehyde 3-phosphate dehydrogenase, C-terminal domain
crystal structure of glyceraldehyde-3-phosphate dehydrogenase from pyrococcus horikoshii ot3
Identifiers
Symbol	Gp_dh_C
Pfam	PF02800
Pfam clan	CL0139
InterPro	IPR020829
PROSITE	PDOC00069
SCOP	1gd1
SUPERFAMILY	1gd1
Available protein structures: Pfam structures PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

Glyceraldehyde 3-phosphate dehydrogenase (abbreviated as GAPDH or less commonly as G3PDH) (EC 1.2.1.12) 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,^[1] ER to Golgi vesicle shuttling, and fast axonal, or axoplasmic transport.^[2]

Metabolic function

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
	NAD+ + Pi	NADH + H+
	NAD+ + Pi	NADH + H+

Compound C00118 at KEGG Pathway Database. Enzyme 1.2.1.12 at KEGG Pathway Database. Reaction R01063 at KEGG Pathway Database. Compound C00236 at KEGG Pathway Database.

Two-step conversion of glyceraldehyde-3-phosphate

The first reaction is the oxidation 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-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 great, 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.

Enzyme regulation

This protein may use the morpheein model of allosteric regulation.^[3]

Interactive pathway map

Click on genes, proteins and metabolites below to link to respective articles. ^{[§ 1]}

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Glycolysis and Gluconeogenesis edit

1. ↑ The interactive pathway map can be edited at WikiPathways: "GlycolysisGluconeogenesis_WP534". 

Additional functions

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. 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

Zheng et al. discovered in 2003 that 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. ^[4]

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.^[5] 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.^[6]

Metabolic switch

GAPDH acts as reversible metabolic switch under oxidative stress.^[7] 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.^[8] 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.^[9]

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.^[10] Bringing several glycolytic enzymes close to each other is expected to greatly increase 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, researchers have reported different regulation of GAPDH under specific conditions.^[11] Therefore, the use of GAPDH as loading control has to be controlled carefully.

References

Further reading

v t e PDB gallery 1j0x: Crystal structure of the rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH)  1u8f: Crystal Structure Of Human Placental Glyceraldehyde-3-Phosphate Dehydrogenase At 1.75 Resolution  1znq: Crsytal Structure of Human Liver GAPDH

v t e Glycolysis Metabolic Pathway

Glucose Hexokinase Glucose 6-phosphate Glucose-6-phosphate isomerase Fructose 6-phosphate phosphofructokinase-1 Fructose 1,6-bisphosphate Fructose-bisphosphate aldolase ATP ADP ATP ADP Dihydroxyacetone phosphate Glyceraldehyde 3-phosphate Triosephosphate isomerase Glyceraldehyde 3-phosphate Glyceraldehyde-3-phosphate dehydrogenase 1,3-Bisphosphoglycerate NAD+ + Pi NADH + H+ + 2 2 Phosphoglycerate kinase 3-Phosphoglycerate Phosphoglycerate mutase 2-Phosphoglycerate Phosphopyruvate hydratase(Enolase) Phosphoenolpyruvate Pyruvate kinase Pyruvate ADP ATP H2O ADP ATP 2 2 2 2 M: MET mt, k, c/g/r/p/y/i, f/h/s/l/o/e, a/u, n, m k, cgrp/y/i, f/h/s/l/o/e, au, n, m, epon m (A16/C10), i (k, c/g/r/p/y/i, f/h/s/o/e, a/u, n, m)