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Glyceraldehyde 3-phosphate dehydrogenase (abbreviated GAPDH) () is an
enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...
of about 37kDa that catalyzes the sixth step of glycolysis and thus serves to break down
glucose Glucose is a sugar with the Chemical formula#Molecular formula, molecular formula , which is often abbreviated as Glc. It is overall the most abundant monosaccharide, a subcategory of carbohydrates. It is mainly made by plants and most algae d ...
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 Apoptosis (from ) is a form of programmed cell death that occurs in multicellular organisms and in some eukaryotic, single-celled microorganisms such as yeast. Biochemistry, Biochemical events lead to characteristic cell changes (Morphology (biol ...
, ER-to-Golgi vesicle shuttling, and fast axonal, or axoplasmic transport. In sperm, a testis-specific isoenzyme GAPDHS is expressed.


Structure

Under normal cellular conditions,
cytoplasm The cytoplasm describes all the material within a eukaryotic or prokaryotic cell, enclosed by the cell membrane, including the organelles and excluding the nucleus in eukaryotic cells. The material inside the nucleus of a eukaryotic cell a ...
ic GAPDH exists primarily as a tetramer. This form is composed of four identical 37- kDa subunits containing a single catalytic thiol group each and critical to the enzyme's catalytic function. Nuclear GAPDH has increased isoelectric point (pI) of pH 8.3–8.7. Of note, the cysteine residue C152 in the enzyme's active site is required for the induction of apoptosis by oxidative stress. Notably, post-translational modifications of cytoplasmic GAPDH contribute to its functions outside of glycolysis. 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 in spermatozoa.


Reaction


Two-step conversion of G3P

The first reaction is the oxidation of glyceraldehyde 3-phosphate (G3P) at the position-1 (in the diagram it is shown as the 4th carbon from glycolysis), in which an aldehyde is converted into a
carboxylic acid In organic chemistry, a carboxylic acid is an organic acid that contains a carboxyl group () attached to an Substituent, R-group. The general formula of a carboxylic acid is often written as or , sometimes as with R referring to an organyl ...
(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 Phosphates are the naturally occurring form of the element phosphorus. In chemistry, a phosphate is an anion, salt, functional group or ester derived from a phosphoric acid. It most commonly means orthophosphate, a derivative of orthop ...
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 In biochemistry, phosphorylation is described as the "transfer of a phosphate group" from a donor to an acceptor. A common phosphorylating agent (phosphate donor) is ATP and a common family of acceptor are alcohols: : This equation can be writ ...
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

GAPDH uses covalent catalysis and general base catalysis to decrease the very large activation energy of the second step (phosphorylation) of this reaction.


1: Oxidation

First, a cysteine residue in the active site of GAPDH attacks the carbonyl group of G3P, creating a hemithioacetal intermediate (covalent catalysis). The hemithioacetal is deprotonated by a
histidine Histidine (symbol His or H) is an essential amino acid that is used in the biosynthesis of proteins. It contains an Amine, α-amino group (which is in the protonated –NH3+ form under Physiological condition, biological conditions), a carboxylic ...
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 a hydride ion. Next, an adjacent, tightly bound molecule of NAD+ accepts the hydride ion, forming NADH while the hemithioacetal is oxidized to a
thioester In organic chemistry, thioesters are organosulfur compounds with the molecular structure . They are analogous to carboxylate esters () with the sulfur in the thioester replacing oxygen in the carboxylate ester, as implied by the thio- prefix ...
. This thioester species is much higher in energy (less stable) than the
carboxylic acid In organic chemistry, a carboxylic acid is an organic acid that contains a carboxyl group () attached to an Substituent, R-group. The general formula of a carboxylic acid is often written as or , sometimes as with R referring to an organyl ...
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).


2: Phosphorylation

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.


Regulation

This protein may use the morpheein model of allosteric regulation.


Function


Metabolic

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.


Adhesion

One of the GAPDH moonlighting functions is its role in adhesion and binding to other partners. Bacterial GAPDH from Mycoplasma and '' Streptococcus'' and fungal GAPDH from '' Paracoccidioides brasiliensis'' are known to bind with the human extracellular matrix component and act in adhesion. GAPDH is found to be surface bound contributing in adhesion and also in competitive exclusion of harmful pathogens. GAPDH from '' Candida albicans'' is found to cell-wall associated and binds to Fibronectin and Laminin. GAPDH from probiotics species are known to bind human colonic
mucin Mucins () are a family of high molecular weight, heavily glycosylated proteins ( glycoconjugates) produced by epithelial tissues in most animals. Mucins' key characteristic is their ability to form gels; therefore they are a key component in ...
and ECM, resulting in enhanced colonization of probiotics in the human gut. Patel D. et al., showed that '' Lactobacillus acidophilus'' GAPDH binds with mucin, acting in adhesion.


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 Metabolism (, from ''metabolē'', "change") is the set of life-sustaining chemical reactions in organisms. The three main functions of metabolism are: the conversion of the energy in food to energy available to run cellular processes; the co ...
. 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 Apoptosis (from ) is a form of programmed cell death that occurs in multicellular organisms and in some eukaryotic, single-celled microorganisms such as yeast. Biochemistry, Biochemical events lead to characteristic cell changes (Morphology (biol ...
. 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 Parkinson's disease (PD), or simply Parkinson's, is a neurodegenerative disease primarily of the central nervous system, affecting both motor system, motor and non-motor systems. Symptoms typically develop gradually and non-motor issues become ...
, strongly reduces the apoptotic action of GAPDH by preventing its S-nitrosylation and might thus be used as a drug.


Metabolic switch

GAPDH acts as a reversible metabolic switch under oxidative stress. When cells are exposed to
oxidant An oxidizing agent (also known as an oxidant, oxidizer, electron recipient, or electron acceptor) is a substance in a redox chemical reaction that gains or "Electron acceptor, accepts"/"receives" an electron from a (called the , , or ''electr ...
s, 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 The endoplasmic reticulum (ER) is a part of a transportation system of the eukaryote, eukaryotic cell, and has many other important functions such as protein folding. The word endoplasmic means "within the cytoplasm", and reticulum is Latin for ...
(ER) to the
Golgi apparatus The Golgi apparatus (), also known as the Golgi complex, Golgi body, or simply the Golgi, is an organelle found in most eukaryotic Cell (biology), cells. Part of the endomembrane system in the cytoplasm, it protein targeting, packages proteins ...
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 In biochemistry, phosphorylation is described as the "transfer of a phosphate group" from a donor to an acceptor. A common phosphorylating agent (phosphate donor) is ATP and a common family of acceptor are alcohols: : This equation can be writ ...
by Src.


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. GAPDH has been described to exhibit higher order multifunctionality in the context of maintaining cellular iron homeostasis, specifically as a chaperone protein for labile heme within cells. 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.


Use 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. Hypoxia also strongly upregulates GAPDH. Therefore, the use of GAPDH as loading control has to be considered carefully.


Cellular distribution

All steps of glycolysis take place in the cytosol and so does the reaction catalysed by GAPDH. In red blood cells, GAPDH and several other glycolytic enzymes assemble in complexes on the inside of the
cell membrane The cell membrane (also known as the plasma membrane or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of a cell from the outside environment (the extr ...
. 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. 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.


Clinical significance


Cancer

GAPDH is overexpressed in multiple human cancers, such as cutaneous melanoma, and its expression is positively correlated with tumor progression. Its glycolytic and antiapoptotic functions contribute to proliferation and protection of tumor cells, promoting tumorigenesis. Notably, GAPDH protects against telomere shortening induced by chemotherapeutic drugs that stimulate the
sphingolipid Sphingolipids are a class of lipids containing a backbone of sphingoid bases, which are a set of aliphatic amino alcohols that includes sphingosine. They were discovered in brain extracts in the 1870s and were named after the mythological sp ...
ceramide Ceramides are a family of waxy lipid molecules. A ceramide is composed of sphingosine and a fatty acid joined by an amide bond. Ceramides are found in high concentrations within the cell membrane of Eukaryote, eukaryotic cells, since they are co ...
. Meanwhile, conditions like oxidative stress impair GAPDH function, leading to cellular aging and death. Moreover, depletion of GAPDH has managed to induce senescence in tumor cells, thus presenting a novel therapeutic strategy for controlling tumor growth.


Neurodegeneration

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. For example, GAPDH interactions with beta-amyloid precursor protein (betaAPP) could interfere with its function regarding the
cytoskeleton The cytoskeleton is a complex, dynamic network of interlinking protein filaments present in the cytoplasm of all cells, including those of bacteria and archaea. In eukaryotes, it extends from the cell nucleus to the cell membrane and is compos ...
or membrane transport, while interactions with huntingtin could interfere with its function regarding apoptosis, nuclear tRNA transport,
DNA replication In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all life, living organisms, acting as the most essential part of heredity, biolog ...
, and
DNA repair DNA repair is a collection of processes by which a cell (biology), cell identifies and corrects damage to the DNA molecules that encode its genome. A weakened capacity for DNA repair is a risk factor for the development of cancer. DNA is cons ...
. In addition, nuclear translocation of GAPDH has been reported in
Parkinson's disease Parkinson's disease (PD), or simply Parkinson's, is a neurodegenerative disease primarily of the central nervous system, affecting both motor system, motor and non-motor systems. Symptoms typically develop gradually and non-motor issues become ...
(PD), and several anti-apoptotic PD drugs, such as
rasagiline Rasagiline, sold under the brand name Azilect among others, is a medication which is used in the treatment of Parkinson's disease. It is used as a monotherapy to treat symptoms in early Parkinson's disease or as an adjunct therapy in more advan ...
, 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. The SNP rs3741916 in the 5' UTR of the ''GAPDH'' gene may be associated with late onset
Alzheimer's disease Alzheimer's disease (AD) is a neurodegenerative disease and the cause of 60–70% of cases of dementia. The most common early symptom is difficulty in remembering recent events. As the disease advances, symptoms can include problems wit ...
.


Interactions


Protein binding partners

GAPDH participates in a number of biological functions through its protein–protein interactions with: * tubulin to facilitate microtubule bundling; *
actin Actin is a family of globular multi-functional proteins that form microfilaments in the cytoskeleton, and the thin filaments in muscle fibrils. It is found in essentially all eukaryotic cells, where it may be present at a concentration of ...
to facilitate actin polymerization; * VDAC1 to induce mitochondrial membrane permeabilization (MMP) and apoptosis; * Inositol 1,4,5-trisphosphate receptor to regulate intracellular Ca2+ signaling; * Oct-1 to form the coactivator complex OCA-S, which is required for histone H2B synthesis during S phase of the
cell cycle The cell cycle, or cell-division cycle, is the sequential series of events that take place in a cell (biology), cell that causes it to divide into two daughter cells. These events include the growth of the cell, duplication of its DNA (DNA re ...
; * p22 to aid microtubule organization; * Rab2 to facilitate
endoplasmic reticulum The endoplasmic reticulum (ER) is a part of a transportation system of the eukaryote, eukaryotic cell, and has many other important functions such as protein folding. The word endoplasmic means "within the cytoplasm", and reticulum is Latin for ...
(ER)– golgi transport; *
Transferrin Transferrins are glycoproteins found in vertebrates which bind and consequently mediate the transport of iron (Fe) through blood plasma. They are produced in the liver and contain binding sites for two Iron(III), Fe3+ ions. Human transferrin is ...
on the surface of diverse cells and in extracellular fluid; * Lactate dehydrogenase; * Lactoferrin; * Apurinic/apyrimidinic endonuclease ( APE1), thus converting oxidized APE1 to its reduced form, to restart its
endonuclease In molecular biology, endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain (namely DNA or RNA). Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically (with regard to sequence), while man ...
activity; * Promyelocytic leukaemia protein (PML) in an
RNA Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself (non-coding RNA) or by forming a template for the production of proteins (messenger RNA). RNA and deoxyrib ...
-dependent fashion; * Rheb to sequester the
GTPase GTPases are a large family of hydrolase enzymes that bind to the nucleotide guanosine triphosphate (GTP) and hydrolyze it to guanosine diphosphate (GDP). The GTP binding and hydrolysis takes place in the highly conserved P-loop "G domain", a ...
during low glucose conditions; * Siah1 to form a complex that translocates to the nucleus, where it ubiquitinates and degrades nuclear proteins during nitrosative stress conditions; * GAPDH's competitor of Siah protein enhances life (GOSPEL) to block GAPDH interaction with Siah1 and, thus, cell death in response to oxidative stress; * p300/ CREB binding protein (CBP), which acetylates GAPDH and, in turn, enhances the acetylation of additional apoptotic targets; * skeletal muscle-specific Ca2+/calmodulin-dependent protein kinase; * Akt; * Beta-amyloid precursor protein (betaAPP); * Huntingtin. * GAPDH can self-associate into homotypic oligomers/aggregates


Nucleic acid binding partners

GAPDH binds to single-stranded RNA and DNA and a number of nucleic acid binding partners have been identified: * tRNA, * Hepatitis A viral RNA, * Hepatitis B viral RNA, *
Hepatitis C Hepatitis C is an infectious disease caused by the hepatitis C virus (HCV) that primarily affects the liver; it is a type of viral hepatitis. During the initial infection period, people often have mild or no symptoms. Early symptoms can include ...
viral RNA, * HPIV3, *
lymphokine Lymphokines are a subset of cytokines that are produced by a type of immune cell known as a lymphocyte. They are protein mediators typically produced by T cells to direct the immune system response by signaling between its cells. Lymphokines have ...
mRNA, * IFN-γ mRNA, * JEV mRNA, and * telomeric DNA.


Inhibitors

* Desmethylselegiline * Koningic acid *
Rasagiline Rasagiline, sold under the brand name Azilect among others, is a medication which is used in the treatment of Parkinson's disease. It is used as a monotherapy to treat symptoms in early Parkinson's disease or as an adjunct therapy in more advan ...
* Selegiline


Interactive pathway map


References


Further reading

* *
diagram of the GAPDH reaction mechanism
from Lodish MCB at NCBI bookshelf
similar diagram
from Alberts The Cell at NCBI bookshelf


External links


PDBe-KB
provides an overview of all the structure information available in the PDB for Human Glyceraldehyde-3-phosphate dehydrogenase {{DEFAULTSORT:Glyceraldehyde 3-Phosphate Dehydrogenase EC 1.2.1 Glycolysis enzymes Glycolysis