Glyceraldehyde 3-phosphate dehydrogenase
Encyclopedia
Glyceraldehyde 3-phosphate dehydrogenase (abbreviated as GAPDH or less commonly as G3PDH) 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
, and ER to Golgi vesicle shuttling
.
as 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 cytosol
of eukaryotic cells. Glyceraldehyde 3-phosphate is converted to D-glycerate 1,3-bisphosphate in two coupled steps. The first is favourable and allows the second unfavourable step to occur.
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.
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, 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.
, 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.
. 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.
. 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 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
.
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.
. 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. Therefore, the use of GAPDH as loading control has to be controlled carefully.
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
of ~37kDa that catalyzes the sixth step of glycolysis
Glycolysis
Glycolysis is the metabolic pathway that converts glucose C6H12O6, into pyruvate, CH3COCOO− + H+...
and thus serves to break down glucose
Glucose
Glucose is a simple sugar and an important carbohydrate in biology. Cells use it as the primary source of energy and a metabolic intermediate...
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
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
activation, initiation of apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
, and ER to Golgi vesicle shuttling
COPI
COPI is a protein complex that coats vesicles transporting proteins from the cis end of the Golgi complex back to the rough endoplasmic reticulum , where they were originally synthesized and between golgi compartments. This type of transport is termed as retrograde transport...
.
Metabolic function
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyses the conversion of glyceraldehyde 3-phosphateGlyceraldehyde 3-phosphate
Glyceraldehyde 3-phosphate, also known as triose phosphate or 3-phosphoglyceraldehyde and abbreviated as G3P, GADP, GAP, TP, GALP or PGAL, is a chemical compound that occurs as an intermediate in several central metabolic pathways of all organisms...
as the name indicates. This is the 6th step of the breakdown of glucose (glycolysis
Glycolysis
Glycolysis is the metabolic pathway that converts glucose C6H12O6, into pyruvate, CH3COCOO− + H+...
), an important pathway of energy and carbon molecule supply located in the cytosol
Cytosol
The cytosol or intracellular fluid is the liquid found inside cells, that is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondrion into compartments....
of eukaryotic cells. Glyceraldehyde 3-phosphate is converted to D-glycerate 1,3-bisphosphate in two coupled steps. The first is favourable and allows the second unfavourable step to occur.
Overall reaction catalyzed
Two-step conversion of glyceraldehyde-3-phosphate
The first reaction is the oxidation of glyceraldehyde 3-phosphateGlyceraldehyde 3-phosphate
Glyceraldehyde 3-phosphate, also known as triose phosphate or 3-phosphoglyceraldehyde and abbreviated as G3P, GADP, GAP, TP, GALP or PGAL, is a chemical compound that occurs as an intermediate in several central metabolic pathways of all organisms...
at the carbon 1 position (the 4th carbon from glycolysis which is shown in the diagram), in which an aldehyde
Aldehyde
An aldehyde is an organic compound containing a formyl group. This functional group, with the structure R-CHO, consists of a carbonyl center bonded to hydrogen and an R group....
is converted into a carboxylic acid
Carboxylic acid
Carboxylic acids are organic acids characterized by the presence of at least one carboxyl group. The general formula of a carboxylic acid is R-COOH, where R is some monovalent functional group...
(ΔG°'=-50 kJ/mol (-12kcal/mol)) and NAD+ is simultaneously reduced endergonically to NADH. The energy released by this highly exergonic
Exergonic
Exergonic means "releasing energy in the form of work". By thermodynamic standards, work, a form of energy, is defined as moving from the system to the surroundings...
oxidation reaction drives the endergonic
Endergonic
Endergonic means "absorbing energy in the form of work." Endergonic reactions are not spontaneous...
second reaction (ΔG°'=+50 kJ/mol (+12kcal/mol)), in which a molecule of inorganic phosphate
Phosphate
A phosphate, an inorganic chemical, is a salt of phosphoric acid. In organic chemistry, a phosphate, or organophosphate, is an ester of phosphoric acid. Organic phosphates are important in biochemistry and biogeochemistry or ecology. Inorganic phosphates are mined to obtain phosphorus for use in...
is transferred to the GAP intermediate to form a product with high phosphoryl-transfer potential: 1,3-bisphosphoglycerate
1,3-Bisphosphoglycerate
1,3-Bisphosphoglyceric acid is a 3-carbon organic molecule present in most, if not all, living organisms. It primarily exists as a metabolic intermediate in both glycolysis during respiration and the Calvin cycle during photosynthesis...
(1,3-BPG). This is an example of phosphorylation
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
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 cysteineCysteine
Cysteine is an α-amino acid with the chemical formula HO2CCHCH2SH. It is a non-essential amino acid, which means that it is biosynthesized in humans. Its codons are UGU and UGC. The side chain on cysteine is thiol, which is polar and thus cysteine is usually classified as a hydrophilic amino acid...
residue in the active site of GAPDH attacks the carbonyl group of GAP, creating a hemithioacetal
Hemithioacetal
Hemithioacetal, also known as hemimercaptal, is an organic functional group with the general formula RCHSR. They form in a spontaneous reaction between a thiol and an aldehyde. Since the formerly carbonyl carbon bears four different substituents, hemiacetals are chiral. Hemithioacetals are usually...
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
Thioester
Thioesters are compounds with the functional group C-S-CO-C. They are the product of esterification between a carboxylic acid and a thiol. Thioesters are widespread in biochemistry, the best-known derivative being acetyl-CoA.-Synthesis:...
intermediate using a molecule of water. This thioester species is much higher in energy than the carboxylic acid
Carboxylic acid
Carboxylic acids are organic acids characterized by the presence of at least one carboxyl group. The general formula of a carboxylic acid is R-COOH, where R is some monovalent functional group...
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, for a living organism). Donation of the hydride ion by the hemithioacetal is facilitated by its deprotonation by a histidine
Histidine
Histidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...
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
Thiol
In organic chemistry, a thiol is an organosulfur compound that contains a carbon-bonded sulfhydryl group...
group of the enzyme's cysteine residue.
Additional functions
GAPDH, like many other enzymes, has multiple functions. In addition to catalysing the 6th step of glycolysisGlycolysis
Glycolysis is the metabolic pathway that converts glucose C6H12O6, into pyruvate, CH3COCOO− + H+...
, 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.
Transcription and apoptosis
Zheng et al. discovered in 2003 that GAPDH can itself activate transcriptionTranscription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
. The OCA-S transcriptional coactivator complex contains GAPDH and lactate dehydrogenase
Lactate dehydrogenase
Lactate dehydrogenase is an enzyme present in a wide variety of organisms, including plants and animals.Lactate dehydrogenases exist in four distinct enzyme classes. Two of them are cytochrome c-dependent enzymes, each acting on either D-lactate or L-lactate...
, two proteins previously only thought to be involved in metabolism
Metabolism
Metabolism is the set of chemical reactions that happen in the cells of living organisms to sustain life. These processes allow organisms to grow and reproduce, maintain their structures, and respond to their environments. Metabolism is usually divided into two categories...
. GAPDH moves between the cytosol
Cytosol
The cytosol or intracellular fluid is the liquid found inside cells, that is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondrion into compartments....
and the nucleus
Cell nucleus
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
and may thus link the metabolic state to gene transcription.
In 2005, Hara et al. showed that GAPDH initiates apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
. This is not a third function, but can be seen as an activity mediated by GAPDH binding to DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
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
Ubiquitin ligase
A ubiquitin ligase is a protein that in combination with an E2 ubiquitin-conjugating enzyme causes the attachment of ubiquitin to a lysine on a target protein via an isopeptide bond; the E3 ubiquitin ligase targets specific protein substrates for degradation by the proteasome...
. 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
Parkinson's disease is a degenerative disorder of the central nervous system...
, 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 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 pathwayPentose phosphate pathway
The pentose phosphate pathway is a process that generates NADPH and pentoses . There are two distinct phases in the pathway. The first is the oxidative phase, in which NADPH is generated, and the second is the non-oxidative synthesis of 5-carbon sugars...
. 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
Glutaredoxin
Glutaredoxins are small redox enzymes of approximately one hundred amino-acid residues that use glutathione as a cofactor. Glutaredoxins are oxidized by substrates, and reduced non-enzymatically by glutathione. In contrast to thioredoxins, which are reduced by thioredoxin reductase, no...
and thioredoxin
Thioredoxin
Thioredoxin is a class of small redox proteins known to be present in all organisms. It plays a role in many important biological processes. In humans, it is encoded by the TXN gene. Loss-of-function mutation of either of the two human thioredoxin genes is lethal at the four-cell stage of the...
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 reticulumEndoplasmic reticulum
The endoplasmic reticulum is an organelle of cells in eukaryotic organisms that forms an interconnected network of tubules, vesicles, and cisternae...
(ER) to the Golgi apparatus
Golgi apparatus
The Golgi apparatus is an organelle found in most eukaryotic cells. It was identified in 1898 by the Italian physician Camillo Golgi, after whom the Golgi apparatus is named....
which is part of shipping route for secreted proteins. It was found that GAPDH is recruited by rab2
Rab (G-protein)
The Rab family of proteins is a member of the Ras superfamily of monomeric G proteins. Approximately 70 types of Rabs have now been identified in humans. Rab GTPases regulate many steps of membrane traffic, including vesicle formation, vesicle movement along actin and tubulin networks, and membrane...
to the vesicular-tubular clusters of the ER where it helps to form COP 1 vesicles
COPI
COPI is a protein complex that coats vesicles transporting proteins from the cis end of the Golgi complex back to the rough endoplasmic reticulum , where they were originally synthesized and between golgi compartments. This type of transport is termed as retrograde transport...
. GAPDH is activated via tyrosine
Tyrosine
Tyrosine or 4-hydroxyphenylalanine, is one of the 22 amino acids that are used by cells to synthesize proteins. Its codons are UAC and UAU. It is a non-essential amino acid with a polar side group...
phosphorylation
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
by Src
Src (gene)
Proto-oncogene tyrosine-protein kinase Src is an enzyme that in humans is encoded by the SRC gene.Src is a proto-oncogene encoding a tyrosine kinase originally discovered by J. Michael Bishop and Harold E. Varmus, for which they won the 1989 Nobel Prize in Physiology or Medicine. It belongs to a...
.
Cellular location
All steps of glycolysis take place in the cytosolCytosol
The cytosol or intracellular fluid is the liquid found inside cells, that is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondrion into compartments....
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
Cell membrane
The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...
. 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.
Miscellaneous
Because the GAPDH gene is often stably and constitutively expressed at high levels in most tissues and cells, it is considered a housekeeping geneHousekeeping gene
A housekeeping gene is typically a constitutive gene that is required for the maintenance of basic cellular function, and are found in all cells of an organism. Although some housekeeping genes are expressed at relatively constant levels , other housekeeping genes may vary depending on...
. For this reason, GAPDH is commonly used by biological researchers as a loading control for western blot
Western blot
The western blot is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide...
and as a control for RT-PCR. However, researchers have reported different regulation of GAPDH under specific conditions. Therefore, the use of GAPDH as loading control has to be controlled carefully.
Further reading
- diagram of the GAPDH reaction mechanism from Lodish MCB at NCBI bookshelf
- similar diagram from Alberts The Cell at NCBI bookshelf