Glyceraldehyde-3-phosphate dehydrogenase

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Glyceraldehyde-3-phosphate dehydrogenase
Glyceraldehyde-3-phosphate dehydrogenase
Surface / ribbon model of the GAPDH tetramer, according to PDB  1U8F

Existing structural data : 1j0x , 1u8f , 1znq , 2feh , 3gpd

Properties of human protein
Mass / length primary structure 334 aa; 35.9 kDa
Secondary to quaternary structure Homotetramer
Cofactor PRKCI, sulfate
Identifier
Gene names GAPDH  ; GAPD
External IDs
Enzyme classification
EC, category 1.2.1.12 oxidoreductase
Response type Phosphorylation
Substrate D -Glyceraldehyde-3-phosphate + phosphate + NAD (+)
Products 3- phospho - D -glyceroyl phosphate + NADH
Occurrence
Homology family CLU_030140_0_3
Parent taxon Creature
Orthologue
human chicken
Entrez 2597 374193
Ensemble ENSG00000111640 ENSGALG00000014442
UniProt P04406 P00356
Refseq (mRNA) NM_002046 NM_204305
Refseq (protein) NP_002037 NP_989636
Gene locus Chr 12: 6.51 - 6.52 Mb Chr 1: 80.09 - 80.09 Mb
PubMed search 2597 374193

The glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) is an enzyme of glycolysis , and therefore indispensable for all living beings. It catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate . During this reaction, an energy-rich phosphate bond is built up, which is transferred to ADP in the subsequent glycolysis step, creating ATP. In addition, an NAD + is converted to NADH / H + in the catalyzed reaction .

structure

Under normal cellular conditions, the cytoplasmic GAPDH exists primarily as a tetramer . This form consists of four identical 37 kDa subunits, each of which contains a single catalytic thiol group and is crucial for the catalytic function of the enzyme. The GAPDH in the cell nucleus has an elevated isoelectric point (pI) at pH 8.3 to 8.7. The cysteine ​​residue Cys152 in the active center of the enzyme is necessary for the induction of apoptosis by oxidative stress . In particular, post-translational modifications of the cytoplasmic GAPDH contribute to its functions outside of glycolysis.

Catalytic Mechanism

The following enzymological experiments have contributed to understanding the reaction mechanism of GAPDH :

  • GAPDH was rendered ineffective by alkylation with stoichiometric amounts of iodoacetate . The presence of carboxymethylcysteine in the hydrolyzate of the resulting alkylated enzyme shows that GAPDH has a cysteine ​​residue in the active site, the thiol group of which plays a role in the mechanism.
Alkylation of GAPDH
  • GAPDH quantitatively transfers a tritium from the C1 atom of GAP to NAD + . This proved that the reaction proceeds via hydride transfer.
Quantitative tritium transfer from substrate to NAD (+)
  • GAPDH catalyzes the exchange of 32 P between P i and acetyl phosphate. Such isotope exchange reactions indicate an acyl-enzyme as an intermediate product , ie that the acyl group forms a covalent complex with the enzyme, similar to the acyl-enzyme intermediate in the reaction mechanism of serine proteases .
Isotope exchange reaction with GAPDH

The main problem of the reaction is the splitting off of the H - ion ( hydride ion ) from the aldehyde group of the glyceraldehyde-3-phosphate. This is energetically unfavorable since the carbon in the aldehyde group has a partial positive charge. Through a sulfhydryl group of a cysteine of the enzyme is via a covalent bond , a nucleophilic residue introduced. The nucleophile attacks the carbonyl carbon of glyceraldehyde-3-phosphate (GAP) to form a thio-hemiacetal. After abstraction of the proton of the hydroxyl group on the C1 atom of GAP by a base, the hydride ion dissolves and a thioester is formed between the enzyme and the substrate (oxidation). This energy-rich compound is used in the subsequent reaction to bind an inorganic phosphate and to convert the intermediate product into 1,3-bisphosphoglycerate (phosphorylation). The hydride ion now binds to an NAD + that is non-covalently bound to a Rossmann fold , so that NADH / H + is formed. This loosens from the bond with the enzyme and is replaced by an NAD + because the positive charge of the NAD + polarizes the thioester intermediate in order to facilitate the attack by the orthophosphate. The catalyzed reaction is energetically very important. The resulting mixed anhydride of phosphoric and carboxylic acids is used in the subsequent reaction to form ATP . From the NADH / H + , in turn, ATP can be formed in the respiratory chain . Because cells only have a limited amount of NAD + , glycolysis would stop if the NADH produced in glycolysis were not continuously reoxidized. Therefore, NAD + is regenerated from the anaerobic breakdown of pyruvate.

Reaction mechanism of glyceraldehyde-3-phosphate dehydrogenase from E. coli .
Active center of glyceraldehyde-3-phosphate dehydrogenase of E. coli , according to PDB  1GAD .

The reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase is actually the sum of two processes: the oxidation of glyceraldehyde-3-phosphate to 3-phosphoglycerate by NAD + and the phosphorylation of 3-phosphoglycerate to 1,3-bisphosphoglycerate ( dehydration ).

Oxidation of glyceraldehyde-3-phosphate to 3-phosphoglycerate (upper reaction) and formation of 1,3-bisphosphoglycerate from 3-phosphoglycerate and orthophosphate (lower reaction)

The first reaction is thermodynamically quite favorable with a change in standard free enthalpy ( ) of about -50 kJ / mol. The second reaction is very unfavorable with a standard free enthalpy of about +50 kJ / mol. A high activation energy would therefore be required for the second reaction, which is why it would not proceed at a biologically significant rate.

Therefore, the two reactions must be coupled so that the overall reaction can take place. The coupling takes place via a thioester intermediate which is bound to the enzyme by a thioester bond. Thioesters are high-energy compounds found in many biochemical metabolic pathways. The free energy of the thioester intermediate is greater than that of the free carboxylic acid. This means that most of the free enthalpy that was released during the oxidation reaction is retained.

Features and function

All glycolysis steps take place in the cytosol, as does the reaction catalyzed by GAPDH. In red blood cells , GAPDH and several other glycolytic enzymes form certain enzyme complexes on the inside of the cell membrane . The process seems to be regulated by phosphorylation and oxygenation. The approach of several glycolytic enzymes is expected to greatly increase the overall rate of glucose breakdown. Recent studies have also shown that GAPDH is iron-dependently expressed on the outside of the cell membrane , where it plays a role in maintaining cellular iron homeostasis , particularly as a chaperone protein for labile heme in cells.

Transcription and apoptosis

GAPDH can activate transcription itself . The OCA-S transcription coactivator complex contains GAPDH and lactate dehydrogenase , two proteins that were previously thought to be only involved in metabolism. GAPDH moves between the cytosol and the cell nucleus and can thus combine the metabolic state with gene transcription.

In 2005, Hara et al. demonstrated that GAPDH initiates apoptosis. Initiation is mediated through GAPDH binding to DNA . The study showed that GAPDH is S -nitrosylated by nitric oxide in response to cell stress , causing it to bind to the protein SIAH1, a ubiquitin ligase . The complex moves into the nucleus where SIAH1 targets core proteins for breakdown, initiating a controlled shutdown of the cells. In a subsequent study, the group showed that selegiline , which was used clinically to treat Parkinson's disease , greatly reduced the apoptotic effects of GAPDH by preventing its S -nitrosylation and could therefore be used as a drug.

Metabolism switch

GAPDH acts as a reversible metabolic switch under oxidative stress. When cells are exposed to certain oxidizing agents , they need excessive amounts of the antioxidant cofactor NADPH . In the cytosol, NADPH is produced from the reduction of NADP + by several enzymes , three of which catalyze the first steps of the pentose phosphate pathway . Oxidant treatments cause inactivation of GAPDH. This inactivation redirects the metabolic flow from glycolysis to the pentose phosphate pathway so that the cell can produce more NADPH. Under stressful conditions, NADPH is required by some antioxidant systems, including glutaredoxin and thioredoxin , and is essential for the recycling of glutathione .

inhibition

The formation of the energy-rich anhydride can be inhibited by arsenic . AsO 4 3− binds to the GAPDH analogously to phosphate . NADH continues to be formed. However, the bond between the carboxylate resulting from the oxidation of the aldehyde and the arsenate is very unstable, so that the mixed anhydride breaks down to 3-phosphoglycerate . As a result, an energy-fixing step in glycolysis is skipped, which contributes to the poisonous effect of arsenic.

More functions

GAPDH also appears to be involved in vesicle transport from the endoplasmic reticulum (ER) to the Golgi apparatus , which is part of the transport route for secreted proteins. GAPDH has been found to be recruited by Rab2 into the ER's vesicular-tubular clusters, where it contributes to the formation of COPI vesicles . GAPDH is activated by tyrosine phosphorylation by tyrosine kinase Src .

Since the GAPDH gene is stably and constitutively expressed to a high degree in most tissues and cells, it is considered to be a household gene . For this reason, GAPDH is widely used by biological researchers as a load control for Western blotting and as a control for qPCR . However, researchers reported different GAPDH regulations under certain conditions. For example, the transcription factor MZF-1 has been shown to regulate the GAPDH gene. Therefore, the use of GAPDH as a cargo control must be carefully considered.

Clinical significance

cancer

GAPDH is overexpressed in several human cancers, such as skin melanoma , and its expression has a positive correlation with tumor progression. Its glycolytic and anti-apoptotic functions contribute to the proliferation and protection of tumor cells and promote carcinogenesis . In particular, GAPDH protects against telomere shortening induced by chemotherapeutic drugs that stimulate the sphingolipid ceramide . Conditions such as oxidative stress also impair GAPDH function and lead to cell aging and death. In addition, the removal of GAPDH induces senescence in tumor cells, which represents a novel therapeutic strategy for controlling tumor growth.

Neurodegenerative Disease

GAPDH is implicated in several neurodegenerative diseases and disorders, primarily through interactions with other proteins specific to that disease or disorder. These interactions can affect not only the energy metabolism but also other GAPDH functions. For example, GAPDH interactions with the β- amyloid precursor protein (BetaAPP) could impair the function of the cytoskeleton or membrane transport , while interactions with huntingtin could impair the function of apoptosis, nuclear tRNA transport, DNA replication and DNA repair could affect. In addition, GAPDH nuclear translocation (transport through the nuclear pores of the cell nucleus) has been reported in Parkinson's disease (PD), and several anti-apoptotic PD drugs, such as rasagiline, have been reported to work by preventing GAPDH nuclear translocation. It is believed that hypometabolism (decreased metabolic rate) contributes to Parkinson's disease. However, the exact mechanisms underlying GAPDH's involvement in neurodegenerative diseases have yet to be clarified. The SNP rs3741916 in the 5'-UTR of the GAPDH gene can be associated with late-onset Alzheimer's disease .

Genes, proteins and metabolites are linked to the respective articles. The metabolic pathway can be edited at WikiPathways :

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Glycolysis and Gluconeogenesis edit

Web links

Wikibooks: Glyceraldehyde-3-phosphate dehydrogenase  - learning and teaching materials
Wikibooks: Glycolysis  - Learning and teaching materials

Individual evidence

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