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From Proteopedia
Contents |
Glyceraldehyde 3-Phosphate Dehydrogenase
Introduction
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| 1vc2, resolution 2.60Å () | |||||||||
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| Activity: | Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating), with EC number 1.2.1.12 | ||||||||
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| Resources: | FirstGlance, OCA, RCSB, PDBsum, TOPSAN | ||||||||
| Coordinates: | save as pdb, mmCIF, xml | ||||||||
Glyceraldehyde 3-Phosphate dehydrogenase (GAPDH) is an Oxidoreductase enzyme involved in many important biochemical reactions and belongs to the Aldehyde Dehydrogenase superfamily[1]. GAPDH has been divided into two large classes and subsequent subclasses. Class 1 consists of eukaryotes and eubacteria whereas class 2 contains archael GAPDHs [2]. It is involved in glycolysis, gluconeogenesis and in the case of photosynthetic organism, the carbon reduction cycle [2]. This protein is responsible for catalyzing the conversion of glyceraldeyde 3-Phosphate into 1,3-Biphosphoglycerate in a two step coupled mechanism. This conversion occurs during step 6 or the beginning of the "payoff phase" of glycolysis (the second half of the entire process) in which ATP and NADH is produced. A total of 2 NADH and 4 ATP are produced during this phase for a net gain of 2 NADH and 2 ATP for the entire glycolysis pathway per glucose.A number of disease causing parasites particularly protists such as Trypanosoma brucei rely on glycolysis to provide the energy for their biochemical functions. Due to this, such parasites will heavily rely on GAPDH due to its intrinsic role in the glycolytic pathway and therefore targeting this enzyme complex can be a promising field of research. Subsequent pharmaceutical drug development and testing can then be conducted to provide protection against deadly viruses and disease. This protein has also been linked as acting as a nitric oxide sensor and plays roles in transcriptional regulation of genes along with translational silencing [3]. Although the exact mechanism of these roles are unknown at the moment it is believed that posttranslational modifications play a part in determining these other functions[3].
Structure & Function
Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH)has carefully been studied in a number of bacterial, parasitic and mammalian species and it has been found that it exists as homotetrameric protein [4]. Each subunit within the protein is 38,151Da (tetramer is 152.4 kDa)and contains seven alpha helices and two beta sheets one of which has seven strands and the other with eight[4] [5]. Two anion binding sites have been found where the two phosphates involved in the reaction will be bound during catalysis. One site is labeled "Pi" and is the location where the inorganic phosphate involved will bind and the other has been labeled "Ps" which is where the C-3 phosphate of Gylceraldeyhde 3-Phosphate will bind [4]. Further experimentation has shown that the "Ps" site has been conserved in numerous GAPDH complexes and that the former may involve two possible sites in which the second or new "Pi" site is located 2.9 Angstroms from the primary "Pi" site[4].
The enzyme contains a functional NAD+ group which functions as a hydrogen acceptor during the course of the reaction which is bound to a Rossman fold. During the catalysis of glyceraldehyde 3-phosphate to 1,3-biphosphoglycerate a hydride ion is enzymatically transferred from the aldehyde group of glyceraldehyde 3-phosphate to the nicotinamide ring of NAD+ reducing it to NADH[4]. The active site of GAPDH contains a cysteine (Cys149) residue which reacts with the glyceraldehyde 3-phosphate molecule through its -SH group. The substrate is covalently bound during the reaction through its aldehyde group to the -SH group of the cysteine residue and the resulting reaction produces a thiohemiacetal intermediate [4]. Note that this reaction occurs through acid base catalysis with aid of a histidine residue (His176).The of the molecule is illustrated to the right.
A simplified illustration of the net reaction is as follows:
D-glyceraldehyde 3-phosphate + phosphate + NAD+ ---------> 1,3 biphospho-D-glycerate + NADH + H+
The inorganic phosphate (Pi) that is involved in the reaction functions to attack by phosphorolysis the thioester intermediate that is formed by the substrate on the cysteine reside after NAD+ has been reduced [4]. The attack by Pi on the carbonyl carbon of C1 is simultaneously followed by the replacement of bound NADH for NAD+ so another turn of the cycle can now commence. The final product is released as 1,3 bisphosphoglycerate in which the second Pi molecule has been incorporated.
Active Site in Detail
Relations to Medicine
Strong structural analysis and in depth studies of parasitic protozoans has allowed the determination of conservation and differences between the parasite and human forms of GAPDH. Trypanosoma cruzi, a protozoan parasite is responsible for causing Chagas' disease in approximately 16-18 million people from southern and central America[7]. This parasite is responsible for causing up to 45,000 deaths per year and can cause severe complications such as neurological disorders and chronic cardiopathy[7]. Although there are preexisting drugs on the market that do show some effectiveness, they are all known to cause severe side effects which defeats the purpose of their production. The role of Glyceraldehyde 3-Phosphate Dehydrogenase in this case is that it has been found that the bloodstream forms of the related protozoan T. bruceiare shown to lack a functional tricarboxylic acid cycle and thus its ultimate ATP source must come from glycolysis[7]. It is this finding that has intrigued scientists to find solutions to the diseases caused by these protozoans. At the moment the most relevant target is the binding site of the adenosine ring of the NAD+ cofactor. It has been studied in great detail and its differences from the human form have been well recorded to allow the outcome of a possible solution[7]. The NAD+ binding region is homologous in both T. brucei and T. cruzi and this site is therefore a suitable target for inhibitors that will provide a solution to Chagas' disease[7]. This step will be advantageous because several adenosine analogues have already been designed and applied as selective and competitive inhibitors to trypanosomatid GAPDHs and have shown to stop the growth of T. brucei within the bloodstream[8].
Research relating oxidative stress to GAPDH has also been conducted and shows very promising results. Nitration of tyrosine residues is a sign that oxidative stress is occuring, and nitration of tyrosine has been known to be linked to neurodegenerative disorders and cancer[3]. It has been documented that nitration of the cysteine (Cys149) residue within the active site of the GAPDH enzyme is responsible for causing loss of enzymatic activity[3]. This loss of enzymatic activity is due to the nitration of two tyrosine residues (Tyr311 and Tyr317) which are in close proximity to the active site cysteine. The ultimate result of this nitration is that it causes the loss of affinity for NAD+ and therefore a loss of NAD+ binding[3]. Other studies have shown that GAPDH is involved in the death of neuron cells as a result of oxidative stress, and that GAPDH is found to be deposited in protein aggregates containing disulfide bonds[9]. Oxidative stress causes the oligomerization of proteins such as GAPDH which result from the formation of intramolecular disulfide bonds. The accumulation of GAPDH as insoluble aggregates has been linked to diseases such as Alzheimer's disease and Parkinson's disease, as insoluble aggregates have indeed been found in both[9]
