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== '''Glyceraldehyde 3-Phosphate Dehydrogenase'''== == Introduction == {{STRUCTURE_1vc2| PDB=1vc2 | Scene =Sandbox_160/Newscene/1'>active site }} Glyceraldehyde 3-Phosphate dehydrogenase (GAPDH) is an Oxidoreductase enzyme involved in many important biochemical reactions and belongs to the Aldehyde Dehydrogenase superfamily<ref name="ref 6">PMID:9497334 </ref>. GAPDH has been divided into two large classes and subsequent subclasses. Class 1 consists of eukaryotes and eubacteria whereas class 2 contains archael GAPDHs <ref name="ref 2">PMID:11846565 </ref>. It is involved in glycolysis, gluconeogenesis and in the case of photosynthetic organism, the carbon reduction cycle <ref name="ref 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 <ref name="ref 3">PMID:20014444 </ref>. == 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 <ref name="reference 1"/>. 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<ref name="reference 1"/> <ref name="ref 5">PMID:15953771</ref>. 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 <ref name="reference 1"/>. 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<ref name="reference 1"/>. 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<ref name="reference 1">PMID:19243605 </ref>. 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 <ref name="reference 1"/>. Note that this reaction occurs through acid base catalysis with aid of a histidine residue (His176).The <scene name='Sandbox_160/Newscene/1'>active site</scene> 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 <ref name="reference 1"/>. 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 == [[Image:1vc2 image.png | left|thumb|upright=2 |Figure 1. Illustration created using PDB software highlighting the NAD+ ligand and Cysteine and Histidine residues within the active site of 1vc2.]] Once Glyceraldehyde 3-phophate comes into contact with the active site it forms a hydrogen bond through its C2 hydroxyl group to Cys149N (Cys149 colored green. The C1 hydroxyl group of the substrate binds to His176NE2 (His176 colored teal)<ref name="ref 4">PMID:10191140 </ref>. Additional hydrogen bonds to the phosphate group of the substrate from additional residues such as Thr1790G1, Arg231NH1(these two residues are not highlighted in the illustration below) along with N7N and 02'N of the NAD+ moeity (NAD colored pink) help stabilize the molecule during the course of the reaction in the active site <ref name="ref 4"/>. The nicotinamide ring of the NAD+ ligand is responsible for orienting the hydrogen atom at C1 towards itself which allows for easier transfer in producing in reducing NAD+ to NADH. The positive charge that arises when NADH is formed helps to stabilize the negatively charged oxygen on the carbonyl group that is present in the active site. Usually the holoenzyme form of GAPDH is found to contain NAD+ in two or three of its active sites, however in the protozoan parasite ''Cryptosporidium parvum'' NAD+ is found to be bound in each subunit<ref name="reference 1"/>. Binding of NAD+ to its designated subunit is known to cause a conformational change in that individual subunit and cause distances between residues to change. This is seen in ''E. coli'' in which it is known that upon the binding of the ligand (NAD+), the distance between the thiol group of the cysteine residue and the NE2 of the histidine atom increases within the active site<ref name="reference 1"/>. This movement and subsequent differences between residues in the active site or at other locations is thought to occur to support the NAD+ molecule and allow for hydrophobic interactions with the its ring<ref name="reference 1"/>. The movement of residues 77-83 in ''C. parvum'' maneuvers an important oxygen of residue K79 so that it is in favorable proximity to hydrogen bond with AN6 located on the ligand molecule<ref name="reference 1"/>. Class II (Archael) GAPDHs have also been studied in detail and show interesting and definite properties. High concentrations of NADP(H) along with ATP and NADH have been found to reduce the enzymes affinity for NAD+, whereas glucose 1-phosphate, fructose 6-phosphate, AMP and ADP show to increase the affinity between the two as evidenced by the studies performed on ''Thermoproteus tenax''<ref name="ref 6"/>. ==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<ref name="ref 7">PMID:9580189 </ref>. 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<ref name="ref 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. brucei''are shown to lack a functional tricarboxylic acid cycle and thus its ultimate ATP source must come from glycolysis<ref name="ref 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<ref name="ref 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<ref name="ref 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 bloodstram<ref name="ref 8">PMID:11405646 </ref>. == References == <references/>

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