• GAPDS
• References
• Images
• Slides

Glyceraldehyde-3-Phosphate Dehydrogenase (PDB ID: 3PFW) from Homo sapiens

Created by: Felicia White

Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (abbreviated GAPDS, PDB ID: 3pfw) is a glycolytic enzyme that catalyzes the oxidative phosphorylation of D-G3P (D-glyceraldehyde 3-phosphate) to form DPG (1,3-diphosphoglycerate). In addition, GAPDS also has other biological functions such as nuclear RNA transport, DNA replication and repair, membrane fusion and apoptosis (1). The human GAPD protein is also associated with several diseases, including Huntington’s or Alzheimer’s among others, that affect the neurological system.  The protein is present in mammals in two different forms that have a significant sequence identity but perform different functions. The somatic counterpart (GAPDH) is active all over the body in various cell types. GAPDH is the protein responsible for functions such as RNA transport, DNA replication during mitosis, and most membrane fusion and cellular apoptosis. The somatic form also influences the neurological diseases. Mammals also possess a sperm-specific counterpart that acts only in the sperm and do not have functions in the rest of the body. This protein, GAPDS, is associated strongly with sperm motility and therefore, fertility, of the male. The movement of the sperm cells, powered by their flagella, requires a large amount of ATP which is generated through glycolysis. The GAPDS shows enzymatic activity in a reaction that eventually leads to ATP formation. This protein contains a unique N-terminal 72-residue polyproline extension, not found in the GAPDH protein, which is needed for the tight association of GAPDS with the flagellum. Through this association of the flagella and the protein, the protein is able to provide the needed ATP for movement via glycolysis. Researchers are interested in studying this particular protein as a form of contraception to provide an alternative to hormone therapies and surgical options that currently exist. If the GAPDS was somehow inhibited from catalyzing the glycolysis reaction (without changing the GADPH enzymatic activity) or if the association of the GAPDS and flagella did not occur then the sperm cells would be left without the required energy for movement, obviously reducing their motility and therefore their ability to fertilize the female ovum.

            The ExPasy server was used to determine the weight and isoelectric point for the human GAPDS protein, the weight is reported at 44501.1 Da and the isoelectric point was found to be pH of 8.39 (3) so the protein is charged at physiological pH. The PSI-Blast yielded three proteins for comparison based on sequence similarity. One protein, a GAPDS from Rattus norvegicus (PDB ID: 2vyn) has an e-value of 0.0 which indicates that this protein has the potential to have 100 percent sequence identity with the query protein (PDB ID: 3pfw). Experimental comparisons of the sequence of the Rattus norvegicus GAPDS and the human GAPDS have confirmed 87% sequence identity and 95% similarity (2). The blast also highlighted two other proteins with very similar sequences. These proteins are from Trypanosoma cruzi (PDB ID: 3dmt) and Trypanosoma brucei (PDB ID: 2x0n) each with very low e-values approaching zero (4). Additionally, the Dali server yielded three proteins based on tertiary structure similarity to the POI. Two also turned up in the blast search, protein 3dmt (z-score = 50.0) and 2vyn (z-score = 56.1), however the third was not high in the list of the blast search (5). This protein (PDB ID: 4o59 z-score = 54.3)  was isolated from Bos taurus. The z-scores are considered significant above a value of 2, so the z-scores yielded from the Dali server all are significant and the larger value for the z-score usually translates to a more similar tertiary structure. Given this information it is prudent to consider this GAPDS from the Rattus norvegicus (PBD ID: 2vyn) as one of the comparison proteins since determining the active site could be done with more confidence given that the Rattus norvegicus GAPDS and the human GAPDS should have very similar, if not the same, active sites with the same functionally important residues present and have very similar tertiary structure which is determined in part by the manner in which the protein folds into secondary components. The GAPDH from Bos taurus (PDB ID: 4o59) will also be considered a comparison protein given the considerable z-score (50.0) as well as the close structural alignment. The difference in comparison proteins lies mostly in ligands as well as the length of the amino acid chain. The GAPDH from Trypanosoma cruzi has the same ligands but has a modified residue and the residues included in the active site are conserved but they are not necessarily in the same numerical positions in the sequence. This somatic GAPDH can be found in either the closed or open conformation, however the closed conformation is of more importance since this resembles the only possible conformation of the POI. Also, the Trypanosoma GAPDH contains 351 amino acids where the human GAPDS contains 346. The Rattus norvegicus GAPDS contains 334 residues. This protein also has formic acid in place of the glycerol ligand but does contain the NAD+ ligands. This difference in ligands could relate to catalysis of different sinlge step reactions in the production of ATP. The Rattus norvegicus GAPDS is the testis-specific protein and is only found in the closed conformation; a trait that is shared with the POI.  Much like the Trypanosoma protein, the residues in the active site of the Rattus norvegicus GAPDS are mostly conserved but may not be in the same numerical location along the amino acid chain.

           The ligands of GAPDS (PBD ID: 3pfw) are NAD+ and glycerol. The function of the NAD+ is to accept a proton and a pair of electrons during the glycolysis reaction. Glycerol serves an altogether different function. Glycerol is a sugar alcohol and an intermediate in carbohydrate and lipid metabolism (6). Glucose is a carbohydrate and the protein is catalyzing part of the glycolysis reaction so glycerol might be present as a sort of inhibitor, preventing the reaction from proceeding too quickly or too far towards the products in accordance with Le Chateliers principle. Additionally, in some organisms, glycerol can enter the glycolysis pathway directly and provide energy for cellular metabolism so glycerol could be attached as a ligand only to further the production of ATP (6). Human GAPDS (PBD ID: 3pfw) in composed of 418 amino acids (including the tail), the sequence is comprised of about 10% acidic residues and 12.9% basic residues lending itself to a higher pI. Also, given the relative amount of hydrophobic residues, the protein adopts a globular shape to shield them from water.Additionally, the protein has regions of alpha helices, beta sheets, and 3/10 helices as well as random coil segments. GAPDS has two subunitsper chain named O and P, for a total of four subunits, and is known as a homotetramer (7). The GAPDS ligands are imbeded in the structure. This protein is not bound to any metals and does not appear to have specifically known ionic interactions or hydrogen bonds among the residues. The structure was of the protein was determined by over expression of human GAPDS and removing the N-terminal polyproline chain. Crystal structures were determined while the protein was in complex with NAD+ and phosphate and NAD+ and glycerol using X-ray diffraction (1,7).  The secondary structure consists of multiple α-helices and β-strands and contains many turns and bends according to the ProtParam tool (8). The PDB only classifies about 56% of the protein into either an α-helix or β-strand so it is probable that some of the turns or bends that the ProtParam tool lists are actually sections of random coil or 3/10 helices. This implies that GAPDS will have segments associated with other subunits or proteins to stabilize it. ProtParam also estimates the half-life of the protein to be about thirty hours.

As mentioned previously, GAPDS (PBD ID: 3pfw) functions to catalyze glycolysis reactions. First and foremost, the N-terminus contains a polyproline tail that is approximately 72 residues long (1,9). The tail binds to the cytoskeletal fibrous sheath of the flagella via anchoring proteins and accounts for most of the sequence dissimilarity between the human GADPS and the somatic counter-part. Without this tail the protein would not be able to attach itself to the sperm and provide it with the energy (ATP) needed for motility, therefore completely neglecting the functional importance of this protein. Residues Asp-106, Lys-151, Tyr-173, Ser-193, and Asn-388 are residues that are functionally important since they are binding sites for NAD+ (binding pocket) and according to the primary citation the reaction that the protein catalyzes is dependent on NAD+ which is converted to NADH over the course of the reaction (1, 9). Additionally, nucleotide binding to NAD takes place at Arg-85 and Ile-86. Residue Cys-224 is the nucleophilic active site (space-filling) that bonds to the inorganic phosphate which becomes bonded to D-glyceraldehyde 3-phosphate during the reaction to yield 3-phospho-D-glyceroyl phosphate as one of the products (9). The D-glyceraldehyde 3-phosphate is bound to residues Thr-254 and Arg-306. An acid-base-nucleophile triad is important for enzymatic catalysis and involves the interaction of an acidic residue with a basic residue to polarize the base. The base will then deprotonate with the nucleophile and increase its reactivity toward an electrophile encouraging bonding. In GAPDS Asp-106 acts as the acidic residue aligning and polarizing the His-251 residue. The Histidine then polarizes and deprotonates the thiol group of Cys-224 to increase its reactivity in binding with the inorganic phosphate which it will later donate to D-glyeraldehyde-3-phosohate. Given all of the residues and reactants that must be in or very near the active site, it is clear that the substrates must fit into the protein site in a specific conformation and that donor for the inorganic phosphate should be rather small. Otherwise, it seems as though the rate of the reaction would be slowed by steric hindrance. Most likely the phosphate donor is not really a donor so much as a free phosphate group. The small NAD binding pocket in which 2 glycerol molecules and all the necessary substrates must fit limits the sort of phosphate that can used as well as the possibility of catalyzing other reactions.