Succinyl

The primary enzyme in the reaction that transforms CoA to acyl-CoA thioesters to free carboxylates is succinyl-CoA (PBD ID: 5DW4). This 514-residue chain serves a significant purpose within the citric acid cycle of multiple species (for the present data, succinyl-CoA derived from Acetobacter aceti was used) (1). The citric acid cycle falls into the category of aerobic pathways. Aerobic pathways are essential for sustaining and facilitating the production of adenosine triphosphate (ATP) throughout an organism. The overall purpose of this particular reaction is the oxidation of carbohydrates and fatty acids in order to derive energy for the organism to use (2). Classified as a transferase, succinyl-CoA is an enzyme that catalyzes the conversion of acetate to acetyl-CoA with an additional succinate side product (3). Figure 1 displays the overall citric acid cycle.

With an overall molecular weight of succinyl-CoA is 1,128,000 Da and isoelectric point of 5.37 (3). Succinyl-CoA is one of the smaller protein compositions. Although the protein itself expands to 514 residues in length, the overall active coenzyme-binding site of succinyl-CoA ranges from residue 269 to 273, approximately 5 residues (3). Binding sites can also be found on residues IIe-364, Asn-384, Gly-388, and Lys-408. Due to the polarity of all of these residues (aside from Ile), the binding site has a strong affinity to coenzyme A, which is also polar. The carbonyl oxygen of IIe-364 contains a dipole, which has an attraction to the polar surface of the coenzyme similar to that of the amide nitrogen on Gly-388 (3)

The active site has four stages of conformation, spanning from fully open to fully closed. All of these conformations, one associated with the 270s loops found on both subunit A and B (5) Both imidazole and acetate ion ligands are prevalent within the active site, due to the association with the coenzyme A substrate. Imidazole binds to the later surface of the active site, while the acetate ion allows for ionic interactions at the active site. When the active site is closed, it allows acetyl-CoA (AarC) to engage, immobilize and desolvate substrates near the protein. Glu-294 is also covalently during this process by AarC (1).

When the substrate binds to the closing conformation of the enzyme, the substrate fills the external oxyanion hole. Once this occurs, hydrogen bonds begin to form in order to close over the substrate itself. Val-270 is located at the top of the two loops on subunit A and B and seems to do the most movement during the closure of the binding site. While its side group can constrain the thioesters, it can also donate hydrogen bonds to the configuration that eventually stabilizes the leaving group of the substrate-enzyme complex (4).

There are several different residues that promote the shape of the protein. Phe-232B creates a loop within the secondary structure of the protein, causing its alpha-4-helix to make contact with subunit A (1). Polar Phe-232 starts the first stated residue for the active site and continues through to Gly-388N. This group of residues acts as the orthorhombic space group. As previously stated, due to the substrate in question, ligands play an important role in succinyl-CoA's function. Ser-71 acts as a hydrogen bond donor, in order to latch onto the now activated acetate ion. Arg-228 and Thr-94 also serve a similar purpose in terms of hydrogen bonding (1).

Some residues are only exposed within the closed conformation or opened conformation. Val-270 is internally bound within the closed conformation; this is parallel to the idea that nonpolar peptides will remain within the internal body of the protein. Arg-228 is only exposed during the opened conformation. Glu-294 is within the center of the active site. Its position allows for a favorable nucleophilic attack on AarC. This is particularly important within this protein because it is due to the ability of the protein to undergo a nucleophilic attack that categorizes it as an enzyme, and further more a transferase (6).

Multiple studies have also been preformed in order to interpret certain residues that if they were replaced, would be detrimental to both the conformation and ability of the enzyme. For example, if Glu-294 were to be replaced with alanine, it would result in the abolishment of the enzymes activity. Glu-294 is key to allowing a nucleophilic attack by the substrate due to its charged, acidic nature. Alanine, on the other hand, is nonpolar, and although it would not alter the structure of the active site, binding by the substrate would not occur, as the charged structure is crucial. Altering Cys-357 or Glu-435 would change the solubility of the protein. By changing Cys-357 to a larger group that is also polar, such as tyrosine, there is a strong impair of protein solubility due to the new occurring pKa and therefore, alternating charge. If Glu-435 is replaced with either a nonpolar group such as alanine or even its polar counterpart, glutamine, the solubility of the protein is completely abolished (1). Glutamic acid has a negative charge after being deprotonated at a pH of 4.3. Since homeostasis of the body keeps the pH typically at an average higher than that, it is expected that that negative charge plays a key role in protein solubility (6).

PSI-BLAST and DALI comparison tests were both ran in order to compare succinyl-coA to alternative proteins. PSI-BLAST looks at the individual primary structure of proteins and compares them to one another in order to determine similar proteins. DALI Server compares the tertiary structure of individual proteins by approximating the changes in distances between molecules intramolecularly. This approximation is done using a sum of squares method and states that when any protein is compared to another protein, any z-score above 2 will yield a similar tertiary structure. While both of these servers identify proteins with similar sequences and tertiary structure, even if a protein has a similar primary sequence or tertiary structure, the functions can still be vastly different. 4-hydroxybutyrate-coA-transferase (PBD ID: 3GK7) from clostridium aminobutyricum returned by Dali to have a Z-score of 35.8 when compared to that of succinyl-coA, therefore, the two structures have an almost identical tertiary structure. This protein also falls within the family of transferases and is associated with several ligands on its active sight such as, 1,2-ethanediol and acetic acid (1). Both proteins have acetic acid as a vital ligand. However, 4-hydroxybutyrate-coA-transferase only contains 497 residues compared to that of succinyl-coA, which has 514. The same amount of beta sheets and alpha sheets can be found intertwined in the secondary structure of both proteins, however, 4-hydroxybutyrate-coA-transferase seems to be shifted more to the right in terms of the pattern of secondary structure (4). The structures do not line up perfectly. BLAST retrieved the protein coenzyme A transferase from pseufomonas aeruginosa (PBD ID: 2G39). The E-value between this protein and that of succinyl-coA was 0.0, which is interpreted as an almost identical protein in terms of primary structure that occurs in an alternative species. Similar to that of the other two proteins in question, coenzyme A transferase also contains ligands at its active site, spermidine and 2-amino-2-hyrdroxymethyl-propane-1,3-diol (1).

Overall, succinyl-CoA is found in numerous organisms in order to catalyze the conversion of acetate to acetyl-CoA, which is a step in the overall metabolic process. Its charged activation site, allows for the substrate to bind effectively. Other metabolic proteins, such as the previously discussed, coenzyme A transferase from pseufomonas aeruginosa, contain similar primary and tertiary structure, only highlighting the fact that structure does indeed qualify function.