Succinyl-CoA synthetase
Created by: Ashley Mills
Succinyl-CoA synthetase (SCS) is an enzyme that catalyzes the reaction in which succinyl-CoA, NDP (adenine or guanine diphosphate), and a phosphate group are converted to succinate, coenzyme A, and a molecule of NTP. This reaction takes place during the citric acid cycle of metabolism in which acetyl CoA is oxidized to carbon dioxide to produce transient energy-carrying triphosphate molecules (Freeman, 158). Succinyl-CoA synthetase (3UFX) isolated from Thermus aquaticus is a biologically significant enzyme because of its function in this role and its preference for guanine diphosphate and guanine triphosphate opposed to the comparable adenine molecules (Joyce, 751). In the event that succinyl-CoA synthetase is absent, the amount of energy-containing molecules that the cell is able to produce decreases substantially. ATP and GTP are the major energy-carrying molecules present in most metabolic systems, they do not exist in adequate quantities to maintain function without being renewed through metabolic pathways (Garrett & Grisham, 69). T. aquaticus SCS differs from SCS enzymes of other species due to a marked preference for GDP/GTP as well as its ability to function under extreme physiological conditions in a thermophilic species. The molecular weight of T. aquaticus SCS is 294199.24 Da and the isolelectric point is 5.90 (Artimo, ExPASy). No alternate conformations for this protein are currently known, nor is the protein the target of drug reactions at this time.
The structure of SCS directly correlates to the function of the enzyme. T. aquaticus SCS exists under physiological conditions as a heterotetramer with two alpha subunits and two beta subunits, one of the alpha subunits and one beta subunit form a dimer and two of these dimers form the enzyme complex (Joyce et al., 751). Each alpha subunit is 296 residues in length with a secondary structure consisting of 35% alpha helices, 22% beta sheets, and the remainder consisting of 3/10 helices, turns, and random coil. Each beta subunit is 397 residues in length with a secondary structure of 36% alpha helices and 26% beta sheets (RCSB PDB: 3ufx). The binding of the substrate, GDP, and the reaction itself occurs in complex with the beta subunit while the alpha subunits act as structural support to the complex, giving integrity to the molecule (Joyce et al., 758).
During the crystallization of SCS the best crystal formed contained the enzyme complexed as an octamer with GDP and Mn2+ ligands. The GDP ligand is a major reactant in the catalyzed reaction and Mn2+ is needed for catalysis to occur. In its physiological function, SCS requires a divalent cation to catalyze the reaction (usually Mg2+) Mn2+ can act in this function (Joyce et al., 751). The enzyme complex contains a cis-peptide bond in each subunit located at turns near catalytic residues; in the alpha subunit where the histidine residue is phosphorylated and in the beta subunit where Mn2+ binds (Joyce et al., 758).
Thermus aquaticus SCS shows a defined preference for binding GDP opposed to ADP resulting from reactions between the nucleotide and the residues within the nucleotide-binding site. The guanine base interacts with the amide nitrogen and carbonyl oxygen of Val-94 beta forming two hydrogen bonds. Further hydrogen bonding and interactions with water molecules link the base to residues Lys-45 beta, Ala-203 beta, and Glu-92 beta. ADP and GDP have different protonation and hydrogen bonding behaviors resulting in the preference for GDP despite ADP still having the ability to bond to the beta subunit (Joyce et al., 758).
The active site histidine residue, His-246 alpha of T. aquaticus SCS is protonated and forms a hydrogen bond with Glu-208 alpha, water molecules also interact with His-246 alpha binding it to the power helices of the alpha and beta subunits making up the active site (Joyce et al., 760). The succinate-binding site of the beta subunit loop contains the residues Gly-312 beta and Thr-314 beta which bind to the carboxylate group of succinate. The glycine residues of the loop provide flexibility in the absence of succinate (Joyce et al., 760).
Thermus aquaticus SCS contains one cysteine residue, Cys-123 alpha, located near the catalytic histidine residue and protected by several other alpha subunit residues, this cysteine residue is highly conserved throughout SCS proteins across species (Joyce et al., 760). The low content of residues such as cysteine, serine, phenylalanine, asparagines, and glutamine correlates with the presence of this SCS protein within a thermophilic organism. Lower content of these residues shows a correlation with an increase in thermostability of the protein and increased resistance to chemical denaturation, characteristics necessary in the harsh environment of Thermus aquaticus (Joyce et al., 760).
The PSI-BLAST database compares proteins based on primary structure similarities, each protein in assigned an E-value corresponding to total sequence homology and assigned gap sequences compared with a protein of interest. An E-value less than .05 is significant for proteins, showing a high degree of sequence homology. No comparison proteins were generated for the PSI-BLAST search that could be used as none were contained in the Protein Data Bank. Several proteins had E-values of 0.0 including SCS subunit alpha of Desulfomicrobium baculatum and Succinyl-CoA ligase of Brevibacillus lateros (NCBI Blast). The Dali server compares proteins based on tertiary structure similarities based on intramolecular distance comparisons. A Z-score is then assigned to each protein compared to a query protein, a Z-score above 2 indicates similar folding patterns among proteins (Holm, Dali).
Succinyl-CoA synthetase isolated from Escheria coli (PDBID: 1JKJ) has a Z-score of 46.7 compared with SCS from T.aquaticus (Holm, Dali). An E-value for this protein was not generated during the PSI-BLAST search. Visual comparison between the sequences of the protein of interest and SCS from E. coli shows a high degree of similarity. The differences that are observed between the primary structures between the two molecules does not translate into large differences between the secondary structures. The secondary structures are very similar with a few differences in which random coil exists in the comparison protein where alpha helical secondary structure is present in the protein of interest (Holm, Dali). Following the high Z-score generated by the Dali server, the superimposition of the two proteins shows very little tertiary structure differences with a high degree of conserved folding patterns (Holm, Dali).
E. coli SCS is responsible for the catalysis of the same reaction as T. aquaticus SCS. This degree of functional similarity leads to a comparable degree of structural similarity. Many analogies can be drawn between the two proteins as a result of these similarities as stated in (Joyce et al., 751-762). The differences between the proteins are important to the understanding of how each protein functions in different physiological environments. E. coli SCS does not show the preference for GDP seen in T. aquaticus and several important residues for the function of the enzyme are not conserved among the two proteins, specifically the beta glutamic acid residues located near the catalytic histidine residue in E. coli (Fraser et al., 537).
The structure of Thermus aquaticus SCS dictates its ability to function as the catalyst in an essential metabolic reaction that takes place in the citric acid cycle. The presence of several binding sites on the surface of the protein, primarily located on the beta subunit allows for the enzyme to consistently maintain contact with all phases of the reaction, GDP substrate, Mn2+ or Mg2+ catalysis aids, and succinate product. The dimeric constitution of the enzyme with alpha-beta dimers attached to one another allows for numerous reaction sites while maintaining the integrity of the molecule. Binding a reaction occurs on the beta subunits while the alpha subunits maintain the structure of the complex. Each residue within the primary structure of the protein plays a role in physiological function and the stability of the enzyme in harsh conditions of Thermus aquaticus (Joyce et al., 751).