Succinyl-CoA synthetase (SCS; EC 184.108.40.206/4; reviewed in Bridger, 1974
; Nishimura, 1986
) is an enzyme of the citric acid cycle, where it catalyzes the only step that involves substrate-level phosphorylation. In the citric acid cycle, the enzyme acts not as a synthetase, but as a thiolase. It uses the energy of the succinyl-CoA thioester to phosphorylate itself on the active-site histidine residue and then transfers the phosphoryl group to nucleotide diphosphate to form nucleotide triphosphate. The reaction requires magnesium or other divalent cations.
The first structure of SCS to be determined was that of the Escherichia coli
enzyme (Wolodko et al.
). The E. coli
enzyme is a heterotetramer of two α-subunits and two β-subunits (Bridger, 1971
). The active-site histidine residue is residue 246 of the α-subunit, which was phosphorylated in the structure (Wolodko et al.
). CoA, which is required for crystallization (Wolodko et al.
), bound to the amino-terminal domain of the α-subunit, with the free thiol group near the phosphorylated histidine residue. Subsequent experiments proved that the nucleotide was bound to the amino-terminal domain of the β-subunit (Joyce et al.
), 30 Å away from the location of the phosphohistidine in the crystal structure (Joyce et al.
). Based on the crystal structures of SCS and of other members of the ATP-grasp family (Murzin, 1996
), it was hypothesized that the phosphohistidine loop swings to shuttle the phosphoryl group from site I, where CoA and presumably succinate bind, to site II, where the nucleotide binds (Fraser et al.
). E. coli
SCS can use ADP or GDP, but in mammals there are two forms of SCS, one specific for ADP/ATP and the other specific for GDP/GTP (Johnson et al.
). The structure of GTP-specific SCS from pig, which is active as an αβ-dimer (Wolodko et al.
), has been determined in both the phosphorylated and dephosphorylated forms (Fraser et al.
), as well as in open conformations without nucleotide and closed conformations with nucleotide, GDP or GTP, bound (Fraser et al.
To date, there are no structures available of a complete SCS heteromultimer from a thermophilic organism. The structure of the α-subunit of Thermus thermophilus
SCS was determined as part of a structural genomics initiative (H. Takahashi, Y. Tokunaga, C. Kuroishi, N. Babayeba, S. Kuramitsu, S. Yokoyama, M. Miyano, M. & T. H. Tahirov, unpublished work) and the model and crystallographic data have been deposited in the Protein Data Bank (Berman et al.
) with code 1oi7
. The crystals diffracted to very high resolution, 1.23 Å, but no electron density was observed for the residues of the phosphohistidine loop (Kleywegt et al.
). This is likely to be because the loop is disordered in the absence of the β-subunit, since in the structure of SCS the phosphohistidine loop interacts with the carboxy-terminal domain of the β-subunit (Wolodko et al.
). It would be interesting to compare the structure of SCS from a thermophile with that from E. coli
in order to gain a better understanding of how the enzyme protects its substrates, in particular succinyl-CoA, from hydrolysis at higher temperatures and to see what makes the thermophilic enzyme stable at higher temperatures. For this purpose, SCS from T. aquaticus
was cloned, overexpressed, purified and crystallized.