Interactions of GTP with the ATP-grasp Domain of GTP-specific Succinyl-CoA Synthetase

Abstract

Two isoforms of succinyl-CoA synthetase exist in mammals, one specific for ATP and the other for GTP. The GTP-specific form of pig succinyl-CoA synthetase has been crystallized in the presence of GTP and the structure determined to 2.1 Å resolution. GTP is bound in the ATP-grasp domain, where interactions of the guanine base with a glutamine residue (Gln-20␤) and with backbone atoms provide the specificity. The ␥-phosphate interacts with the side chain of an arginine residue (Arg-54␤) and with backbone amide nitrogen atoms, leading to tight interactions between the ␥-phosphate and the protein. This contrasts with the structures of ATP bound to other members of the family of ATP-grasp proteins where the ␥-phosphate is exposed, free to react with the other substrate. To test if GDP would interact with GTP-specific succinyl-CoA synthetase in the same way that ADP interacts with other members of the family of ATP-grasp proteins, the structure of GDP bound to GTP-specific succinyl-CoA synthetase was also determined. A comparison of the conformations of GTP and GDP shows that the bases adopt the same position but that changes in conformation of the ribose moieties and the ␣-and ␤-phosphates allow the ␥-phosphate to interact with the arginine residue and amide nitrogen atoms in GTP, while the ␤-phosphate interacts with these residues in GDP. The complex of GTP with succinyl-CoA synthetase shows that the enzyme is able to protect GTP from hydrolysis when the active-site histidine residue is not in position to be phosphorylated.The enzyme succinyl-CoA synthetase (SCS) 4 uses ATP or GTP to catalyze the formation of succinyl-CoA from succinate and coenzyme A. In animals, two different isoforms exist, one specific for ATP and the other specific for GTP (1). The two isoforms include the same ␣-subunit, but different ␤-subunits (2). The amino-terminal domain of the ␤-subunit has an ATP-grasp fold (3-5), and Mg 2ϩ -ADP was shown to bind in this domain of the Escherichia coli SCS using labeling experiments and site-directed mutagenesis (6) and by soaking the nucleotide into crystals and determining the structure of the resulting complex (7). The ATP-grasp fold has been found in a number of other enzymes (8) (24), and RNA ligase 2 (25). The determinations of the structures of several of these enzymes in complex with nucleotides, nucleotide analogues, and their other substrates has led to a good understanding of the interactions that are important in their binding and catalysis.The biological roles of the ATP-and GTP-specific SCS have not been fully delineated. Originally it was thought that the primary role for SCS was in the citric acid cycle, where it was responsible for the breakdown of succinyl-CoA to succinate and coenzyme A accompanied by the phosphorylation of nucleotide diphosphate to nucleotide triphosphate (26). This step provides the only substrate-level phosphorylation of the citric acid cycle. It was thought that some species had SCS that could use either ADP or GDP, e.g. E. coli (27), while others had SCS that coul...

Structure of NDP-forming Acetyl-CoA synthetase ACD1 reveals a large rearrangement for phosphoryl transfer

“…To gain insight into the potential binding mode of Gua nucleotides, we superimposed ckcACD1 with the ATP-grasp domain containing enzyme CK2 from Zea mays, which is known to metabolize Ade and Gua nucleotides (42)(43)(44). We found that (i) the Ade moiety of ATP bound to CK2 [Protein Data Bank (PDB) ID code 1DAW] is oriented comparable to ckcACD1 and (ii) the Gua moiety of GTP bound to CK2 (PDB ID code 1DAY) is shifted in its position and compares quite well with the binding mode found in the structure of GTP-specific ssSCS (16). Hence, we expect a comparable binding mode for Guo nucleotides in ckcACD1.…”

“…At the corresponding sequence position, other members of the ACD family feature either Arg or Lys residues. In the case of GTP bound to ssSCS, the amino function of the side chain of Lys222β interacts with the gamma-phosphate in a similar manner (16), which might indicate a specific role for this residue.…”

“…This phenomenon, termed "domain shuffling," is one of the key features of this superfamily (8). Superposition of several structures of SCS from Escherichia coli (ecSCS) (12)(13)(14), Thermus aquaticus (15), the mammalian GTP-specific SCS from pig (16), and a truncated form of human ACLY (17,18) revealed that subdomains 1-5 share a common arrangement in these enzymes. From detailed studies of the reaction mechanism of ecSCS, a crucial enzyme tightly connected to the TCA cycle, a three-step mechanism was proposed, which involves the phosphorylation of a highly conserved His residue at the first active site (site I) as an intermediate step (12,13).…”

A Novel ADP-forming Succinyl-CoA Synthetase in Thermococcus kodakaraensis Structurally Related to the Archaeal Nucleoside Diphosphate-forming Acetyl-CoA Synthetases

“…It has been pointed out that other members in this superfamily also commonly harbor these five domains, although the order and distribution of the domains between the two subunits display variation (18). Although all previously characterized classical SCSs share the same subunit and domain structure (domain order: ␣-subunit, 1-2; and ␤-subunit, 3-4-5) (23,24,27,28), the SCS identified here from T. kodakaraensis exhibits a distinct structure: domain 5 is fused to the ␣-subunit (␣-subunit, domains 1-2-5; and ␤-subunit, domains 3-4) (Fig. 2).…”

“…ADP/GDP-forming succinyl-CoA synthetase (SCS) also belongs to this superfamily and catalyzes the reversible conver-sion of succinyl-CoA to succinate and CoA concomitant with substrate level phosphorylation of ADP/GDP (23,24). Generally, SCS functions in the tricarboxylic acid cycle of aerobic organisms, and it has been suggested that the mammalian ATPdependent enzyme serves a catabolic role, whereas the GTPdependent enzyme is involved in succinyl-CoA synthesis (24). Many Archaea also possess closely related homologs of SCS on their genomes.…”