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...

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