ATP-specificity of succinyl-CoA synthetase from Blastocystis hominis

Succinyl-CoA synthetase (SCS) catalyzes the only step of the tricarboxylic acid cycle that leads to substrate-level phosphorylation. Some forms of SCS are specific for ADP/ATP or for GDP/GTP, while others can bind all of these nucleotides, generally with different affinities. The theory of 'gatekeeper' residues has been proposed to explain the nucleotide-specificity. Gatekeeper residues lie outside the binding site and create specific electrostatic interactions with incoming nucleotides to determine whether the nucleotides can enter the binding site. To test this theory, the crystal structure of the nucleotide-binding domain in complex with Mg2+-ADP was determined, as well as the structures of four proteins with single mutations, K46 beta E, K114 beta D, V113 beta L and L227 beta F, and one with two mutations, K46 beta E/K114 beta D. The crystal structures show that the enzyme is specific for ADP/ATP because of interactions between the nucleotide and the binding site. Nucleotide-specificity is provided by hydrogen-bonding interactions between the adenine base and Gln20 beta, Gly111 beta and Val113 beta. The O atom of the side chain of Gln20 beta interacts with N6 of ADP, while the side-chain N atom interacts with the carbonyl O atom of Gly111 beta. It is the different conformations of the backbone at Gln20 beta, of the side chain of Gln20 beta and of the linker that make the enzyme ATP-specific. This linker connects the two subdomains of the ATP-grasp fold and interacts differently with adenine and guanine bases. The mutant proteins have similar conformations, although the L227 beta F mutant shows structural changes that disrupt the binding site for the magnesium ion. Although the K46 beta E/K114 beta D double mutant of Blastocystis hominis SCS binds GTP better than ATP according to kinetic assays, only the complex with Mg2+-ADP was obtained.