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 β E, K114 β D, V113 β L and L227 β F, and one with two mutations, K46 β E/K114 β 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 β , Gly111 β and Val113 β . The O atom of the side chain of Gln20 β interacts with N6 of ADP, while the side-chain N atom interacts with the carbonyl O atom of Gly111 β . It is the different conformations of the backbone at Gln20 β , of the side chain of Gln20 β 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 an...
Source: Acta Crystallographica Section D - Category: Biochemistry Authors: Tags: nucleotide specificity ATP-grasp fold thermal stability acyl-CoA synthetase (ADP-forming) differential scanning fluorimetry research papers Source Type: research