ATP-specificity of succinyl-CoA synthetase fromBlastocystis hominis

Abstract

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 Mg 2+ -ADP was determined, as well as the structures of four proteins with single mutations, K46E, K114D, V113L and L227F, and one with two mutations, K46E/K114D. 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, Gly111 and Val113. The O atom of the side chain of Gln20 interacts with N6 of ADP, while the sidechain N atom interacts with the carbonyl O atom of Gly111. It is the different conformations of the backbone at Gln20, of the side chain of Gln20 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 L227F mutant shows structural changes that disrupt the binding site for the magnesium ion. Although the K46E/K114D double mutant of Blastocystis hominis SCS binds GTP better than ATP according to kinetic assays, only the complex with Mg 2+ -ADP was obtained.

Second distinct conformation of the phosphohistidine loop in succinyl-CoA synthetase

“…The residues of this loop are likely to undergo large conformational changes when shuttling the phosphohistidine between sites I and II. Crystal structures of SCSs from different species show the binding of Mg 2+ -succinate (Huang & Fraser, 2016), ADP (Joyce et al, 2000;Huang et al, 2019), GDP (Joyce et al, 2012), GDP and GTP (Fraser et al, 2006), and both phosphorylated and dephosphorylated SCSs (Fraser et al, 2000), but the active-site histidine has yet to be seen in site II.…”