Browsing by Author "Huang, Ji"

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    Succinate-binding site of succinyl-CoA synthetase
    (2014-12-15) Huang, Ji; Fraser, Marie
    Succinyl-CoA synthetase (SCS) is an essential enzyme that catalyzes substrate-level phosphorylation in the citric acid cycle. In my study, the pig GTP-specific SCS (GTPSCS) was overproduced in E. coli BL21(DE3), and purified through three different columns: Ni-NTA affinity, Superdex-200 prep grade size exclusion and Hitrap Blue High Performance affinity. A crystal of GTPSCS in complex with coenzyme A (CoA) diffracted to 2.1 Å. The CoA-binding site was determined to be located in the N-terminal domain of the α-subunit of the enzyme. A crystal of GTPSCS in complex with Mg2+-succinate and CoA was grown in polyethylene glycol 3350, ammonium succinate and Hepes (pH 7.0) and diffracted to 2.2 Å. The succinate-binding site of GTPSCS was determined to be located in the C-terminal domain of the β-subunit of the enzyme. The discovery of the succinate-binding site of SCS supports the view that the succinyl phosphate complex can be formed during the catalytic reaction.
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    Understanding the Mechanism of Succinyl-CoA Synthetase Catalysis from Structural Studies
    (2020-08-24) Huang, Ji; Fraser, Marie E.; Howell, P. Lynne; MacCallum, Justin L.; Zimmerly, Steven J.; Lewis, Ian A.
    The catalytic mechanism of succinyl-CoA synthetase (SCS) was investigated using X-ray crystallography. Two nucleotide-specific forms of SCS exist in mammals, of which one is ATP-specific (ATPSCS) and the other is GTP-specific (GTPSCS). ATPSCS and GTPSCS share the same α-subunit, but have a different ß-subunit, which determines the nucleotide specificity. In order to understand why ATPSCS can only use ATP/ADP in the catalytic reaction, the ATP-binding domain of Blastocystis hominis SCS (BhSCS) was co-crystallized with Mg2+-ADP. The 2.2 Å-resolution structure showed that this enzyme is ATP-specific due to the conformations of the backbone at Gln20ß, of the side chain of Gln20ß, and of the linker that connects the two subdomains of the ATP-grasp fold. While investigating the structure of human GTPSCS, tartryl-CoA was observed in the 1.52 Å-resolution structure. The structure revealed the tartryl portion of tartryl-CoA bound in the phosphate-binding site and showed why tartryl-CoA is an inhibitor of SCS. In order to support the hypothesis that the phosphohistidine loop of the α-subunit shuttles the phosphoryl group ~30 Å during catalysis, pig GTPSCS was co-crystallized with Mg2+-GDP or Mg2+-GMPPNP or Mg2+-GMPPCP. The structures of the complexes revealed the conformation of the phosphohistidine loop in site II, the nucleotide-binding site, for the first time. In an attempt to crystallize succinyl-phosphate bound to pig GTPSCS, the structure of GTPSCS complexed with Mg2+-succinate and desulfo-CoA revealed a second succinate and magnesium ion binding site, located at the interface of the α- and ß-subunits. These additional succinate and magnesium ions may regulate the conformational change of the phosphohistidine loop. In order to better understand the biological roles of human ATPSCS and GTPSCS, their kinetics and structures were compared. Human ATPSCS was co-crystallized with ADP or GDP, and the structures were determined at 3.1 Å-resolution. Both enzymes displayed Michaelis-Menten kinetics for all substrates except succinate, for which human ATPSCS gave a sigmoidal curve, while GTPSCS gave the expected asymptotic curve. This may indicate regulation of human ATPSCS.