Understanding the Mechanism of Succinyl-CoA Synthetase Catalysis from Structural Studies

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dc.contributor.advisorFraser, Marie E.
dc.contributor.authorHuang, Ji
dc.contributor.committeememberHowell, P. Lynne
dc.contributor.committeememberMacCallum, Justin L.
dc.contributor.committeememberZimmerly, Steven J.
dc.contributor.committeememberLewis, Ian A.
dc.dateFall Convocation
dc.date.accessioned2021-11-19T22:02:58Z
dc.date.available2021-11-19T22:02:58Z
dc.date.issued2020-08-24
dc.description.abstractThe 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.
dc.identifier.citationHuang, J. (2020). Understanding the Mechanism of Succinyl-CoA Synthetase Catalysis from Structural Studies (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.
dc.identifier.doihttp://dx.doi.org/10.11575/PRISM/39384
dc.identifier.urihttp://hdl.handle.net/1880/114120
dc.language.isoenen
dc.language.isoEnglish
dc.publisher.facultyGraduate Studiesen
dc.publisher.facultyScience
dc.publisher.institutionUniversity of Calgaryen
dc.rightsUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.en
dc.subject.classificationBiology--Molecular
dc.subject.classificationBiophysics--General
dc.subject.classificationChemistry--Biochemistry
dc.titleUnderstanding the Mechanism of Succinyl-CoA Synthetase Catalysis from Structural Studies
dc.typedoctoral thesis
thesis.degree.disciplineBiological Sciences
thesis.degree.grantorUniversity of Calgaryen
thesis.degree.grantorUniversity of Calgary
thesis.degree.nameDoctor of Philosophy (PhD)
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