Fraser, MarieHuang, Ji2014-12-152015-02-232014-12-152014http://hdl.handle.net/11023/1962Succinyl-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.engUniversity 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.BiochemistrySuccinate-binding siteGTP-specific succinyl-CoA synthetaseCrystalStructuremaster thesis10.11575/PRISM/27933