Succinate-binding site of succinyl-CoA synthetase
Date
2014-12-15
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Abstract
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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Biochemistry
Citation
Huang, J. (2014). Succinate-binding site of succinyl-CoA synthetase (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/27933