Browsing by Author "Puri, Rishika"

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    Mutations of succinyl-CoA synthetase to study the swinging of the phosphohistidine loop
    (2022-07-22) Puri, Rishika; Fraser, Marie; Moorhead, Gregory
    Succinyl-CoA synthetase (SCS), an enzyme of the citric acid cycle, has two active sites 35 Ȧ apart. A histidine in the α-subunit gets phosphorylated during the reaction and is on a loop that swings between the active sites. The goal was to gain insights into the mechanism of the swinging of the phosphohistidine loop. To test whether a previously published mutation of a second histidine in the α-subunit (Luo, G. X., & Nishimura, J. S. (1991). Site-directed mutagenesis of Escherichia coli succinyl-CoA synthetase: Histidine 142α is a facilitative catalytic residue. J. Biol. Chem. 266, 20781-20785) affected the swinging of the phosphohistidine loop, the mutation was made in human GTP-specific succinyl-CoA synthetase (G-SCS) by site-directed mutagenesis. The mutant protein was produced in E. coli and purified by nickel-nitrilotriacetic acid, size exclusion and ion exchange chromatography. Its specific activity and apparent Michaelis constants (Kmapp) were measured, with the expectation that the specific activity would be low compared to the wild-type, but the Kmapp values would be similar. When the human G-SCS mutant protein had high specific activity, the mutation was made in E. coli SCS, as done by Luo and Nishimura. Unexpectedly, the E. coli SCS mutant protein also had high specific activity, contradicting the previously published results. In case a different histidine had been mutated by Luo and Nishimura, the remaining four histidine residues in the α-subunit were mutated, and all four mutant proteins had high specific activity. It was concluded that the previous publication had a second, accidental mutation present in their mutant protein.