Molecular Basis for Allosteric Control of Escherichia Coli Glycerol Kinase by Fructose 1,6-Bisphosphate and IIAglc
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There has been progress towards elucidating the mechanism of Escherichia coli glycerol kinase (EcGK) control by its allosteric effectors fructose-1,6-bisphosphate (FBP) and IIAglc (a member of the phosphoenolpyruvate:glycose phosphotransferase system). Determining the mechanism requires analysis of the interaction between these effectors and the substrates of EcGK. In this study, a structural and kinetic approach was used to determine inhibition by both the effectors. For this work, the use of fluorescence anisotropy to observe ligand binding was investigated. Also, a foundation was laid for future NMR experiments with EcGK. For fluorescence studies, E36C EcGK was labeled with fluorescein and tested for changes in anisotropy in the presence of different ligands. To ensure that E36C was an appropriate representative of wildtype protein, initial velocity, inhibition, and heterotropic coupling assays were performed. Groundwork for future NMR experiments required analyzing substitutions of the native EcGK cysteines by initial velocity and inhibition studies. By comparing wildtype enzyme and E36C (variant of wildtype with an engineered cysteine residue at position 36), it was found that E36C is a suitable substitute and was not drastically affected by labeling with fluorescein. Anisotropy values differed upon binding of different ligands and enabled titrations of the enzyme substrate complexes with both effectors to obtain dissociation constants. This supports using the stopped-flow method to assess the on- and off- rates of substrates and to obtain values for Q coupling. Furthermore, the results for FBP showed that inhibition by FBP is K-type (affects affinity) with respect to ATP and V-type (affects enzyme velocity) with respect to ADP. The findings presented also showed that native cysteine substitutions effect some of the catalytic and allosteric parameters of EcGK and would be powerful reporters for ligand binding in NMR. However, the enzymes are unstable and new protocols for protein isolation will need to be drafted.
Mayorov, Shanna Quinn (2011). Molecular Basis for Allosteric Control of Escherichia Coli Glycerol Kinase by Fructose 1,6-Bisphosphate and IIAglc. Master's thesis, Texas A&M University. Available electronically from