Calorimetric and spectroscopic investigation of the interaction between the C-terminal domain of Enzyme I and its ligands.

Enzyme I initiates a series of phosphotransfer reactions during sugar uptake in the bacterial phosphotransferase system. Here, we have isolated a stable recombinant C-terminal domain of Enzyme I (EIC) of Escherichia coli and characterized its interaction with the N-terminal domain of Enzyme I (EIN) and also with various ligands. EIC can phosphorylate EIN, but their binding is transient regardless of the presence of phosphoenolpyruvate (PEP). Circular dichroism and NMR indicate that ligand binding to EIC induces changes near aromatic groups but not in the secondary structure of EIC. Binding of PEP to EIC is an endothermic reaction with the equilibrium dissociation constant (K(D) ) of 0.28 mM, whereas binding of the inhibitor oxalate is an exothermic reaction with K(D) of 0.66 mM from calorimetry. The binding thermodynamics of EIC and PEP compared to that of Enzyme I (EI) and PEP reveals that domain-domain motion in EI can contribute as large as ∼-3.2 kcal/mol toward PEP binding.