Conformational stability changes of the amino terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sUgar phosphotransferase system produced by substituting alanine or glutamate for the active‐site histidine 189: Implications for

The amino terminal domain of enzyme I (residues 1‐258 + Arg; EIN) and full length enzyme I (575 residues; EI) harboring active‐site mutations (H189E, expected to have properties of phosphorylated forms, and H189A) have been produced by protein bioengineering. Differential scanning calorimetry (DSC) and temperature‐induced changes in ellipticity at 222 nm for monomeric wild‐type and mutant EIN proteins indicate two‐state unfolding. For EIN proteins in 10 mM K‐phosphate (and 100 mM KCl) at pH 7.5ΔH ≅ 140± 10 (160) kcal mol⁻¹ and ΔC_p ≅ 2.7 (3.3) kcal K⁻¹ mol⁻¹. Transition temperatures (T_m) are 57 (59), 55 (58), and 53 (56) °C for wild‐type, H189A, and H189E forms of EIN, respectively. The order of conformational stability for dephospho‐His 189, phospho‐His189, and H189 substitutions of EIN at pH7.5 is: His > Ala > Glu > His‐PO₃²⁻ due to differences in conformational entropy. Although H189E mutants have decreased T_m values for overall unfolding the amino terminal domain, a small segment of structure (3 to 12%) is stabilized (T_m ∼ 66–68 °C). This possibly arises from an ion pair interaction between the γ‐carboxyl of Glu189 and the ϵ‐amino group of Lys69 in the docking region for the histidine‐containing phosphocarrier protein HPr. However, the binding of HPr to wild‐type and active‐site mutants of EIN and EI is temperature‐independent (entropically controlled) with about the same affinity constant at pH 7.5: K'_A = 3 ± 1 × 10⁵ M⁻¹ for EIN and ∼1.2 × 10⁵ M⁻¹ for EI.