Phosphorylation destabilizes the amino-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

Thermal stabilities of enzyme I (63 562 Mr subunit), in the Escherichia coli phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), and a cloned amino-terminal domain of enzyme I (EIN; 28 346 Mr) were investigated by differential scanning calorimetry (DSC) and far-UV circular dichroism (CD) at pH 7.5. EIN expressed in a Δpts E. coli strain showed a single, reversible, two-state transition with Tm = 57 °C and an unfolding enthalpy of ∼140 kcal/mol. In contrast, monomeric EIN expressed in a wild-type strain (pts+) had two endotherms with Tm ≅ 50 and 57 °C and overall ΔH = 140 kcal/mol and was converted completely to the more stable form after five DSC scans from 10 to 75 °C (without changes in CD:  ∼58% α-helices). Thermal conversion to a more stable form was correlated with dephosphorylation of EIN by mass spectral analysis. Dephospho-enzyme I (monomer ⇄ dimer) exhibited endotherms for C- and N-terminal domain unfolding with Tm = 41 and 54 °C, respectively. Thermal unfolding of the C-terminal dom...