Interdomain interaction and substrate coupling effects on dimerization and conformational stability of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

The bacterial PEP:sugar phosphotransferase system couples the phosphorylation and translocation of specific sugars across the membrane. The activity of the first protein in this pathway, enzyme I (EI), is regulated by a monomer−dimer equilibrium where a Mg2+-dependent autophosphorylation by PEP requires the dimer. Dimerization constants for dephospho- and phospho-EI and inactive mutants EI(H189E) and EI(H189A) (in which Glu or Ala is substituted for the active site His189) have been measured under a variety of conditions by sedimentation equilibrium at pH 7.5 and 4 and 20 °C. Concurrently, thermal unfolding of these forms of EI has been monitored by differential scanning calorimetry and by changes in the intrinsic tryptophanyl residue fluorescence. Phosphorylated EI and EI(H189E) have 10-fold increased dimerization constants [∼2 × 106 (M monomer)-1] compared to those of dephospho-EI and EI(H189A) at 20 °C. Dimerization is strongly promoted by 1 mM PEP with 2 mM MgCl2 [KA‘ ≥ 108 M-1 at 4 or 20 °C], as demo...