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 degreesC. Concurrently, thermal unfolding of these forms of El 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 [similar to2 x 10(6) (M monomer)-(1)] compared to those of dephospho-EI and EI(H189A) at 20 degreesC. Dimerization is strongly promoted by 1 mM PEP with 2 mM MgCl2 [K-A' greater than or equal to 10(8) M-1 at 4 or 20 degreesC], as demonstrated with EI(H189A) which cannot undergo autophosphorylation. Together, 1 mM PEP and 2 mM Mg2+ also markedly stabilize and couple the unfolding of C- and N-terminal domains of EI(HI89A), increasing the transition temperature (T-m) for unfolding the C-terminal domain by similar to18 degreesC and that for the N-terminal domain by similar to9 degreesC to T-max congruent to 63 degreesC, giving a value of K-D' congruent to 3 muM PEP at 45 degreesC. PEP alone also promotes the dimerization of EI(H189A) but only increases T-m similar to5 degreesC for C-terminal domain unfolding without affecting N-terminal domain unfolding, giving an estimated value of K-D' congruent to 0.2 mM for PEP dissociation in the absence of Mg2+ at 45 degreesC. In contrast, the dimerization constant of phospho-EI at 20 degreesC is the same in the absence and presence of 5 mM PEP and 2 MM MgCl2. Thus, the separation of substrate binding effects from those of phosphorylation by studies with the inactive EI(H189A) has shown that intracellular concentrations of PEP and Mg2+ are important determinants of both the conformational stability and dimerization of dephospho-EI.