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 phosphorylation effects

The amino terminal dom:lin 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 wil d-type and mutant EIN proteins indicate two-state unfolding. For EIN protei ns in 10 mM K-phosphate (and 100 mM KCl) at pH 7.5, Delta H congruent to 14 0 +/- 10 (160) kcal mol(-1) and Delta C-p congruent to 2.7 (3.3) kcal K-1 m ol(-1) Transition temperatures (T-m) are 57 (59), 55 (58), and 53 (56) degr ees C for wild-type, H189A, and H189E forms of EIN, respectively. The order of conformational stability for dephospho-His189, phospho-His189, and H189 substitutions of EIN at pH 7.5 is: His > Ala > Glu > His-PO32- due to diff erences in conformational entropy. Although H189E mutants have decreased T- m values for overall unfolding the amino terminal domain, a small segment o f structure (3 to 12%) is stabilized (T-m similar to 66-68 degrees C). This possibly arises from an ion pair interaction between the gamma-carboxyl of Glu189 and the epsilon-amino group of Lys69 in the docking region for the histidine-containing phosphocarrier protein HPr. However, the binding of HP r to wild-type and active-site mutants of EIN and EI is temperature-indepen dent (entropically controlled) with about the same affinity constant at pH 7.5: K-A' = 3 +/- 1 x 10(5) M-1 for EIN and similar to 1.2 x 10(5) M-1 for EI.