On the conformational stability and dimerization of phosphotransferase enzyme I from Escherichia coli

Abstract

The activity of enzyme I (EI), the first protein in the bacterial PEP:sugar phosphotransferase system, is regulated by a monomer–dimer equilibrium where a Mg 2+-dependent autophosphorylation by PEP requires the homodimer. Using inactive EI(H189A), in which alanine is substituted for the active-site His189, substrate binding effects can be separated from those of phosphorylation. Whereas 1 mM PEP (with 2 mM Mg 2+) strongly promotes dimerization of EI(H189A) at pH 7.5 and 20 °C, 5 mM pyruvate (with 2 mM Mg 2+) has the opposite effect. A correlation between the coupling of N- and C-terminal domain unfolding, measured by differential scanning calorimetry, and the dimerization constant for EI, determined by sedimentation equilibrium, is observed. That is, when the coupling between N- and C-terminal domain unfolding produced by 0.2 or 1.0 mM PEP and 2 mM Mg 2+ is inhibited by 5 mM pyruvate, the dimerization constant for EI(H189A) decreases from >10 8 to <5 × 10 5 or 3 × 10 7 M −1, respectively. With 2 mM Mg 2+ at 15–25 °C and pH 7.5, PEP has been found to bind to one site/monomer of EI(H189A) with K A ′≅10 6 M −1 (Δ G′=−33.7±0.2 kJ mol −1 and Δ H=+16.3 kJ mol −1 at 20 °C with Δ C p =−1.4 kJ K −1 mol −1). The binding of PEP to EI(H189A) is synergistic with that of Mg 2+. Thus, physiological concentrations of PEP and Mg 2+ increase, whereas pyruvate and Mg 2+ decrease the amount of dimeric, active, dephospho-enzyme I.