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 Mg2+-dependent autophosphorylation by PEP requires the homodimer. Using inactive EI(H189A), in which alanine is substituted for the active-site His 189, substrate binding effects can be separated from those of phosphorylation. Whereas 1 mM PEP (with 2 MM Mg2+) strongly promotes dimerization of EI(H189A) at pH 7.5 and 20degreesC, 5 mM pyruvate (with 2 mM Mg2+) 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 Mg2+ is inhibited by 5 mM pyruvate, the dimerization constant for EI(H189A) decreases from > 10(8) to <5 x 10(5) or 3 x 10(7) M-1, respectively. With 2 mM Mg2+ at 15-25degreesC and pH 7.5, PEP has been found to bind to one site/monomer of EI(H189A) with K'(A) congruent to 10(6) M-1 (DeltaG' = -33.7 +/- 0.2 kJ mol(-1) and DeltaH = +16.3 kJ mol(-1) at 20degreesC with DeltaC(p) = 1.4 kJ K-1 mol(-1)). The bindine of PEP to EI(H189A) is synergistic with that of Mg2+. Thus, physiological concentrations of PEP and Mg2+ increase, whereas pyruvate and Mg2+ decrease the amount of dimeric, active, dephospho-enzyme 1. (C) 2004 Elsevier B.V. All rights reserved.