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Opposing effects of phosphoenolpyruvate and pyruvate with Mg²⁺ on the conformational stability and dimerization of phosphotransferase enzyme I from Escherichia coli

¹ Section on Protein Chemistry, Laboratory of Biochemistry, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, Maryland 20892-8012, USA
² Laboratory of Cell Biology (LCB), National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, Maryland 20892-8017, USA

Ann Ginsburg, National Heart, Lung, and Blood Institute, National Institutes of Health, 50 South Drive, Room 2339, Bethesda, MD 20892-8012, USA; e-mail: ginsbura{at}nhlbi.nih.gov; fax: (301) 480-5492.

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 His189, 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 20°C, 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 > 10⁸ to < 5 x 10⁵ or 3 x 10⁷ M^-1, respectively. Incubation of the wild-type, dephospho-enzyme I with the transition-state analog phosphonopyruvate and 2 mM Mg²⁺ also increases domain coupling and the dimerization constant 42-fold. With 2 mM Mg²⁺ at 15–25°C and pH 7.5, PEP has been found to bind to one site/monomer of EI(H189A) with K_A' 10⁶ M^-1 (G' = -8.05 ± 0.05 kcal/mole and H = +3.9 kcal/mole at 20°C); C_p = -0.33 kcal K^-1 mole^-1. The binding 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 I.

Keywords: Enzyme I; E. coli phosphoenolpyruvate:sugar phosphotransferase system; active-site mutant (EI[H189A]); phosphoenolpyruvate; pyruvate; Mg²⁺; phosphonopyruvate; differential scanning calorimetry; Trp fluorescence; sedimentation equilibrium; dimerization; isothermal titration calorimetry

Abbreviations: PEP, phosphoenolpyruvate • Pyr, pyruvate • PPyr, phosphonopyruvate • PTS, phosphoenolpyruvate:sugar phosphotransferase system • EI, enzyme I (575 amino acid residues) of the PTS • dephospho-EI, nonphosphorylated enzyme I • phospho-EI, phosphorylated enzyme I containing phospho-His189 • EI(H189A), EI in which the active-site His189 has been replaced by alanine • HPr, histidine-containing phosphocarrier protein (9 kD) • ME, 2-mercaptoethanol • 2,4-DNPH, 2,4-dinitrophenylhydrazine • Hepes, N-(2-hydroxyethyl)piperazine-N'-2-ethane-sulfonic acid • Trp fluorescence, intrinsic tryptophanyl residue fluorescence • DSC, differential scanning calorimetry • ITC, isothermal titration calorimetry • K1,2, dimerization constant expressed per mole monomer of dephospho-EI or EI(H189A)

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