title: Opposing effects of phosphoenolpyruvate and pyruvate with Mg(2+) 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 degrees 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 x 10(5) or 3 x 10(7) M(-1), respectively. Incubation of the wild-type, dephospho-enzyme I with the transition-state analog phosphonopyruvate and 2 mM Mg(2+) also increases domain coupling and the dimerization constant approximately 42-fold. With 2 mM Mg(2+) at 15-25 degrees C and pH 7.5, PEP has been found to bind to one site/monomer of EI(H189A) with K(A)' approximately 10(6) M(-1) (deltaG' = -8.05 +/- 0.05 kcal/mole and deltaH = +3.9 kcal/mole at 20 degrees C); deltaC(p) = -0.33 kcal K(-1) mole(-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.
authors.affiliation: Section on Protein Chemistry, Laboratory of Biochemistry, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, Maryland 20892-8012, USA.
authors.forename: Mariana N
authors.initials: MN
authors.lastname: Dimitrova
authors.forename: Alan
authors.initials: A
authors.lastname: Peterkofsky
authors.forename: Ann
authors.initials: A
authors.lastname: Ginsburg
mesh_terms: D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000737:chemistry*; D010728:Phosphoenolpyruvate / Q000494:pharmacology*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D019289:Pyruvic Acid / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D014461:Ultracentrifugation
chemical_list: D010728:Phosphoenolpyruvate; D019289:Pyruvic Acid; D010770:Phosphotransferases; D008274:Magnesium