Domain Stability in Enzyme I of the E. coli PEP:Sugar Phosphotransferase System

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The widespread bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) couples the translocation and phosphorylation of numerous sugars. The system is composed of two cytoplasmic proteins (enzyme I and HPr) that are used for all sugars as well as sugar specific, membrane-bound components known as enzymes II and III. PEP is the phosphoryl donor in the Mg(II)-dependent autophosphorylation of enzyme I on histidine 189.Thermal stabilities of enzyme I (63,562 Da subunit) of the E. coli PTS and a cloned amino terminal domain of enzyme I (EIN; 28,346 Da) have been investigated by differential scanning calorimetry (DSC) and far UV circular dichroism (CD) at pH 7.5. Previously, we showed that phosphorylation of the active-site His 189 destabilizes the amino terminal domain of enzyme I by ca. 7 C. A decrease in the conformational stability of the amino terminal domain by phosphorylation of His 189 promotes phosphotransfer to HPr (the next protein of the PTS). In order to investigate the effects of phosphorylation on the stability of full-length enzyme I and EIN further, we have produced active-site mutations (H189E, expected to have the properties of phosphorylated forms, and H189A) by protein bioengineering. DSC and temperature-induced changes in ellipticity at 222 nm for wild-type EIN(H189) and mutant EIN proteins show two-state unfolding for all three proteins in 10 mM K-phosphate (and 100 mM KCl), pH 7.5 with an unfolding enthalpy of 140 (160) kcal/mol and heat capacity change of 2.7 (3.3) kcal/(K mol). Corresponding transition temperature (Tm) values are 57 (59), 55 (58), and 53 (56) C for EIN(wt), EIN(H189A), and EIN(H189E), respectively. The order of overall conformational stability of dephospho- and phospho-His189 and His189 substitutions in EIN at pH 7.5 is His > Ala > Glu > His-P due to differences in conformational entropy, Thus, the introduction of a negative charge at the active site of EIN is the most destabilizing, and neutral salt has the greatest shielding effect on phospho-EIN and EIN(H189E). The binding of HPr produced a 3 degree stabilization in each case. Long-range interactions between the N- and C-terminal domains of intact enzyme I are being investigated. The C-terminal domain is necessary for dimerization of enzyme I and preliminary sedimentation equilibrium studies suggest that the monomer-dimer equilibrium is affected by phosphorylation of His 189 or by substitution of Glu for His at position 189 in the N-terminal domain. DSC results indicate that thermal unfolding and refolding reactions of the N- and C-terminal domains in the absence of substrates are weakly coupled energetically. Having the inactive EI(H189A) allows us to study the effects of binding the substrate Mg(II)-PEP to the C-terminal domain on the overall stability and dimerization potential of enzyme I. We are finding that in the presence of Mg(II)-PEP, the unfolding of N- and C-terminal domains is tightly coupled and both domains are markedly stabilized with large Tm increases. Moreover, the monomer-dimer association constant of EI(H189A) is increased 4-6 orders of magnitude by Mg(II)-PEP binding. This suggests that intracellular concentrations of Mg(II)-PEP determine the amount of enzyme I dimer for autophosphorylation.