Properties of the C-terminal Domain of Enzyme I of the Escherichia coli Phosphotransferase System

Abstract

The bacterial phosphoenolpyruvate (PEP):glycose phosphotransferase system (PTS) mediates uptake/phosphorylation of sugars. The transport of all PTS sugars requires Enzyme I (EI) and a phosphocarrier histidine protein of the PTS (HPr). The PTS is stringently regulated, and a potential mechanism is the monomer/dimer transition of EI, because only the dimer accepts the phosphoryl group from PEP. EI monomer consists of two major domains, at the N and C termini (EI-N and EI-C, respectively). EI-N accepts the phosphoryl group from phospho-HPr but not PEP. However, it is phosphorylated by PEP(Mg 2؉ ) when complemented with EI-C. Here we report that the phosphotransfer rate increases ϳ25-fold when HPr is added to a mixture of EI-N, EI-C, and PEP(Mg 2؉ ). A model to explain this effect is offered. Sedimentation equilibrium results show that the association constant for dimerization of EI-C monomers is 260-fold greater than the K a for native EI. The ligands have no detectable effect on the secondary structure of the dimer (far UV CD) but have profound effects on the tertiary structure as determined by near UV CD spectroscopy, thermal denaturation, sedimentation equilibrium and velocity, and intrinsic fluorescence of the 2 Trp residues. The binding of PEP requires Mg 2؉ . For example, there is no effect of PEP on the T m , an increase of 7°C in the presence of Mg 2؉ , and ϳ14°C when both are present. Interestingly, the dissociation constants for each of the ligands from EI-C are approximately the same as the kinetic (K m ) constants for the ligands in the complete PTS sugar phosphorylation assays.The best characterized function of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) 4 is in the uptake of sugars by bacterial cells, which are phosphorylated during their translocation across the cytoplasmic membrane (for reviews see Refs. 1-3). There are many variants of the PTS, but all of them require the first two proteins in the phosphotransfer process, Enzyme I (EI) and the low molecular weight phosphocarrier protein, HPr. The first two steps in the reaction sequence are depicted in Reactions 1 and 2.The phosphoryl group is then transferred from P-HPr to the sugarspecific proteins. The properties of EI have been reviewed previously (4, 5).As discussed in an earlier report (6), the phosphotransfer potential of PEP is so high that the PTS must be stringently regulated or cells would burst with accumulated sugar phosphate. One potential mechanism for this regulation is dependent on the properties of the monomer/dimer transition of EI. The dimer accepts the phosphoryl group from PEP, whereas the monomer does not, and association/dissociation is a very slow process relative to the phosphotransfer reactions. A report on the kinetics of EI phosphorylation and dimerization has recently been published (7), and the hydrodynamic properties of the monomer/dimer transition are described in the companion paper (36).Early work on thermal unfolding of EI, using high sensitivity scanning calorimetry and partial proteolysis,...

Calorimetric and spectroscopic investigation of the interaction between the C‐terminal domain of Enzyme I and its ligands

“…The absence of proteolysis of EIC 270-575 is possibly due to its formation of an inclusion body before proteolysis took place during the overexpression. Our result is consistent with previous studies, 12,13 and indicates that keeping the linker helix is critical to confer proteolytic stability on EIC, while the short b bridge is important for proper folding of EIC. EIC 231-575 was stable during the purification using chromatography at a room temperature, and used for all experimental measurements hereafter.…”

“…It has been reported that EIC can phosphorylate EIN from a biochemical assay using 32 P-PEP as a substrate. 12,16 When we added PEP to the mixture of EIN and EIC 231-575 after titration, we could immediately observe the chemical shift changes due to the phosphorylation reaction of EIN [red cross-peaks in Fig. 2(A)].…”

“…It is known that the dimeric form of EI is the active form and the monomer-dimer equilibrium can vary with experimental conditions. 12,20,21 Previously, the K D for the dimerization of EIC 256-575 in the presence of 5 mM MgCl 2 and 100 mM NaCl at pH 7.5 and 25 C was reported as $6 nM from analytical ultracentrifugation. 12 When we loaded a serial dilution of Fig.…”

“…Erni group determined the first crystal structure of EIC of a thermophilic bacterium, Thermo tengcongensis, 11 and then Roseman group successfully isolated EIC of Escherichia coli using an intein system and characterized its interaction with the ligands by spectroscopic and kinetic analysis. 12 Isolation of EIC of E. coli, however, still suffered from proteolysis during expression, and protein ligation was the only method of choice. 12 Here we have expressed multiple constructs of EIC of E. coli varying the linker region between EIN and EIC, and obtained a construct that expresses stable EIC in an intact form.…”

“…12 Isolation of EIC of E. coli, however, still suffered from proteolysis during expression, and protein ligation was the only method of choice. 12 Here we have expressed multiple constructs of EIC of E. coli varying the linker region between EIN and EIC, and obtained a construct that expresses stable EIC in an intact form. We have characterized the interaction of EIC with its substrates and inhibitor, and also with EIN using calorimetry, circular dichroism (CD), and NMR spectroscopy.…”