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,...

The first two reactions in the phosphotransfer sequence of bacterial phosphoenolpyruvate:glycose phosphotransferase systems are the autophosphorylation of Enzyme I by phosphoenolpyruvate followed by the transfer of the phospho group to the low-molecular weight protein, HPr. Transient state kinetic methods were used to estimate the second-order rate constants for both phosphotransfer reactions. These measurements support previous conclusions that only the dimer of Enzyme I, EI2, is autophosphorylated, and that the rate of formation of dimer is slow compared to the rate of its phosphorylation. The rate constants of the two autophosphorylation reactions of EI2 by PEP are 6.6 x 10(6) M(-1) s(-1), and differ from one another by a factor of less than 3. The rate constant for the transfer reaction between phospho-EI2 and HPr is unusually large for a covalent reaction between two proteins (220 x 10(6) M(-1) s(-1)), while the constant for the reverse reaction is 4.2 x 10(6) M(-1) s(-1). Using the previously reported equilibrium constant for the autophosphorylation reaction, 1.5, the overall equilibrium constant for phosphotransfer from PEP to HPr is 80, somewhat higher than that previously reported. The results also show that EI2 can phosphorylate multiple molecules of HPr without dissociating to a monomer (EI), and that EI can accept a phospho group from phospho-HPr. These results are directly applicable to predicting the rates of phosphoenolpyruvate phosphotransferase system sugar uptake in whole cells.

The levels of histidine-containing protein HPr and enzyme I of the phosphoenolpyruvate-sugar phosphotransferase system of Escherichia coli strains 1100, NC3, W3110, and P650 and Salmonella typhimurium strains SB3507 and LJ144 have been determined by quantitative sugar phosphorylation assay and immunochemically. The levels have been determined for cells grown on minimal salts with glucose, fructose, mannitol, glycerol, and lactate and on nutrient broth. All determinations indicate a two- to three-fold change in the levels of enzyme I and HPr between growth on hexoses, which gave the higher levels, and the other growth substrates. The highest levels were not always found in glucose-grown cells. Antibodies were produced in rabbits using purified proteins from E. coli P650. The activity measurements and immunochemically determined enzyme I protein gave specific activities in the crude extracts of E. coli strains which were similar to that of the pure enzyme. The wild-type S. typhimurium enzyme I in crude extracts did not have the same immunochemical reactivity, although there was a considerable cross-reaction and the specific activity appeared to be half that of pure enzyme I. The HPr from both E. coli and S. typhimurium behaved identically and, although the immunoprecipitation was weak, it did indicate that HPr assays may not be as reliable as the enzyme I assays. The relative amounts of enzyme I and HPr found indicate that there are between 10- and 20-fold more HPr molecules in a cell than enzyme I subunits which form active dimers.

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