Sugar transport by the bacterial phosphotransferase system. Phosphoryl transfer reactions catalyzed by enzyme I of Salmonella typhimurium.

The phosphorylation of Enzyme I is the first step in the phosphotransfer reaction sequence catalyzed by the phosphoenolpyruvate:glycose phosphotransferase system (PTS) from Salmonella typhimurium. The characterization of phospho approximately Enzyme I and the reactions in which it participates are described in this report. About 1 mol of phosphoryl group was incorporated per mol of Enzyme I monomer when the homogeneous enzyme was incubated with [32]phosphoenolpyruvate and Mg2+. The phosphoryl group in phospho approximately Enzyme I is linked at the N-3 position in the imidazole ring of a histidine residue. Phospho approximately Enzyme I donates its phosphoryl group to pyruvate (to form phosphoenolpyruvate (P-enolpyruvate)) and to the histidine-containing phosphocarrier protein of the phosphotransferase system (HPr) (to form phospho approximately HPr). In the presence of HPr and appropriate sugar-specific proteins, the phosphoryl group can be transferred from Enzyme I to methyl alpha-glucoside (to form sugar-phosphate). The phosphorylation of Enzyme I by phosphoenolpyruvate requires divalent cation, but the phosphoryl group is transferred from phospho approximately Enzyme I to HPr in the presence of 20 mM EDTA. Kinetic studies show a biphasic rate for Enzyme I phosphorylation, suggesting that the enzyme is phosphorylated in the associated state. Equilibrium experiments were conducted on the following Reactions A and C. (formula: see text). The apparent K' for Reaction B was calculated from K'A and K'C. K'C was found to be about 11. K'A was studied both at very low and high substrate (P-enolpyruvate and pyruvate) concentrations relative to their respective Km values. At low substrate concentrations, the reaction appeared independent of pH in the range of 6.5 to 8.0, and when analyzed according to the simplest expression that could be written for total species of each component (Reaction A), the apparent average K' was 1.5. At high substrate concentrations, about 50% of the Enzyme I was phosphorylated, and this value changed only slightly with large changes in the P-enolpyruvate to pyruvate ratio. Expressions for K'A are derived which partially explain these results by including enzyme-substrate complexes in the equilibrium expression. The K' values were used to derive apparent standard free energy changes for the hydrolysis of the phosphoproteins of the PTS. Since these are similar to those for the hydrolysis of P-enolpyruvate, the phosphate transfer potentials of the PTS phosphoproteins are among the highest of known biological phosphate derivatives. In addition, unlike the reactions which occur during anaerobic glycolysis and electron transport, the high phosphate transfer potential is conserved in the PTS reaction sequence until the last step, the translocation of the sugar substrate across the membrane concomitant with its phosphorylation. Potential regulation of the PTS, in particular the effect of the intracellular ratio of P-enolpyruvate to pyruvate, is considered.