No abstract

Enzyme I of the phosphoenolpyruvate - sugar phosphotransferase system (PTS) has been purified to homogeneity from Escherichia coli. A merodiploid strain P650 which had an extra copy of the gene for enzyme I resulting in a twofold increase in the amount of activity was used. The enzyme is a dimer of 67 000 +/- 5000 molecular weight subunits. At low protein concentration and 4 degrees C the monomer predominates, while at room temperature the dimer predominates. At higher protein concentrations (2 to 10 mg) this reversible temperature-dependent association-dissociation is not found. Enzyme I has a pH optimum of pH 7.2, a Km for HPr of 9 +/- 3 microM, a Km for phosphoenolpyruvate of 0.18 +/- 0.04 mM, and kinetics that are consistent with a bi bi Ping-Pong mechanism. No allosteric regulation of kinetic activity has been found. The amino acid composition has been determined and the epsilon 1% 280 nm is 4.4. Evidence suggests that the phosphorylated form of enzyme I is more stable.

The phosphocarrier protein HPr is a central component of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) that is responsible for carbohydrate uptake in many bacterial species. A number of three-dimensional structures of HPrs from both Gram-positive and Gram-negative bacteria have been determined; the overall folding topology of HPr is an open-faced beta-sandwich composed of three alpha-helices and a beta-sheet. A detailed structural comparison of these HPrs has been carried out. Besides the overall main chain folding, many detailed structural features are well conserved in all HPr structures. The three x-ray structures of HPrs from Escherichia coli, Streptococcus faecalis, and Bacillus subtilis show considerable overall similarity with respect to the positions of the C alpha atoms. A significant structural difference between HPrs from Gram-positive and Gram-negative bacteria is found in the region of Gly54, owing to the steric effects of Tyr37 in HPrs from the Gram-positive species. The region around Gly54 is involved in the binding of HPr to other PTS proteins and the differences in this region may be responsible for some of the poor functional complementation between HPrs from Gram-positive and Gram-negative species. The active center region, residues 12-18, appears to have significant differences in the comparisons between the overall structures. These differences support the proposal that phosphorylation and dephosphorylation of the active site His15 is accompanied by conformational changes. However, a local structural comparison of residues 12-18 from the x-ray structures of HPrs from E. coli and B. subtilis, and the two-dimensional nuclear magnetic resonance structure of B. subtilis HPr suggests that there is a conserved active center involving residues His15, Arg 17, and Pro18, which shows little conformational change during the phosphorylation cycle. The results of other experimental approaches, including site-directed mutagenesis and NMR spectroscopy, are in some cases difficult to rationalize with some of the details of the structures, but do appear to favour the conclusion that little conformational change occurs.

The histidine-containing phosphocarrier protein (HPr) of the phosphoenolpyruvate:sugar phosphotransferase system, when phosphorylated, contains a 1-phosphohistidinyl (1-P-histidinyl) residue (His-15). The properties of this 1-P-histidinyl residue were investigated by using phospho-HPr (P-HPr), P-HPr-1, and P-HPr-2. HPr-1 and HPr-2 are deamidated forms of HPr produced by boiling. In addition, HPr-1 produced during frozen storage was investigated. Both pH and temperature dependencies of the rate of hydrolysis of the phosphoryl group of the 1-P-histidinyl residue were investigated. The results show that the 1-P-histidinyl residue in HPr and HPr-1 has significantly different properties from free 1-P-histidine and that these differences are attributable to the active-site residues Glu-66 and Arg-17 and the pK of the imidazole group of the 1-P-histidinyl residue in P-HPr. The 1-P-histidinyl residue in P-HPr and P-HPr-1 shows a greater lability at physiological pH than the free amino acid. A proposal for the active site of P-HPr is made on the basis of these results and the recently obtained tertiary structure. In contrast, the hydrolysis properties of the 1-P-histidinyl residue in P-HPr-2 were similar to those obtained for either free 1-P-histidine or denatured P-HPr. The loss of activity that is associated with boiling HPr was shown to be due to HPr-2 formation as HPr-1 was found to be fully active.