Non-vectorial phosphorylation by the bacterial PEP-dependent phosphotransferase system is an artifact of spheroplast and membrane vesicle preparation procedures

These data demonstrate that the phenomenon of non-vectorial phosphorylation [Saier, M.H. jr and Schmidt, M.R. (1981) J. Bacteriol. 145, 391–397] is an experimental artifact which arises during the conversion of whole cells to spheroplasts or membrane vesicles. It can be explained by a certain population of particles which are not properly sealed. There is no need to envoke two orientations of enzyme II in the membrane, one of which would have no physiological significance.     

Escherichia coli phosphoenolpyruvate dependent phosphotransferase system. NMR studies of the conformation of HPr and P-HPr and the mechanism of energy coupling.

1H and 31P nuclear magnetic resonance investigations of the phosphoprotein intermediate P-HPr and the parent molecule HPr of the E. coli phosphoenolpyruvate dependent phosphotransferase system (PTS) show that HPr can exist in two conformations. These conformations influence the protonation state of the reactive histidine residue, thereby determining the reaction pathway in the phosphoryl group transfer step. A general mechanism is proposed for the energy-coupling process in the PTS.     

Details of mannitol transport in Escherichia coli elucidated by site-specific mutagenesis and complementation of phosphorylation site mutants of the phosphoenolpyruvate-dependent mannitol-specific phosphotransferase system

The mannitol transport protein (EIImtl) carries out translocation with concomitant phosphorylation of mannitol from the periplasm to the cytoplasm, at the expense of phosphoenolpyruvate (PEP). The phosphoryl group which is needed for this group translocation is sequentially transferred from PEP via two phosphorylation sites, located exclusively on the C-terminal cytoplasmic domain, to mannitol. Oligonucleotide-directed mutagenesis was used to investigate the precise role of these sites in phosphoryl group transfer, by producing specific amino acid substitutions. The first phosphorylation site, His-554 (P1), was replaced by Ala, which renders the EII-H554A completely inactive in PEP-dependent mannitol phosphorylation, but not in mannitol/mannitol 1-phosphate exchange. The P2 site mutant, EII-C384S, was inactive both in the mannitol phosphorylation reaction and in the exchange reaction, due to replacement of the essential Cys-384 by Ser. Although EII-H554A and EII-C384S were both catalytically inactive in the PEP-dependent phosphorylation, EII-C384S was able to restore up to 55% of the wild-type mannitol phosphorylation activity with the EII-H554A mutant, indicating a direct phosphotransfer between two subunits. These phosphorylation data together with the data obtained from mannitol/mannitol phosphate exchange kinetics, after mixing EII-H554A and EII-C384S, indicated the formation of functionally stable heterodimers, which consist of an EII-H554A and an EII-C384S monomer.