31phospho-NMR demonstration of phosphocysteine as a catalytic intermediate on the Escherichia coli phosphotransferase system EIIMtl.
The mannitol-specific phosphotransferase system transport protein, Enzyme IIMtl, contains two catalytically important phosphorylated amino acid residues, both present on the cytoplasmic part of the enzyme. Recently, this portion has been subcloned, purified, and shown to be an enzymatically active domain. The N-terminal half has also been subcloned and shown to be the mannitol-binding domain. When combined the two domains catalyze mannitol phosphorylation at the expense of phospho-HPr (van Weeghel, R. P., Meyer, G. H., Pas, H. H., Keck, W. H., and Robillard, G. T., Biochemistry in press). The phospho-NMR spectrum of the purified phosphorylated cytoplasmic domain, taken at pH 8.0, shows two signals, one at -6.9 ppm compared with inorganic phosphate resulting from phosphohistidine and one at +11.9 ppm originating from phosphocysteine. Addition of mannitol plus membranes containing the N-terminal mannitol-binding domain results in the formation of mannitol 1-phosphate and the disappearance of the two signals at -6.9 and +11.9 ppm.
Pas
H H
HH
Department of Chemistry, Groningen, The Netherlands.
Meyer
G H
GH
Kruizinga
W H
WH
Tamminga
K S
KS
van Weeghel
R P
RP
Robillard
G T
GT
D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D003545:Cysteine / Q000031:analogs & derivatives* / Q000032:analysis; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009004:Monosaccharide Transport Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010758:Phosphorus; D010766:Phosphorylation; D010957:Plasmids
D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; C057779:S-phosphocysteine; D010758:Phosphorus; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli; D003545:Cysteine