Characterization of the protonation and hydrogen bonding state of the histidine residues in IIAmtl, a domain of the phosphoenolpyruvate-dependent mannitol-specific transport protein.
The A domain of the mannitol-specific EII, IIAmtl, was subcloned and proven to be functional in the isolated form (Van Weeghel et al., 1991). It contains a histidine phosphorylation site, the first of two phosphorylation sites in the parent protein. In this paper, we describe the characterization of the three histidine residues in IIAmtl with respect to their protonation and hydrogen bonding state, using 1H[15N] heteronuclear NMR techniques and protein selectively enriched with [delta 1,epsilon 2-15N]histidine. The active site residue has a low pKa (less than 5.8) and shows no hydrogen bond interactions. The proton in the neutral ring is located at the N epsilon 2 position, which also proved to be the site of phosphorylation. The phosphorylation raises the pKa of the active site histidine considerably but does not change the hydrogen bond situation. The other two histidine residues, one of which is probably located on the surface of the protein, were also characterized. Both show hydrogen bond interactions in the unphosphorylated protein, but these are disturbed by the phosphorylation process. These observations, combined with small changes in pKa and titration behavior, indicate that the IIAmtl changes its conformation upon phosphorylation.
Van Dijk
A A
AA
Department of Biochemistry, State University of Groningen, The Netherlands.
Scheek
R M
RM
Dijkstra
K
K
Wolters
G K
GK
Robillard
G T
GT
D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006639:Histidine / Q000737:chemistry*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008353:Mannitol / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry*; D011487:Protein Conformation
D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D008353:Mannitol; D006639:Histidine; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli