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Abstract

1H NMR studies of the phosphocarrier protein HPr and its three nitrotyrosyl derivatives revealed some structural features which may finally lead to an explanation of the mechanism of the phospho-transfer reaction. Titration studies on mononitrated, dinitrated, and trinitrated derivatives--i.e., derivatives with Tyr-56, Tyr-56 and Tyr-37, and Tyr-56, Tyr-37, and Tyr-6 modified--have been performed. The three tyrosyl residues seem to be in positions completely different from each other with respect to their solvent accessibility; Tyr-56 seems to be located near the surface of the protein, Tyr-6 seems to be completely buried, and Tyr-37 takes an intermediate position. Tyr-6 contributes to the core structure of the protein. A resonance at -0.18 ppm could be shown to correspond to a CH3 group of a valine. Nuclear Overhauser experiments revealed its being close to Tyr-6. One of the resonances tentatively assigned to methionine SCH3 groups titrates in the dinitrated derivative with the same pK as nitrotyrosyl residue 37. The titration behavior of the active-center histidyl residue suggests a hydrogen bond to the imidazole ring, possibly from Tyr-56 or Arg-17.