The Phosphoenolpyruvate‐Dependent Phosphotransferase System of Staphylococcus aureus

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Abstract
Upon nitration of the phosphocarrier protein HPr, 3 nitrated derivatives of the protein were isolated: mononitrated HPr, dinitrated HPr, dinitrated HPr and trinitrated HPr. Tryptic digestion of the derivatives leads to nitrotyrosine-containing peptides which were isolated and characterized by amino acid analysis. This resulted in the determination of the positions of the nitrated tyrosyl residues in the amino acid sequence. In mononitrated HPr only Tyr-56 was modified, in dinitrated HPr both Tyr-56 and Tyr-37 reacted with the nitrating agent; modification of all 3 tyrosyl residues in trinitrated HPr required more drastic reaction conditions. The NMR spectra of the 3 derivatives allowed the assignments of the tyrosine resonances as follows: Tyr-A and Tyr-B with pK values of 10.5 and 11.5 were designated Tyr-56 and Tyr-37 whereas Tyr-C, whose protons are not titratable before denaturation of the protein, was assigned to Tyr-6 in the amino acid sequence. The nitration studies, together with the titration behavior of the 3 tyrosines, indicate the topology of the tyrosyl residues to be as follows: Tyr-56 is located at the surface, Tyr-37 is slightly buried and Tyr-6 is deeply buried. The nitrotyrosyl derivatives retain their biological activity.

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