Photoinactivation of the Staphylococcus aureus Lactose-Specific EIICB Phosphotransferase Component with p-azidophenyl-ß-D-Galactoside and Phosphorylation of the Covalently Bound Substrate.

ABSTRACT

BACKGROUND: The phosphoenolpyruvate (PEP)lactose phosphotransferase system of Staphylococcus aureus transports and phosphorylates lactose and various phenylgalactosides. Their phosphorylation is catalyzed by the Cys476-phosphorylated EIIB domain of the lactose-specific permease enzyme IICB (EIICBLac). Phosphorylation causes the release of galactosides bound to the EIIC domain into the cytoplasm by a mechanism not yet understood. RESULTS: Irradiation of a reaction mixture containing the photoactivatable p-azidophenyl-ß-D-galactopyranoside and EIICBLac with UV light caused a loss of EIICBLac activity. Nevertheless, photoinactivated EIICBLac could still be phosphorylated with [32P]PEP. Proteolysis of photoinactivated [32P]P-EIICBLac with subtilisin provided an 11-kDa radioactive peptide. Only the sequence of its first three amino acids (-H-G-P-, position 245-247) could be determined. They are part of the substrate binding pocket in EIICs of the lactose/cellobiose PTS family. Surprisingly, while acid treatment caused hydrolysis of the phosphoryl group in active [32P]P∼EIICBLac, photoinactivated [32P]P-EIICBLac remained strongly phosphorylated. CONCLUSION: Phosphorylation of the -OH group at C6 of p-nitrenephenyl-ß-D-galactopyranoside covalently bound to EIICLac by the histidyl-phosphorylated [32P]P∼EIIBLac domain is a likely explanation for the observed acid resistance. Placing p-nitrenephenyl-ß-D-galactopyranoside into the active site of modelled EIICLac suggested that the nitrene binds to the -NH- group of Ser248, which would explain why no sequence data beyond Pro247could be obtained.