Enzyme IIMtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: identification of the activity-linked cysteine on the mannitol carrier.
The cysteines of the membrane-bound mannitol-specific enzyme II (EIIMtl) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system have been labeled with 4-vinylpyridine. After proteolytic breakdown and reversed-phase HPLC, the peptides containing cysteines 110, 384, and 571 could be identified. N-Ethylmaleimide (NEM) treatment of the native unphosphorylated enzyme results in incorporation of one NEM label per molecule and loss of enzymatic activity [Roossien, F. F., & Robillard, G. T. (1984) Biochemistry 23, 211-215]. NEM treatment and inactivation prevented 4-vinylpyridine incorporation into the Cys-384-containing peptide, identifying this residue as the activity-linked cysteine. Both oxidation and phosphorylation of the native enzyme protected the enzyme against NEM labeling of Cys-384. Positive identification of the activity-linked cysteine was accomplished by inactivation with [14C]iodoacetamide, proteolytic fragmentation, isolation of the peptide, and amino acid sequencing.
D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology; D005033:Ethylmaleimide; D008353:Mannitol / Q000378:metabolism; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D010766:Phosphorylation; D013329:Structure-Activity Relationship
D010446:Peptide Fragments; D008353:Mannitol; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D003545:Cysteine; D005033:Ethylmaleimide