The mannose transporter of bacterial phosphotransferase system mediates uptake of mannose, glucose, and related hexoses by a mechanism that couples translocation with phosphorylation of the substrate. Protein phosphorylation plays an important role in energy transduction, signal transduction and enzyme regulation. Ptype ATPases transport cations across the cell membrane and are transiently phosphorylated on an aspartic acid during turnover (1). Protein kinases and protein phosphatases regulate the activity of enzymes and membrane bound receptors by phosphorylation and dephosphorylation (2, 3). In the phosphoenolpyruvate-dependent phosphotransferase system of bacteria (PTS) 1 active transport and signaling are two functions of a protein phosphorylation cascade comprising four phosphoprotein units. The four components, enzyme I, HPr, IIA, and IIB sequentially transfer phosphoryl groups from phosphoenolpyruvate to carbohydrates that are accumulated across the cell membrane by a mechanism coupling translocation to phosphorylation. Whereas enzyme I and HPr are two energy coupling components, IIA and IIB together with the transmembrane IIC (and sometimes an additional IID) units form the sugar-specific transport complexes. IIA, IIB, and IIC occur either as protein subunits or as domains of a multidomain protein. Escherichia coli for example has over 30 genes for transporter complexes that differ in substrate specificity, amino acid sequence, and subunit/domain composition. The phosphorylation sites are histidines in enzyme I, HPr and the different IIA components, cysteines in the IIBs belonging to the glucose, mannitol, and lactose family, and histidines in the IIBs belonging to the mannose family of PTS transporters (for reviews see Refs. 4 and 5).Phosphate-binding sites and phosphate-catalytic sites frequently contain arginines, which by their guanidino group can stabilize phosphate through hydrogen bonding and electrostatic interactions (6). Amino acid sequence comparisons with respect to invariant arginines in PTS proteins from different bacteria reveal the following picture. The N-terminal domain of enzyme I has , which are completely invariant in 17 sequences. are close to the phosphorylation site His-189 (7). Arg-131 is conserved in 14 and Arg-136 is conserved in 15 sequences. The C-terminal domain has 7 completely invariant arginines (Protein Domain Data Base: http://protein.toulouse.inra.fr/prodom/ prodom.html). The functional role of these residues in enzyme I has not been explored. HPr is a 9-kDa open faced fourstranded antiparallel -sheet with three ␣-helices on one face. Arg-17, which is close to the active site His-15, is invariant in all 22 known sequences. When Arg-17 is replaced by Ser or Glu, phospho-donor and -acceptor activity of HPr are reduced to between 6% and less than 0.1% of the control (8). The phosphoryl group bound to His-15 of HPr is most likely complexed by the guanidino group of Arg-17, as inferred from its restricted conformational freedom observed upon phosphorylation of HPr, and fr...