The histidine phosphocarrier protein (HPr) is an essential element in sugar transport by the bacterial phosphoenolpyruvate:sugar phosphotransferase system. Ligand fishing, using surface plasmon resonance, was used to show the binding of HPr to a nonphosphotransferase protein in extracts of Escherichia coli; the protein was subsequently identified as glycogen phosphorylase (GP). The high affinity (association constant ϳ10 M ؊1), species-specific interaction was also demonstrated in electrophoretic mobility shift experiments by polyacrylamide gel electrophoresis. Equilibrium ultracentrifugation analysis indicates that HPr allosterically regulates the oligomeric state of glycogen phosphorylase. HPr binding increases GP activity to 250% of the level in control assays. Kinetic analysis of coupled enzyme assays shows that the binding of HPr to GP causes a decrease in the K m for glycogen and an increase in the V max for phosphate, indicating a mixed type activation. The stimulatory effect of E. coli HPr on E. coli GP activity is species-specific, and the unphosphorylated form of HPr activates GP more than does the phosphorylated form. Replacement of specific amino acids in HPr results in reduced GP activation; HPr residues Arg-17, Lys-24, Lys-27, Lys-40, Ser-46, Gln-51, and Lys-72 were established to be important. This novel mechanism for the regulation of GP provides the first evidence directly linking E. coli HPr to the regulation of carbohydrate metabolism.

The unphosphorylated form of enzyme IIAglc of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system inhibits transport catalyzed by lactose permease. We (Seok et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 13515-13519) previously characterized the area on the cytoplasmic face of lactose permease that interacts with enzyme IIAglc, using radioactive enzyme IIAglc. Subsequent studies (Sondej et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 3525-3530) suggested consensus binding sequences on proteins that interact with enzyme IIAglc. The present study characterizes a region on the surface of enzyme IIAglc that interfaces with lactose permease. Acetylation of lysine residues by sulfosuccinimidyl acetate treatment of enzyme IIAglc, but not lactose permease, reduced the degree of interaction between the two proteins. To localize the lysine residue(s) on enzyme IIAglc that is(are) involved in the regulatory interaction, selected lysine residues were mutagenized. Conversion of nine separate lysines to glutamic acid resulted in proteins that were still capable of phosphoryl acceptance from HPr. Except for Lys69, all the modified proteins were as effective as the wild-type enzyme IIAglc in a test for binding to lactose permease. The Lys69 mutant was also defective in phosphoryl transfer to glucose permease. To derive further information concerning the contact surface, additional selected residues in the vicinity of Lys69 were mutagenized and tested for binding to lactose permease. On the basis of these studies, a model for the region of the surface of enzyme IIAglc that interacts with lactose permease is proposed.