Effects of mutations and truncations on the kinetic Behavior of IIAGlc, a phosphocarrier and regulatory protein of the phosphoenolpyruvate phosphotransferase system of Escherichia coli

IIA(Glc), a component of the glucose-specific phosphoenolpyruvate: phosphotransferase system ( PTS) of Escherichia coli, is important in regulating carbohydrate metabolism. In Glc uptake, the phosphotransfer sequence is: phosphoenolpyruvate -> Enzyme -> I HPr -> IIA(Glc) -> IICBGlc -> Glc. ( HPr is the first phosphocarrier protein of the PTS.) We previously reported two classes of IIAGlc mutations that substantially decrease the P-transfer rate constants to/from IIA(Glc). A mutant of His(75) which adjoins the active site ( His(90)), ( H75Q), was 0.5% as active as wild-type IIAGlc in the reversible P-transfer to HPr. Two possible explanations were offered for this result: ( a) the imidazole ring of His75 is required for charge delocalization and ( b) H75Q disrupts the hydrogen bond network: Thr73, His75, phospho-His90. The present studies directly test the H-bond network hypothesis. Thr73 was replaced by Ser, Ala, or Val to eliminate the network. Because the rate constants for phosphotransfer to/from HPr were largely unaffected, we conclude that the H-bond network hypothesis is not correct. In the second class of mutants, proteolytic truncation of seven residues of the IIAGlc N terminus caused a 20-fold reduction in phosphotransfer to membrane-bound IICBGlc from Salmonella typhimurium. Here, we report the phosphotransfer rates between two genetically constructed N-terminal truncations of IIAGlc ( Delta 7 and Delta 16) and the proteins IICBGlc and IIBGlc ( the soluble cytoplasmic domain of IICBGlc). The truncations did not significantly affect reversible P-transfer to IIBGlc but substantially decreased the rate constants to IICBGlc in E. coli and S. typhimurium membranes. The results support the hypothesis ( Wang, G., Peterkofsky, A., and Clore, G. M. ( 2000) J. Biol. Chem. 275, 39811-39814) that the N-terminal 18-residue domain docks IIAGlc to the lipid bilayer of membranes containing IICBGlc.