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: PEP Enzyme I HPr IIA^Glc IICB^Glc Glc. We previously reported two classes of IIA^Glc mutations that substantially decrease the P-transfer rate constants to/from IIA^Glc. (1) His75 adjoins the active site (His90), and the mutant H75Q was 0.5% as active as wild type IIA^Glc 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. (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. Since the rate constants for phosphotransfer to/from HPr were largely unaffected, we conclude that the H-bond network hypothesis is not correct. (2) In the second class of mutants, proteolytic truncation of seven residues of the IIA^Glc NH2-terminus caused a twenty-fold reduction in phosphotransfer to membrane-bound IICB^Glc from Salmonella typhimurium. Here, we report the phosphotransfer rates between two genetically constructed NH2-terminal truncations of IIA^Glc (delta7 and delta16) and the proteins IICB^Glc and IIB^Glc (the soluble cytoplasmic domain of IICB^Glc). The truncations did not significantly affect reversible P-transfer to IIB^Glc, but substantially decreased the rate constants to IICB^Glc in E. coli and S. typhimurium membranes. The results support the hypothesis that the N-terminal 18 residue domain "docks" IIA^Glc to the lipid bilayer of membranes containing IICB ^Glc. Wang, G., Peterkofsky, A., and Clore, G. M. (2000) J.Biol.Chem. 275, 39811-39814