Results of operative and nonoperative treatment were equivalent.

No abstract

The transmembrane subunit of the glucose transporter, IICB(Glc), mediates vectorial transport with concomitant phosphorylation of glucose. Glucose phosphorylation proceeds through a cystein phosphate intermediate of the cytosolic IIB domain of IIC(Glc), which is phosphorylated by the IIA(Glc) subunit of the glucose transporter. Two- and three-dimensional NMR experiments were used to characterize the phosphorylation of the 10 kDa subclonal IIB domain and the complementary binding interfaces of [15N]IIB and [15N]IIA(Glc). The largest chemical shift perturbations and the only NOE differences accompanying IIB phosphorylation are confined to the active site residue Cys35, as well as Ile36, Thr37, Arg38, Leu39, and Arg40, which are all located in the turn between strands beta1 and beta2 and on beta2 itself. The significant increase of the amide cross-peak intensities of Ile36, Thr37, and Arg38 upon phosphorylation suggests that the conformational freedom of these groups becomes restrained, possibly due to hydrogen bonding to the oxygens of the bound phosphate and to interactions between the guanidinium group of Arg38 and the phosphoryl group. The residues of IIB which experience chemical shift perturbations upon binding of IIA are located on a protruding surface formed by residues of strands beta1, beta2, and beta4, the beta4/alpha3 loop, and residues from the first two turns of alpha3. The corresponding binding surface of the IIA(Glc) domain is comprised of residues on five adjacent beta-strands and two short helices surrounding the active site His90. The binding surface of IIA(Glc) for IIB coincides with the binding surface for HPr, the phosphoryl carrier protein by which IIA(Glc) is phosphorylated [Chen, Y., Reizer, J., Saier, M. H., Fairbrother, W. J., & Wright, P. E. (1993) Biochemistry 32, 32-37].

The glucose transporter of the bacterial phosphotransferase system (PTS) consists of a hydrophilic (IIAGlc ) and a transmembrane subunit (IICB Glc ). IICB Glc has two domains (C and B), which are linked by a highly invariant sequence. Transport of glucose by IIC and phosphorylation by IIB are tightly coupled processes. Three motifs that are strongly conserved in 12 homologous PTS transporters, namely two invariant arginines (Arg-424 and Arg-426) adjacent to the phosphorylation site (Cys-421), the invariant interdomain sequence KT-PGRED, and two conserved histidines (His-211 and His-212) in the IIC domain were mutated and the mutant proteins characterized in vivo and in vitro for transport and phosphorylation activity. Replacement of the strongly ␤-turn favoring residues Thr and Gly of the linker by ␣-helix favoring Ala results in strong reduction of activity, whereas the substitutions of the other residues have only minor effects. The R424K and R426K mutants can be phosphorylated by IIA Glc but can no longer donate the phosphoryl group to glucose. The H211Q and H212Q mutants continue to phosphorylate glucose at a reduced rate but H212Q can no longer transport glucose. Mixtures of purified R424K/H212Q and R426K/H212Q have 10% of wild-type phosphorylation activity and when coexpressed in Escherichia coli support glucose transport.