ANALYSIS OF MUTATIONS THAT UNCOUPLE TRANSPORT FROM PHOSPHORYLATION IN ENZYME-IIGLC OF THE ESCHERICHIA-COLI PHOSPHOENOLPYRUVATE-DEPENDENT PHOSPHOTRANSFERASE SYSTEM

Mutations that uncouple glucose transport from phosphorylation were isolated in plasmid-encoded Escherichia coli enzyme II(Glc) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The uncoupled enzymes II(Glc) were able to transport glucose in the absence of the general phosphoryl-carrying proteins of the PTS, enzyme I and HPr, although with relatively low affinity. K(m) values of the uncoupled enzymes II(Glc) for glucose ranged from 0.5 to 2.5 mM, 2 orders of magnitude higher than the value of normal II(Glc). Most of the mutant proteins were still able to phosphorylate glucose and methyl alpha-glucoside (a non-metabolizable glucose analog specific for II(Glc)), indicating that transport and phosphorylation are separable functions of the enzyme. Some of the uncoupled enzymes II(Glc) transported glucose with a higher rate and lower apparent K(m) in a pts+ strain than in a DELTA-ptsHI strain lacking the general proteins enzyme I and HPr. Since the properties of these uncoupled enzymes II(Glc) in the presence of PTS-mediated phosphoryl transfer resembled those of wild-type II(Glc), these mutants appeared to be conditionally uncoupled. Sequencing of the mutated ptsG genes revealed that all amino acid substitutions occurred in a hydrophilic segment within the hydrophobic N-terminal part of II(Glc). These results suggest that this hydrophilic loop is involved in binding and translocation of the sugar substrate.