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 IIGlc of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The uncoupled enzymes IIGlc 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. Km values of the uncoupled enzymes IIGlc for glucose ranged from 0.5 to 2.5 mM, 2 orders of magnitude higher than the value of normal IIGlc. Most of the mutant proteins were still able to phosphorylate glucose and methyl alpha-glucoside (a non-metabolizable glucose analog specific for IIGlc), indicating that transport and phosphorylation are separable functions of the enzyme. Some of the uncoupled enzymes IIGlc transported glucose with a higher rate and lower apparent Km 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 IIGlc in the presence of PTS-mediated phosphoryl transfer resembled those of wild-type IIGlc, 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 IIGlc. These results suggest that this hydrophilic loop is involved in binding and translocation of the sugar substrate.
Ruijter
G J
GJ
E. C. Slater Institute for Biochemical Research, University of Amsterdam, The Netherlands.
van Meurs
G
G
Verwey
M A
MA
Postma
P W
PW
van Dam
K
K
D001483:Base Sequence; D001665:Binding Sites; D001692:Biological Transport; D002874:Chromosome Mapping; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D005947:Glucose / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation / Q000235:genetics*; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics*; D010766:Phosphorylation
D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C031629:phosphoenolpyruvate-glucose phosphotransferase; D005947:Glucose