Phosphorylation site mutants of the mannitol transport protein enzyme IImtl of Escherichia coli: studies on the interaction between the mannitol translocating C-domain and the phosphorylation site on the energy-coupling B-domain.
Mannitol binding and translocation catalyzed by the C domain of the Escherichia coli mannitol transport protein enzyme IImtl is influenced by domain B. This interaction was studied by monitoring the effects of mutating the B domain phosphorylation site, C384, on the kinetics of mannitol binding to the C domain. The dissociation constants for mannitol to the C384 mutants in inside-out membrane vesicles varied from 45 nM for the wild-type enzyme to 306 nM for the mutants. The rate constants pertinent to the binding equilibrium were also altered by the mutations. The association rate of mannitol to the cytoplasmic binding site in the mutants was accelerated for all mutants. The exchange rate of bound mannitol on the wild-type enzyme was shown to be pH dependent with a pKa of approximately 8 and increasing rates at higher pH. This rate was increased for all the mutants, but the pKas differed for the various mutants. The exchange rate for binding to the isolated IICmtl, however, was not pH dependent and exhibited a low rate. Exchange measured at 4 degrees C showed that, of the two steps, binding and occlusion, involved in binding to wild-type EIImtl in inside-out vesicles, only one could be detected for the C384E and C384L mutants. This suggests that the mutations increased the rate of the occlusion step so that it was no longer separable from the initial binding step or that the mutations eliminated the occlusion step altogether. The change in the mannitol binding kinetics of the C domain indicates that the B and C domains of EIImtl influence each other's conformation.(ABSTRACT TRUNCATED AT 250 WORDS)
Boer
H
H
Department of Biochemistry, University of Groningen, The Netherlands.
ten Hoeve-Duurkens
R H
RH
Lolkema
J S
JS
Robillard
G T
GT
D001483:Base Sequence; D001665:Binding Sites; D001693:Biological Transport, Active; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008353:Mannitol / Q000378:metabolism*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D013816:Thermodynamics
D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D008353:Mannitol; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli