Stoichiometry and Substrate Affinity of the Mannitol Transporter, EnzymeII^sup mtl^, from Escherichia coli

ABSTRACT

Uptake and consecutive phosphorylation of mannitol in Escherichia coli is catalyzed by the mannitol permease Enzymell^sup mtl^. The substrate is bound at an extracellular-oriented binding site, translocated to an inward-facing site, from where it is phosphorylated, and subsequently released into the cell. Previous studies have shown the presence of both a high- and a lowaffinity binding site with K^sub D^-values in the nano- and micromolar range, respectively. However, reported K^sub D^-values in literature are highly variable, which casts doubts about the reliability of the measurements and data analysis. Using an optimized binding measurement system, we investigated the …