EXPRESSION, PURIFICATION, AND KINETIC CHARACTERIZATION OF THE MANNITOL TRANSPORT DOMAIN OF THE PHOSPHOENOLPYRUVATE-DEPENDENT MANNITOL PHOSPHOTRANSFERASE SYSTEM OF ESCHERICHIA-COLI - KINETIC EVIDENCE THAT THE ESCHERICHIA-COLI MANNITOL TRANSPORT PROTEIN IS A FUNCTIONAL DIMER
H. Boer et al., EXPRESSION, PURIFICATION, AND KINETIC CHARACTERIZATION OF THE MANNITOL TRANSPORT DOMAIN OF THE PHOSPHOENOLPYRUVATE-DEPENDENT MANNITOL PHOSPHOTRANSFERASE SYSTEM OF ESCHERICHIA-COLI - KINETIC EVIDENCE THAT THE ESCHERICHIA-COLI MANNITOL TRANSPORT PROTEIN IS A FUNCTIONAL DIMER, The Journal of biological chemistry, 269(27), 1994, pp. 17863-17871
The overexpression of the membrane-bound C domain of the mannitol tran
sport protein EII(Mtl) of Escherichia coli has been achieved. This pro
tein, IICMtl, consisting of the first 346 amino acids, was purified fr
om membrane vesicles and still bound mannitol with a high affinity. Ge
l filtration experiments showed that purified IICMtl was a dimer, conf
irming that the interaction within the EII(Mtl) dimer occurs between t
he membrane bound portions of the protein. IICMtl in combination with
a chimeric protein consisting of the membrane-bound EII(Glc) C domain
and the cytoplasmic EII(Mtl) BA domain could restore both phosphoenolp
yruvate-dependent phosphorylation and mannitol/mannitol-P exchange act
ivity. The interaction in this complex was comparable to that of IICMt
l with soluble IIBA(Mtl) in as much as there appeared to be no specifi
c interaction between IICMtl and the membrane-bound EII(Glc) C domain;
the K-m of IICMtl for the chimer was so low that saturation could not
be achieved. In contrast, a very high affinity with a K-m of 2 nM was
measured between purified IICMtl and purified EII(Mtl). This interact
ion was manifested in a IICMtl-dependent stimulation of the EII(Mtl) c
atalyzed phosphoenolpyruvate dependent mannitol phosphorylation reacti
on and the mannitol/mannitol-P exchange reaction. The high affinity of
IICMtl for the wild type enzyme can be explained by the formation of
heterodimers consisting of a IICMtl monomer and an EII(Mtl) monomer wh
ich interact at the level of the membrane-bound domains. The 2-fold in
crease in mannitol phosphorylation activity of the hetero versus homod
imer is an indication that the individual subunits in the homodimer ar
e functionally coupled and work at only half their maximum rate. It is
known that the EII(Mtl) dimer, but not the monomer, catalyzes the man
nitol/mannitol-P exchange reaction. Since the heterodimer also catalyz
es this reaction, it appears that only one functional B domain per dim
er.