Qp. Weng et Gr. Jacobson, ROLE OF A CONSERVED HISTIDINE RESIDUE, HIS-195, IN THE ACTIVITIES OF THE ESCHERICHIA-COLI MANNITOL PERMEASE, Biochemistry, 32(41), 1993, pp. 11211-11216
The mannitol permease, an enzyme II of the phosphoenolpyruvate-depende
nt carbohydrate phosphotransferase system (PTS) of Escherichia coli, c
arries out the transport and phosphorylation of D-mannitol in this org
anism. Previous studies have shown that His-554 and Cys-384 in the man
nitol permease are sequentially phosphorylated in reactions necessary
for the transport and phosphorylation of the substrate. These residues
are located in a large cytoplasmic domain of the protein. Interaction
of the permease with mannitol, and its membrane translocation, howeve
r, must involve the N-terminal, transmembrane domain (EIIC domain) of
the protein. In this report, we use site-directed mutagenesis and muta
nt complementation to investigate the role of His-195 in the EIIC doma
in of the mannitol permease, a residue that is conserved in many PTS p
ermeases. In a previous report [Weng, Q.-P., Elder, J., & Jacobson, G.
R. (1992) J. Biol. Chem. 267, 19529-19535], we inferred a role for Hi
s-195 that involves its hydrogen-bonding ability. Here we show that Hi
s-195 plays a role in high-affinity mannitol binding. Moreover, mutant
complementation studies show that a functional His-195 must be on the
same subunit as a functional Cys-384 in a permease dimer for phosphot
ransfer to mannitol to occur. These results and kinetic studies of His
-195 mutant proteins imply that His-195 also may play an important rol
e in this phosphotransfer reaction. His-195 is predicted to be in a cy
toplasmic ''loop'' in the EIIC domain of the mannitol permease, in whi
ch several other residues have been shown to have roles in mannitol pe
rmease activity.