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 pK(a), of approximately 8 and increasing rates at higher pH. This rate was increased for all the mutants, but the pK(a)s 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 EII(mtl) 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 EII(mtl) influence each other's conformation. Residues on either the B or C domain close to the second phosphorylation site, C384, play an important role in this process and may provide a mechanism by which the energy coupling within this enzyme takes place.