THERMODYNAMIC EVIDENCE FOR CONFORMATIONAL COUPLING BETWEEN THE B-DOMAIN AND C-DOMAIN OF THE MANNITOL TRANSPORTER OF ESCHERICHIA-COLI, ENZYME IIMTL

The transport across the cytoplasmic membrane and concomitant phosphor ylation of mannitol in Escherichia coli is catalyzed by the mannitol-s pecific transport protein from the phosphoenolpyruvate-dependent phosp hotransferase system, enzyme IImtl. Interactions between the cytoplasm ic B and the membrane embedded C domain play an important role in the catalytic cycle of this enzyme, but the nature of this interaction is largely unknown, We have studied the thermodynamics of binding of (i) mannitol to enzyme IImtl, (ii) the substrate analog perseitol to enzym e IImtl, (iii) perseitol to phosphorylated enzyme IImtl, and (iv) mann itol to enzyme IImtl treated with trypsin to eliminate the cytoplasmic domains. Analysis of the heat capacity increment of these reactions s howed that approximately 50-60 residues are involved in the binding of mannitol and perseitol, but far less in the phosphorylated state or a fter removal of the B domain, A model is proposed in which binding of mannitol leads to the formation of a contact interface between the two domains, either by folding of unstructured parts or by docking of pre existing surfaces, thus positioning the incoming mannitol close to the phosphorylation site on the B domain to facilitate the transfer of th e phosphoryl group.