University of Groningen 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 pKa of approximately 8 and increasing rates at higher pH. This rate was increased for all the mutants, but the pKas 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 "C showed that, of the two steps, binding and occlusion, involved in binding to wild-type EIImtl 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"" 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. The mannitol transport system of Escherichia coli is a phosphoenolpyruvate-dependent group-translocation system able to couple the transport of mannitol to its phosphorylation (Lolkema & Robillard, 1992; Postma et al., 1993). The transport protein EII"" consists of three domains, a membrane-bound C domain and two cytoplasmic domains, B and A. It is known that phosphorylated intermediates of the transport protein exist, His554 on the A domain and Cys384 on the B domain being the two residues which are phosphorylated (Pas & Robillard, 1988). The cysteine at position 384 is the second phosphorylation site; it receives ' This research was supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for Scientific Research (NWO). * To whom correspondence should be addressed (Telephone, 3150-634321; telefax, 31-50-634165; Email, G.T.ROBILLARD@CHEM. RUG.NL). * Present address: Department of Microbiology, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands. @ Abstract published in Advance ACS Abstrucfs, February 15, 1995. EII"" nomenclature: When refemng to domains which are covalently attached, we use the terminology "A domain, B domain, C domain, BA domain, etc.". When refemng to the domains which have been subcloned and expressed separately, we use the nomenclature IIA"' " for domain A of the mannitol-specific enzyme 11, IIBmtl for domain B of the mannitol-specific enzyme 11, IICmtl for domain C of the mannitol-specific enzyme 11, and IIBA"" for domain BA of the mannitol-specific enzyme 11. * Abbreviations: mtl, mannitol; man, mannose; glc, glucose; HPr, histidine-containing protein; EI, enzyme I of the phosphoenolpyruvatedependent carbohydrate transport system; DTT, dithiothreitol; decylPEG, decylpoly(ethy1ene glycol) 300; PEP, phosphoenolpyruvate; iso, inside out; rso, right side out. 0006-2960/95/0434-3239$09.00/0 0 its phosphoryl group via an internal transfer from the first site, His554, which is phosphorylated by a general cytoplasmic PTS protein, P-HF'r. The phosphoryl group transfer from P-HPr to mannitol-P is shown in Scheme 1.