PMID:8841134

Citation	Boer, H, ten Hoeve-Duurkens, RH and Robillard, GT (1996) Relation between the oligomerization state and the transport and phosphorylation function of the Escherichia coli mannitol transport protein: interaction between mannitol-specific enzyme II monomers studied by complementation of inactive site-directed mutants. Biochemistry 35:12901-8
Abstract	Previous experiments with the mannitol-specific enzyme II of Escherichia coli, EIImtl, have demonstrated that (1) the enzyme is a dimer, (2) the dimer is necessary for maximum activity, and (3) phosphoryl groups could be transferred between EIImtl subunits [van Weeghel et al. (1991) Biochemistry 30, 1768-1773; Weng et al. (1992) J. Biol. Chem. 267, 19529-19535; Weng & Jacobson (1993) Biochemistry 32, 11211-11216; Stolz et al. (1993) J. Biol. Chem. 268, 27094-27099]. The experiments in this article address the mechanistic role of the dimer. They indicate that the A, B, and C domains of EIImtl preferentially interact within the same subunit. Site-directed mutants in each of the three domains of EIImtl were used to study phosphoryl group transfer by the EIImtl dimer in vitro and mannitol transport in vivo. The C domain mutant, EIImtl-G196D, which was unable to bind mannitol, and the separated C domain, IICmtl, which was unable to phosphorylate mannitol, formed a heterodimer which was capable of mannitol phosphorylation in vitro and mannitol transport in vivo. The rates of phosphorylation were approximately 10-fold lower in heterodimers containing two inactive subunits relative to the rates in heterodimers containing one inactive and one wild type subunit; phosphoryl group transfer through one subunit is kinetically preferred to intersubunit transfer. Heterodimers formed in vivo between one wild type EIImtl subunit and the CB domain double mutant, EIImtl-G196D/C384S, transported mannitol as rapidly as wild type EIImtl alone; the presence of the inactive double mutant subunit did not reduce the transport rate. Thus, only one active A, B, and C domain in the dimer is sufficient for transport and phosphorylation activity, and if all three domains are situated on the same subunit, maximum rates are achieved.
Links	PubMed Online version:10.1021/bi9611016
Keywords	Biological Transport; Dimerization; Electrophoresis, Polyacrylamide Gel; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins; Genetic Complementation Test; Kinetics; Mannitol/metabolism; Models, Genetic; Monosaccharide Transport Proteins; Mutagenesis, Site-Directed; Phosphates/metabolism; Phosphoenolpyruvate/metabolism; Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry; Phosphoenolpyruvate Sugar Phosphotransferase System/genetics; Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism; Phosphorylation; Protein Conformation
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