Abstract
Enzyme I (EI), the first protein of the bacterial phosphotransferase system (PTS), exists in a monomer/dimer (M/D) equilibrium. We have proposed that the two species are functionally different and that their interconversion may regulate sugar transport via the PTS. The C-terminal Cys of Escherichia coli EI was reacted with pyrene maleimide (Han, M. K., Roseman, S., and Brand, L. (1990) J. Biol. Chem. 265, 1985-1995), and the pyrene conjugate used to characterize the M/D equilibrium by fluorescence anisotropy. The properties of unlabeled and pyrene-labeled EI are indistinguishable. Values for the apparent association constant, K'eq, and the steady-state anisotropy of the monomer and the dimer were obtained under a variety of conditions. K'eq increases 23-fold, from 0.45 x 10(5) to 10.7 x 10(5) M-1, as the temperature increases from 6 to 30 degrees C; the association appears to be entropically driven. Under all conditions tested, the K'eq for phospho-EI is 6-12-fold less than for dephospho-EI. For phospho-EI, PEP and Mg2+ induce a 240-fold increase of K'eq when both ligands are present. Based on these data, EI was preincubated under conditions that change K'eq, and the initial activities of the different species were determined at 37 degrees C in a PTS sugar phosphorylation assay with PEP as the phosphoryl donor. The initial rate depends on the M/D ratio; it is maximal when EI is 100% dimer, and zero when EI is 100% monomer. In the latter case, the rate gradually increases in the assay mixture. The results have important implications for how the PTS regulates sugar transport and other physiological phenomena.
Highlights
Enzyme I (EI), the first proteinof the bacterial phos- sugar uptake for about 1.5 min, followed by essentially no upphotransferase system (PTS), exists in a monomer/ditmakeerfor about 2 min, followed byuptake ata rate that thceells (M/D) equilibrium
We have proposed that the two spe- can handlemetabolically [6]. ( b )Transport of the non-metabocies are functionally different and that their intercon- lizable glucose analogue, methyl a-glucoside, by whole cells of version may regulate sugar transport via thePTS.The Salmonella typhimurium[7] and Staphylococcus aureus [8]ex
Enzyme I was purified from an overproducing strain of E. coli, accordingto a procedure described previously [22].The protein concentration was determined by the Lowry method [23],using bovine serum albumin as a standard,and by DTNB titrations (4 cysteinedmonomer), using the methods of Ellman [24]and Habeeb [25]as described by Han et al [15].In all cases, the "dimer fraction"( f D ) and "monomerfraction" (f,) relate to the proportion ofprotornersinvolved in the species, so thatf, + f, = 1.Enzymatic activity was determined as described by Weigel et al [22], in the presence of the other PTS proteins, where Enzyme I
Summary
Enzyme I (EI), the first proteinof the bacterial phos- sugar uptake for about 1.5 min, followed by essentially no upphotransferase system (PTS), exists in a monomer/ditmakeerfor about 2 min, followed byuptake ata rate that thceells (M/D) equilibrium. ( b )Transport of the non-metabocies are functionally different and that their intercon- lizable glucose analogue, methyl a-glucoside, by whole cells of version may regulate sugar transport via thePTS.The Salmonella typhimurium[7] and Staphylococcus aureus [8]ex-. A brief, initial rapid rate of uptake is pyrene maleimide Fluorescenceanisotropy.Theproperties of unlabeled When methyl a-glucoside is taken upby S. typhimurium memand pyrene-labeledE1 are indistinguishable.Values for brane vesicles, a system withfewer variables, similar data are the apparent association constant,K&, and the steady- obtained, and thereis no obvious explanation for the results[9]. PTS inEscherichia coli and S. typhimurium may itself be regu-
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