Sugar transport by the bacterial phosphotransferase system. Characterization of the Escherichia coli enzyme I monomer/dimer equilibrium by fluorescence anisotropy.
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.
Chauvin
F
F
McCollum-Pratt Institute, Johns Hopkins University, Baltimore, Maryland 21218.
Brand
L
L
Roseman
S
S
D001692:Biological Transport; D050260:Carbohydrate Metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005454:Fluorescence Polarization; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011721:Pyrenes; D013696:Temperature
D011721:Pyrenes; C030984:pyrene; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System