Sugar transport by the bacterial phosphotransferase system. Characterization of the Escherichia coli enzyme I monomer/dimer transition kinetics by fluorescence anisotropy.
Year: 1994
Type: article
Abstract: Enzyme I of the bacterial phosphoenolpyruvate: glycose phosphotransferase system (PTS) exists in a monomer/dimer (MID) equilibrium.These two forms are functionally different, and their interconversion may be a means of regulating the PTS.The M/D equilibrium was studied by fluorescence anisotropy of a pyrene derivative (Chauvin, F., Brand, L., and Roseman, S. (1994) J. Biol.Chem.269,20263-20269).In this paper, the kinetics of the transition is investigated.The following apparent rate constants were found for the M/D transition of phospho-Enzyme I in the presence of Mg2' and PEP at 6 "C: kX = 3.4 x lo3 M -~ s-l and kg = 1.04 x lo-' s-l.The association rate is especially slow, 2-3 orders of magnitude slower than the average dimerization rate determined for other proteins.Furthermore, the rate of quaternary structure changes matches that of enzymatic activity changes, as well as that of tertiary structure changes (Chauvin, F., Toptygin, D.,Roseman, S., and Brand, L. (1992) Biophys.Chen.44,163-173).Finally, the effect of two ligands is shown; PEP increases the relaxation rate by %fold at 23 "C, and Mg2' addition causes a 4-fold increase in the relaxation rate.The bacterial phosphotransferase system (PTS)' catalyzes sugar uptake by the cell.Previous studies have shown that PTS-sugar uptake is stringently regulated (1-5) and that Enzyme I may have a role in the regulation (1, 6-9).The accompanying paper (11) describes the M/D equilibrium of an Enzyme I-pyrene conjugate by fluorescence anisotropy.The aim of the present paper is t o describe the use of the same techniques to measure the rates of association and dissociation of Enzyme I and the influence of PTS ligands on these rates.EXPERIMENTAL PROCEDURES Materials-Pyrene maleimide was purchased from Molecular Probes.HPr was a gift from P. Coyle.The membranes from a Salmonella typhimurium deletion mutant, SB2950, were obtained as de- scribed (10).Methods-Enzyme I was purified, derivatized, phosphorylated, and dephosphorylated as described previously (11).The minimal buffer used throughout this study was: 10 mM KP,, 100 m M KC1,l mM EDTA, 0.2 m M dithiothreitol, pH 6.5 (Buffer B).In most of the studies, the following
Source: Journal of Biological Chemistry
Authors: F. Chauvin, L. Brand, S. Roseman
Institution: Johns Hopkins University
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
Awards: G2108027020, G2424284077
OpenAlex ID: W4239107634