Sugar transport by the bacterial phosphotransferase system. Fluorescence studies of subunit interactions of enzyme I.
Enzyme I of the bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) exhibits a temperature-dependent monomer/dimer equilibrium. The accompanying paper (Han, M. K., Roseman, S., and Brand, L. (1990) J. Biol. Chem. 265, 1985-1995) shows that the C-terminal -SH residue (Cys-575) can be modified specifically with fluorescent probes such as pyrene maleimide. The derivative retains full enzyme activity, and is capable of forming dimers at room temperature. In the present studies, Enzyme I labeled in this way is found to exhibit a temperature-, concentration-, and pH-dependent monomer/dimer association. The kinetics of dimer formation of Enzyme I is measured in the following way. A derivatized Enzyme I sample is prepared with a pyrene moiety irreversibly attached to the C-terminal -SH residue and 5,5'-dithiobis-2-nitrobenzoic acid reversibly attached to the other 3 -SH residues. This modified enzyme does not form dimers at room temperature. Addition of dithiothreitol results in total release of the thionitrobenzoate anion within 2 min. After the three -SH groups are unblocked, steady-state and nanosecond time-resolved emission anisotropy measurements indicate the dimer is formed over a period of 30 min. In a similar experiment, little dimer formation is observed at 3 degrees C, at temperature at which the native enzyme also does not form dimers. Tryptophan fluorescence is also examined during the release of the thionitrobenzoate. After the completion of thionitrobenzoate release, additional slow steady-state tryptophan fluorescence changes are observed. These results suggest that dimer formation may be preceded by a conformational change following thionitrobenzoate release.
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
M K
MK
Han
J R
JR
Knutson
S
S
Roseman
L
L
Brand
D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008301:Maleimides / Q000378:metabolism; D009579:Nitrobenzoates / Q000494:pharmacology; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D017852:Phosphotransferases (Nitrogenous Group Acceptor); D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents / Q000378:metabolism; D013816:Thermodynamics
D046911:Macromolecular Substances; D008301:Maleimides; D009579:Nitrobenzoates; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C011136:thionitrobenzoic acid; D004228:Dithionitrobenzoic Acid; C003944:N-(3-pyrene)maleimide; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase