Fluorescence Quenching Studies on Enzyme I of the Bacterial Phosphotransferase System
Enzyme I is the first protein involved in the series of phosphoryl transfer steps catalyzed by the glycose: pyruvate phosphotransferase system (PTS) (Meadow et al., 1984). The PTS is responsible for the phosphoryla-tion and concomitant transport of its sugar substrates across the bacterial membrane, and in this process phosphoenolpyruvate (PEP) serves as the phosphoryl donor. It has been previously observed (Kukuruzinska et al., 1982) that the subunits of Enzyme I associate in a temperature-dependent manner to form dimers. The enzyme contains two tryptophans per subunit and their intrinsic emission have been characterized at 2°C for the monomer and 23°C for the dimer. The decay of the fluorescence intensity has been resolved into a double exponential decay for both the conformations of the protein (Neyroz et al., 1984). At 2°C a short lifetime of 3 ns and a long one of 7.5 ns were found that account for the 17% and 83% of the total emitted light, respectively. At 23°C the recovered decay constants were 3.7 ns and 7.5 ns with an increase of the fractional contribution of the short lifetime to 43% of the total intensity. Two different decay associated spectra (DAS) (Knutson et al., 1982) were obtained for each decay component. A spectrum with its maximum at 330 nm was found for the short lifetime and a second spectrum centered at 345 nm was resolved for the longer lifetime. Changes in the proportions of the two spectra obtained at 2°C and 23°C well reproduced the difference of amplitudes reported above when the protein was excited at 295 nm and the fluorescence observed at 340 nm.
KeywordsFluorescence Lifetime Tryptophan Residue Short Lifetime Fluorescence Decay Decay Component
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- Birks, J. B., 1970, Photophysics of Aromatic Molecules, New York, N.Y., Wiley-Interscience, pp. 433–447.Google Scholar
- Meadow, N. D., Kukuruzinska, M. A., and Roseman, S., 1984, in: “Enzymes of Biological Membranes”, A. Martonosi, ed., Plenum Press, New York, 3:523.Google Scholar
- Neyroz, P., Beechem, J. M., Roseman, S., and Brand, L., 1984, Photochem. and Photobiol., 39:41s.Google Scholar