Time-resolved fluorescence of the single tryptophan of Bacillus stearothermophilus phosphofructokinase.
The fluorescence of the single tryptophan in Bacillus stearothermophilus phosphofructokinase was characterized by steady-state and time-resolved techniques. The enzyme is a tetramer of identical subunits, which undergo a concerted allosteric transition. Time-resolved emission spectral data were fitted to discrete and distributed lifetime models. The fluorescence decay is a double exponential with lifetimes of 1.6 and 4.4 ns and relative amplitudes of 40 and 60%. The emission spectra of both components are identical with maxima at 327 nm. The quantum yield is 0.31 +/- 0.01. The shorter lifetime is independent of temperature; the longer lifetime has weak temperature dependence with activation energy of 1 kcal/mol. The fluorescence intensity and decay are the same in H2O and D2O solutions, indicating that the indole ring is not accessible to bulk aqueous solution. The fluorescence is not quenched significantly by iodide, but it is quenched by acrylamide with bimolecular rate constant of 5 x 10(8) M-1 s-1. Static and dynamic light scattering measurements show that the enzyme is a tetramer in solution with hydrodynamic radius of 40 A. Steady-state and time-resolved fluorescence anisotropies indicate that the tryptophan is immobile. The allosteric transition has little effect on the fluorescence properties. The fluorescence results are related to the x-ray structure.
Kim
S J
SJ
Department of Chemistry, Louisiana State University, Baton Rouge 70803.
Chowdhury
F N
FN
Stryjewski
W
W
Younathan
E S
ES
Russo
P S
PS
Barkley
M D
MD
D000495:Allosteric Site; D055592:Biophysical Phenomena; D001703:Biophysics; D005454:Fluorescence Polarization; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D008027:Light; D010732:Phosphofructokinase-1 / Q000737:chemistry*; D011487:Protein Conformation; D012542:Scattering, Radiation; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry*
D012996:Solutions; D014364:Tryptophan; D010732:Phosphofructokinase-1