TIME-RESOLVED FLUORESCENCE OF THE SINGLE TRYPTOPHAN OF BACILLUS-STEAROTHERMOPHILUS PHOSPHOFRUCTOKINASE

The fluorescence of the single tryptophan in Bacillus stearothermophil us phosphofructokinase was characterized by steady-state and time-reso lved techniques. The enzyme is a tetramer of identical subunits, which undergo a concerted allosteric transition. Time-resolved emission spe ctral data were fitted to discrete and distributed lifetime models. Th e 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 yiel d 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 sam e in H2O and D2O solutions, indicating that the indole ring is not acc essible to bulk aqueous solution. The fluorescence is not quenched sig nificantly by iodide, but it is quenched by acrylamide with bimolecula r rate constant of 5 x 10(8) M-1 s-1. Static and dynamic light scatter ing measurements show that the enzyme is a tetramer in solution with h ydrodynamic radius of 40 angstrom. Steady-state and time-resolved fluo rescence anisotropies indicate that the tryptophan is immobile. The al losteric transition has little effect on the fluorescence properties. The fluorescence results are related to the x-ray structure.