Time-resolved fluorescence of the single tryptophan of Bacillus stearothermophilus phosphofructokinase

Abstract

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.

Reevaluation of the accepted allosteric mechanism of phosphofructokinase from Bacillus stearothermophilus

“…For E. coli PFK, the interactions between substrates and effectors have been studied extensively in this manner (20)(21)(22). However, in the case of wild-type BsPFK, the single tryptophan is for the most part unresponsive to conformational changes induced by ligand binding (23). The only change in the fluorescence properties of wild-type BsPFK is a 10% decrease in the intensity upon the binding of the inhibitor PEP to the allosteric site (23).…”

“…However, in the case of wild-type BsPFK, the single tryptophan is for the most part unresponsive to conformational changes induced by ligand binding (23). The only change in the fluorescence properties of wild-type BsPFK is a 10% decrease in the intensity upon the binding of the inhibitor PEP to the allosteric site (23). This change has proven to be sufficient, in wild type and in the E161A͞R162A mutant, to assess the coupling interactions between Fru-6-P and PEP by monitoring the change in intensity upon PEP binding as a function of Fru-6-P concentration.…”

On the involvement of electron transfer reactions in the fluorescence decay kinetics heterogeneity of proteins

“…The origin of the complex fluorescence kinetics of Trp in proteins was a subject of extensive debate during the last two decades. The most interesting interpretations are the rotamer model (Szabo and Rayner 1980; Petrich et al 1983; Kim et al 1993; Dahms and Szabo 1997) and the recently proposed energy transfer model (Bajzer and Prendergast 1993). Nevertheless, some points remain unclear.…”

The effect of introducing small cavities on the allosteric inhibition of phosphofructokinase from Bacillus stearothermophilus

“…Many studies have attempted to uncover the source of increased thermal stability in thermophilic enzymes, but it appears that there is not a single molecular or thermodynamic explanation, although the added stability is generally evident at all temperatures [35]. In the case of BsPFK, Kim et al showed that the native tryptophan position is quite rigid [36]. Additional fluorescence studies using tryptophan-shifted mutants have revealed that a majority of the BsPFK structure is rigid [16, 37, 38].…”