Tryptophan fluorescence reveals the conformational state of a dynamic loopin recombinant porcine fructose-1,6-bisphosphatase

Wild-type porcine fructose-1,6-bisphosphatase (FBPase) has no tryptophan re sidues. Hence, the mutation of Try57 to tryptophan places a unique fluoresc ent probe in the structural element (loop 52-72) putatively responsible for allosteric regulation of catalysis. On the basis of steady-state kinetics, circular dichroism spectroscopy, and X-ray crystallography, the mutation h as little effect on the functional and structural properties of the enzyme. Fluorescence intensity from the Trp57 mutant is maximal in the presence of divalent cations, fructose 6-phosphate and orthophosphate, which together stabilize an R-state conformation in which loop 52-72 is engaged with the a ctive site. The level of fluorescence emission decreases monotonically with increasing levels of AMP, an allosteric inhibitor, which promotes the T-st ate, disengaged-loop conformation. The titration of various metal-product c omplexes of the Trp57 mutant with fructose 2,6-bisphosphate (F26P(2)) cause s similar decreases in fluorescence, suggesting that F26P(2) and AMP indivi dually induce similar conformational states in FBPase. Fluorescence spectra , however, are sensitive to the type of divalent cation (Zn2+, Mn2+, or Mg2 +) and suggest conformations in addition to the R-state, loop-engaged and T -state, loop-disengaged forms of FBPase. The work presented here demonstrat es the utility of fluorescence spectroscopy in probing the conformational d ynamics of FBPase.