Campus Units
Biochemistry, Biophysics and Molecular Biology
Document Type
Article
Publication Version
Published Version
Publication Date
9-2000
Journal or Book Title
Biochemistry
Volume
39
Issue
36
First Page
11100
Last Page
11106
DOI
10.1021/bi000609c
Abstract
Wild-type porcine fructose-1,6-bisphosphatase (FBPase) has no tryptophan residues. Hence, the mutation of Try57 to tryptophan places a unique fluorescent 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 has 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 active site. The level of fluorescence emission decreases monotonically with increasing levels of AMP, an allosteric inhibitor, which promotes the T-state, disengaged-loop conformation. The titration of various metal−product complexes of the Trp57 mutant with fructose 2,6-bisphosphate (F26P2) causes similar decreases in fluorescence, suggesting that F26P2 and AMP individually 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 demonstrates the utility of fluorescence spectroscopy in probing the conformational dynamics of FBPase.
Copyright Owner
American Chemical Society
Copyright Date
2000
Language
en
File Format
application/pdf
Recommended Citation
Nelson, Scott W.; Iancu, Cristina Valeria; Choe, Jun-yong; Honzatko, Richard B.; and Fromm, Herbert J., "Tryptophan Fluorescence Reveals the Conformational State of a Dynamic Loop in Recombinant Porcine Fructose-1,6-bisphosphatase" (2000). Biochemistry, Biophysics and Molecular Biology Publications. 80.
https://lib.dr.iastate.edu/bbmb_ag_pubs/80
Comments
Reprinted (adapted) with permission from Biochemistry 39 (2000): 11100, doi: 10.1021/bi000609c. Copyright 2000 American Chemical Society.