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Environment of Tryptophan 57 in Porcine Fructose‐1,6‐bisphosphatase Studied by Time‐resolved Fluorescence and Site‐directed Mutagenesis ¶
Author(s) -
Wen Jin,
Nelson Scott W.,
Honzatko Richard B.,
Fromm Herbert J.,
Petrich Jacob W.
Publication year - 2001
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1562/0031-8655(2001)0740679eotipf2.0.co2
Subject(s) - fructose 1,6 bisphosphatase , tryptophan , fluorescence , mutagenesis , chemistry , site directed mutagenesis , fructose , biochemistry , biophysics , biology , mutation , optics , physics , gene , mutant , amino acid
The environment of Trp 57 , introduced by the mutation of a tyrosine in the dynamic loop of porcine liver fructose‐1,6‐bisphosphatase (FBPase), was examined using time‐resolved fluorescence and directed mutation. The Trp 57 enzyme was studied previously by X‐ray crystallography and steady‐state fluorescence, the latter revealing an unexpected redshift in the wavelength of maximum fluorescence emission for the R‐state conformer. The redshift was attributed to the negative charge of Asp 127 in contact with the indole side chain of Trp 57 . Time‐resolved fluorescence experiments here reveal an indole side chain less solvent exposed and more rigid in the R‐state, than in the T‐state of the enzyme, consistent with X‐ray crystal structures. Replacement of Asp 127 with an asparagine causes a 6 nm blueshift in the wavelength of maximum fluorescence emission for the R‐state conformer, with little effect on the emission maximum of the T‐state enzyme. The data here support the direct correspondence between X‐ray crystal structures of FBPase and conformational states of the enzyme in solution, and provide a clear example of the influence of microenvironment on the fluorescence properties of tryptophan.