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Environment of Tryptophan 57 in Porcine Fructose‐1,6‐bisphosphatase Studied by Time‐resolved Fluorescence and Site‐directed Mutagenesis ¶

The environment of Trp57, 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 Trp57 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 Asp127 in contact with the indole side chain of Trp57. 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 Asp127 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.