Fluorescent properties of the Escherichia coli D-xylose isomerase active site.
The consequences of active site mutations of the Escherichia coli D-xylose isomerase (E.C. 5.3.1.5) on substrate binding were examined by fluorescence spectroscopy. Site-directed mutagenesis of conserved tryptophan residues in the E. coli enzyme (Trp49 and Trp188) reveals that fluorescence quenching of these residues occurs during the binding of xylose by the wild-type enzyme. The fluorescent properties of additional active site substitutions at His101 were also examined. Substitutions of His101 which inactivate the enzyme were shown to have altered spectral characteristics, which preclude detection of substrate binding. In the case of H101S, a mutant protein with measurable isomerizing activity, substrate binding with novel fluorescent properties was observed, possibly the bound pyranose form of xylose under steady-state conditions.
Jamieson
A C
AC
Institute of Food Science, Cornell University, Ithaca, NY 14853.
Batt
C A
CA
D019747:Aldose-Ketose Isomerases; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014364:Tryptophan / Q000737:chemistry; D014961:X-Ray Diffraction; D014994:Xylose / Q000378:metabolism
D014364:Tryptophan; D004247:DNA; D014994:Xylose; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase