Fluorogenic substrates for the screening assay of transketolase through beta-elimination of umbelliferone—Development, scope and limitations

5-O-Coumarinyl-d-xylulose was studied as a fluorogenic substrate for the stereospecific assay of transketolase enzyme. Enzymatic C2–C3 cleavage released an α-hydroxyl, β-coumarinyl substituted aldehyde. Although the subsequent β-elimination step was rate limiting under chemical or enzymatic catalysis, we detected a TK activity as low as 0.7 mIU. To improve the fluorescence signal release, kinetic and product distribution analyses of this reaction were performed by LC/UV/MS coupling.