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 alpha-hydroxyl, beta-coumarinyl substituted aldehyde. Although the subsequent beta-elimination step was rate limiting under chemical or enzymatic catalysis, we detected a TK activity as low as 0.7mIU. To improve the fluorescence signal release, kinetic and product distribution analyses of this reaction were performed by LC/UV/MS coupling.