title: Fluorogenic substrates for the screening assay of transketolase through beta-elimination of umbelliferone--Development, scope and limitations.
abstract: 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.
authors.affiliation: Clermont Université, Université Blaise Pascal, Laboratoire SEESIB, BP 10448, F-63000 Clermont-Ferrand, France. Franck.CHARMANTRAY@univ-bpclermont.fr
authors.forename: F
authors.initials: F
authors.lastname: Charmantray
authors.forename: B
authors.initials: B
authors.lastname: Légeret
authors.forename: V
authors.initials: V
authors.lastname: Hélaine
authors.forename: L
authors.initials: L
authors.lastname: Hecquet
mesh_terms: D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D057230:Limit of Detection; D013058:Mass Spectrometry; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013237:Stereoisomerism; D014153:Transaldolase / Q000378:metabolism; D014174:Transketolase / Q000378:metabolism*; D014468:Umbelliferones / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D014468:Umbelliferones; D012710:Serum Albumin, Bovine; C031477:7-hydroxycoumarin; D014174:Transketolase; D014153:Transaldolase