A bifunctionalized fluorogenic tetrasaccharide as a substrate to study cellulases.
Cellulases are usually classified as endoglucanases and cellobiohydrolases, but the heterogeneity of cellulose, in terms of particle size and crystallinity, has always represented a problem for the biochemical characterization of the enzymes. The synthesis of a bifunctionalized tetrasaccharide substrate suitable for measuring cellulase activity by resonance energy transfer is described. The substrate, which carries a 5-(2-aminoethylamino)-1-naphthalenesulfonate group on the non-reducing end and an indolethyl group on the reducing end, was prepared from beta-lactosyl fluoride and indolethyl beta-cellobioside by a chemoenzymatic approach using the transglycosylating activity of endoglucanase I of Humicola insolens as the key step. The bifunctionalized substrate has been used for the determination of the catalytic constants of H. insolens endoglucanase I and cellobiohydrolases I and II; this substrate could be of general use to measure the kinetic constants of cellulases able to act on oligomers of degree of polymerization <5. The data also provide evidence that cellobiohydrolases I and II are able to degrade an oligosaccharide substrate carrying non-carbohydrate substituents at both ends.
Armand
S
S
Centre de Recherches sur les Macromolécules Végétales, F-38041 Grenoble cedex 9, France.
Drouillard
S
S
Schülein
M
M
Henrissat
B
B
Driguez
H
H
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002480:Cellulase / Q000378:metabolism*; D004735:Energy Transfer; D007202:Indicators and Reagents; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D003904:Mitosporic Fungi / Q000201:enzymology; D008969:Molecular Sequence Data; D009282:Naphthalenesulfonates / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D009844:Oligosaccharides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D007202:Indicators and Reagents; D009282:Naphthalenesulfonates; D009844:Oligosaccharides; C104421:indolethyl 6-N-6-(5-(2-aminoethylamino)-1-naphthalenesulfonate)-6-deoxygalactopyranosyl-1-4-glucopyranosyl-1-4-glucopyranosyl-1-4-glycopyranoside; D002480:Cellulase