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Article Dans Une Revue RSC Advances Année : 2019

Monitoring glycosidase activity for clustered sugar substrates, a study on β-glucuronidase

Résumé

Determination of glycosidase hydrolysis kinetics for a monovalent sugar substrate is relatively straightforward and classically achieved by monitoring the fluorescence signal released from the sugar-conjugated probe after enzymatic hydrolysis. Naturally occuring sugar epitopes are, however, often clustered on biopolymers or at biological surfaces, and previous reports have shown that glycosidase hydrolytic rates can differ greatly with multivalent presentation of the sugar epitopes. New probes are needed to make it easier to interpret the importance of substrate clustering towards a specific enzyme activity. In this work, we developed multivalent glucuronide substrates attached to fluorescent amino-coumarines through self-immolative linkers to enable real time-monitoring of the hydrolysing activity of E.coli b-glucuronidases (GUS) towards clustered substrates. GUS are exoglycosidases of considerable therapeutic interest cleaving b-D-glucuronides and are found in the lysosomes, in the tumoral microenvironment, and are expressed by gut microbiota. GUS showed a much lower catalytic efficiency in hydrolysing clustered glucuronides due to a significantly lower enzymatic velocity and affinity for the substrates. GUS was 52-fold less efficient in hydrolysing GlcA substrates presented on an octameric silsequioxane (COSS) compared with a monovalent GlcA of similar chemical structure. Thus, kinetic and thermodynamic data of GUS hydrolysis towards multivalent glucuronides were easily obtained with these new types of enzymatically-triggered probes. More generally, adapting the substrate nature and valency of these new probes, should improve understanding of the impact of multivalency for a specific enzyme.
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Dates et versions

hal-02992513 , version 1 (08-12-2020)

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Yoan Brissonnet, Guillaume Compain, Brigitte Renoux, Eva-Maria Krammer, Franck Daligault, et al.. Monitoring glycosidase activity for clustered sugar substrates, a study on β-glucuronidase. RSC Advances, 2019, 9 (69), pp.40263-40267. ⟨10.1039/c9ra08847d⟩. ⟨hal-02992513⟩
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