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Monitoring glycosidase activity for clustered sugar substrates, a study on β-glucuronidase
Yoan Brissonnet1, Guillaume Compain2, Brigitte Renoux2
1Université de Nantes, CEISAM, Chimie Et Interdisciplinarité, Synthèse, Analyse, Modélisation, UMR CNRS 6230, UFR des Sciences et des Techniques 2, rue de la Houssinière, BP 92208 44322 Nantes Cedex 3 France.
RSC Advances
|May 11, 2022
Summary
New fluorescent probes reveal that clustered sugar substrates significantly reduce the activity of E. coli beta-glucuronidases (GUS). This finding is crucial for understanding enzyme kinetics and developing targeted therapies.
Area of Science:
- Biochemistry
- Enzymology
- Chemical Biology
Background:
- Glycosidase hydrolysis kinetics are typically studied using monovalent sugar substrates.
- Naturally occurring sugars are often clustered on biopolymers, affecting enzyme activity.
- Existing methods lack probes to easily study the impact of substrate clustering on enzyme kinetics.
Purpose of the Study:
- To develop novel multivalent glucuronide substrates for real-time monitoring of E. coli beta-glucuronidases (GUS) activity.
- To investigate the effect of clustered substrate presentation on GUS hydrolytic rates.
- To obtain kinetic and thermodynamic data for GUS hydrolysis of multivalent glucuronides.
Main Methods:
- Synthesis of multivalent glucuronide substrates linked to fluorescent amino-coumarines via self-immolative linkers.
- Real-time monitoring of GUS enzymatic activity using the developed fluorescent probes.
- Kinetic analysis comparing hydrolysis rates of monovalent and multivalent substrates.
Main Results:
- E. coli beta-glucuronidases (GUS) exhibited significantly lower catalytic efficiency towards clustered glucuronide substrates.
- GUS showed a 52-fold decrease in efficiency when hydrolyzing octameric silsequioxane-presented glucuronides compared to monovalent ones.
- Reduced enzymatic velocity and affinity were observed for clustered substrates.
Conclusions:
- The developed probes enable straightforward kinetic and thermodynamic analysis of GUS hydrolysis on multivalent substrates.
- Substrate clustering substantially impacts GUS enzyme kinetics, reducing its efficiency.
- This approach can be adapted to study the influence of multivalency on other enzyme-substrate interactions.
