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CUPRA-ZYME: An Assay for Measuring Carbohydrate-Active Enzyme Activities, Pathways, and Substrate Specificities
Zhixiong Li1, Pavel I Kitov1, Elena N Kitova1
1Department of Chemistry , University of Alberta , Edmonton , Alberta , Canada T6G 2G2.
Analytical Chemistry
|January 22, 2020
Summary
A new assay, CUPRA-ZYME, uses mass spectrometry to quantify Carbohydrate-Active enZymes (CAZymes) activities and substrate specificities, overcoming challenges with glycosyltransferases and enabling new biomedical applications.
Area of Science:
- Biochemistry
- Enzymology
- Mass Spectrometry
Background:
- Carbohydrate-Active enZymes (CAZymes) are crucial for biological processes, industrial applications, and disease diagnostics.
- Measuring glycosyltransferase activity is challenging, unlike spectrophotometric methods for glycosyl hydrolases.
- Understanding CAZyme kinetics and substrate specificity is vital for biological and biotechnological exploitation.
Purpose of the Study:
- To introduce CUPRA-ZYME, a novel assay for quantifying CAZyme kinetic parameters, reaction pathways, and substrate specificities.
- To validate the CUPRA-ZYME assay using a known glycosyl hydrolase and demonstrate its utility for profiling enzyme specificities.
- To explore the influence of natural substrate structure and protein environment on CAZyme activity.
Main Methods:
- Development and implementation of the Competitive Universal Proxy Receptor Assay (CUPRA) in a time-resolved manner.
- Utilizing electrospray ionization mass spectrometry (ESI-MS) for quantitative measurements.
- Application to glycosyl hydrolases and glycosyltransferases using synthetic and natural substrates.
Main Results:
- CUPRA-ZYME accurately measures kinetic parameters and catalytic pathways, validated with human neuraminidase 3.
- The assay enables quantitative profiling of substrate specificities for both glycosidases and glycosyltransferases.
- Simultaneous measurement of CUPRA and natural substrates reveals the impact of glycoconjugate structure on CAZyme activity.
Conclusions:
- CUPRA-ZYME is a versatile and quantitative ESI-MS assay for comprehensive CAZyme characterization.
- This method addresses limitations in studying glycosyltransferases and offers insights into enzyme-substrate interactions.
- The assay holds significant potential for advancing CAZyme research in biomedical and biotechnological fields.
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