Enzyme activity fingerprinting with substrate cocktails.
In the postgenomic era, emphasis is shifting from protein identification to protein functional analysis. Enzyme function can be characterized by measuring activity across series of substrates, which generates an activity profile or fingerprint. Activity fingerprinting is particularly useful to differentiate closely related enzymes. Previously reported fingerprinting methods use series of parallel measurements, which are complex and difficult to reproduce. Here we report a new method for fingerprinting enzyme activities based on using mixtures of substrates, or substrate cocktails, in a single reaction that is then analyzed by HPLC. The fingerprints produced are highly reproducible and allow functional differentiation and classification of closely related enzymes, as demonstrated for a series of lipases and esterases. The method is practical, general, and flexible in terms of reaction conditions and can be adapted to any reaction type.
Goddard
Jean-Philippe
JP
Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, 3012 Berne, Switzerland.
Reymond
Jean-Louis
JL
D004798:Enzymes / Q000032:analysis / Q000378:metabolism*; D004950:Esterases / Q000032:analysis / Q000378:metabolism; D008049:Lipase / Q000032:analysis / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods*
D004798:Enzymes; D004950:Esterases; D008049:Lipase