Novel Fluorescent Phosphonic Acid Esters for Discrimination of Lipases and Esterases

摘要

Lipases and esterases are responsible for carboxylester hydrolysis inside and outside cells and are useful biocatalysts for (stereo)-selective modification of synthetic substrates.Here we describe novel fluorescent suicide inhibitors that differ in structure and polarity for screening and discrimination of lipolytic enzymes in enzyme preparations.The inhibitors covalently react with the enzymes to form fluorescent lipid-protein complexes that can be resolved by gel electrophoresis.The selectivities of the inhibitors were determined by using different (phospho)lipase,esterase and cholesterol esterase preparations.The results indicate that formation of an inhibitor-enzyme complex is highly dependent on the chemical structure of the inhibitor.We identified inhibitors with very low specificity,and other derivatives that were highly specific for certain subgroups of lipolytic enzymes such as lipases and cholesterol esterases.A combination of these substrate-analogous activity probes represents a useful toolbox for rapid identification and classification of serine hydrolase enzymes.