PROFILING OF PROTEASE SPECIFICITY USING COMBINATORIAL FLUOROGENIC SUBSTRATE LIBRARIES

摘要

Fluorogenic peptide substrates allow for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of enzymes, such as proteases. The substrates contain a fluorogenic-leaving group, such as 7-amino-4-carbamoylmethyl-coumarin (ACC). Substrates incorporating the ACC leaving group show comparable kinetic profiles as those with the traditionally used 7-amino-4-methyl-coumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries using solid-phase synthesis techniques. The approximately 3-fold increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations, so that a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Employing this screening method, the substrate specificities of a diverse array of proteases were profiled, including serine proteases and cysteine proteases.