A continuous fluorimetric assay for tail-specific protease

Abstract

A continuous fluorimetric assay for tail-specific protease (Tsp) has been developed using a fluorescence donor/quencher system, in which 5-[(2-aminoethyl) amino]naphthalene-1-sulfonic acid (EDANS) and 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) are attached to the N-terminus and the lysyl side chain of peptide AARAAK-(6-aminocaproyl)2-ENYALAA, respectively. Tsp-mediated cleavage of the Ala-Arg peptide bond separates the quencher, DABCYL, from the donor, EDANS, and results in a large increase in the fluorescent yield of EDANS (>50-fold). Using this sensitive assay, Escherichia coli tail-specific protease was shown to exhibit typical Michaelis-Menten kinetics with a kcat of 0.086 +/- 0.002 s-1, KM of 4.0 +/- 0.3 microM, and kcat/KM of 2.2 x 10(4) M-1 s-1. A control substrate, which only differs from the above substrate by having a charged residue (glutamate) at the C-terminus, showed drastically reduced activity to Tsp (kcat/KM = 58 M-1 s-1). A peptide containing the C-terminal sequence of the substrate, GRGYALAA, was shown to be a competitive inhibitor of Tsp with a KI value of 31 microM. These results demonstrate the utility of this assay for the rapid assessment of Tsp activity.