A Continuous Fluorimetric Assay for Tail-Specific Protease

✍ Scribed by Kirk D. Beebe; Dehua Pei

• Publisher: Elsevier Science
• Year: 1998
• Tongue: English
• Weight: 116 KB
• Volume: 263
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1998.2797

✦ Synopsis

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-(4dimethylaminophenylazo)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 k cat of 0.086 ؎ 0.002 s ؊1 , K M of 4.0 ؎ 0.3 M, and k cat /K M of 2.2 ؋ 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 (k cat /K M ‫؍‬ 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 K I value of 31 M. These results demonstrate the utility of this assay for the rapid assessment of Tsp activity.