A Continuous Fluorimetric Assay for Tumor Necrosis Factor-α Converting Enzyme

Abstract

Fluorogenic peptide substrates with fluorophore/quencher-capped ends have found extensive use in monitoring protease activity in the screening of small-molecule libraries for protease inhibitors. We report here the identification and characterization of a fluorogenic substrate for tumor necrosis factor-α converting enzyme (TACE). This substrate is a 10-amino-acid peptide (LAQAVRSSSR) capped with an o-aminobenzoyl group on the N-terminal end and with a 3-(2,4-dinitrophenyl)-l-2,3-diaminopropionic amide group on the C-terminal end. Exhaustive enzymatic conversion of the substrate to products resulted in a fluorescence enhancement of ∼11-fold. A single cleavage occurred at the A-V scissile bond of the peptide. The validity of this fluorimetric assay for TACE was corroborated by an independent HPLC method. Interestingly, the hydrolysis of the substrate displayed positive cooperativity with a Hill coefficient of 1.5, while the hydrolysis of the corresponding uncapped peptide displayed Michaelis–Menten kinetics. A kcat value of 21.6 s−1 and an S0.5 value of 342 μM were obtained for the fluorogenic substrate. The addition of the two capping groups on the two ends of the peptide enhanced the kcat value by 64-fold. Nine additional decapeptides that contained the same capping groups on the two ends and substitutions at the P1 and P1′ sites were also tested. TACE appears to slightly prefer the A-V scissile bond. The enzyme also cleaves scissile bonds such as F-V, A-I, and A-L efficiently.