Real‐time <scp>FRET</scp> assay for monitoring detyrosination by <scp>TMCP1</scp> and <scp>VASH2</scp>

Tubulin detyrosination is an important α-tubulin specific posttranslational modification which has been implicated in various disorders including neurodegeneration and cancer. As such, the enzymes involved in the generation of this modification emerged as promising therapeutic targets. Previous studies have identified the members of the vasohibin family, VASH1 and VASH2, as the first class of enzymes involved in the generation of detyrosination. Recently, we have discovered Tubulin MetalloCarboxyPeptidase 1 (TMCP1) as the second class of enzymes catalyzing this modification. Here we describe the development of a highly sensitive FRET-based enzymatic assay to study and monitor the activity of TMCP1 and VASH2. The originality of this assay lies in the use of 3-nitrotyrosine as a quencher, which not only restores fluorescence upon cleavage but also closely mimics the natural tyrosine substrate, ensuring optimal enzyme recognition. The selected fluorogenic substrate, named FS2, exhibited strong quenching efficiency and a high signal-to-noise ratio, allowing for real-time kinetic monitoring of TMCP1 and VASH2 activity. Enzyme kinetics, competition assays, and metal ion dependency studies confirmed the assay's specificity, robustness, and physiological relevance. This optimized assay provides a powerful and reliable tool for the future identification and characterization of inhibitors of α-tubulin detyrosination.