On Terminal Alkynes That Can React with Active-Site Cysteine Nucleophiles in Proteases
Active-site directed probes are powerful in studies of enzymatic function. We report an active-site directed probe based on a warhead so far considered unreactive.
- Ekkebus R., Kasteren S.I. van, Kulathu Y., Scholten A., Berlin I., Geurink P.P., Jong A. de, Goerdayal S., Neefjes J., Heck A.J.R., Komander D., Ovaa H.
- 06 February 2013
- DOI link
By replacing the C-terminal carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of de-ubiquitinating enzymes was observed. The resulting product was shown to be a quaternary vinyl thioether, as determined by X-ray crystallography. Proteomic analysis of proteins bound to an immobilized Ub alkyne probe confirmed the selectivity toward de-ubiquitinating enzymes. The observed reactivity is not just restricted to propargylated Ub, as highlighted by the selective reaction between caspase-1 (interleukin converting enzyme) and a propargylated peptide derived from IL-1β, a caspase-1 substrate.