On Terminal Alkynes That Can React with Active-Site Cysteine Nucleophiles in Proteases

TitleOn Terminal Alkynes That Can React with Active-Site Cysteine Nucleophiles in Proteases
Publication TypeJournal Article
Year of Publication2013
AuthorsEkkebus, R., S.I. van Kasteren, Y. Kulathu, A. Scholten, I. Berlin, P.P. Geurink, A. de Jong, S. Goerdayal, J. Neefjes, A.J.R. Heck, D. Komander, H. Ovaa
JournalJournal of the American Chemical Society
Volume135
Issue8
Pagination2867-2870
Date PublishedFeb 27
ISBN Number0002-7863
Accession NumberWOS:000315618900001
Keywordsazides, conjugation, cycloaddition, deubiquitinating enzymes, identification, ligation, molecular-basis, proteomics, reveals, ubiquitin-based probes
Abstract

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. By replacing the C-terminal carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of deubiquitinating 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-1 beta, a caspase-1 substrate.

DOI10.1021/Ja309802n
Alternate JournalJ Am Chem Soc

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