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

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• 作者：Reggy Ekkebus ; Sander I. van Kasteren ; Yogesh Kulathu ; Arjen Scholten ; Ilana Berlin ; Paul P. Geurink ; Annemieke de Jong ; Soenita Goerdayal ; Jacques Neefjes ; Albert J. R. Heck ; David Komander ; Huib Ovaa
• 刊名：The Journal of the American Chemical Society
• 出版年：2013
• 出版时间：February 27, 2013
• 年：2013
• 卷：135
• 期：8
• 页码：2867-2870
• 全文大小：297K
• 年卷期：v.135,no.8(February 27, 2013)
• ISSN：1520-5126

文摘

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 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-1尾, a caspase-1 substrate.