On terminal alkynes that can react with active-site cysteine nucleophiles in proteases

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

doi:10.1021/ja309802n

On terminal alkynes that can react with active-site cysteine nucleophiles in proteases

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

Research output: Contribution to journal › Article › peer-review

296 Citations (Scopus)

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 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-1ß, a caspase-1 substrate.

Original language	English
Pages (from-to)	2867-70
Number of pages	4
Journal	Journal of the American Chemical Society
Volume	135
Issue number	8
DOIs	https://doi.org/10.1021/ja309802n
Publication status	Published - 27 Feb 2013

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title = "On terminal alkynes that can react with active-site cysteine nucleophiles in proteases",

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 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{\ss}, a caspase-1 substrate.",

author = "Reggy Ekkebus and \{van Kasteren\}, \{Sander I\} and Yogesh Kulathu and Arjen Scholten and Ilana Berlin and Geurink, \{Paul P\} and \{de Jong\}, Annemieke and Soenita Goerdayal and Jacques Neefjes and Heck, \{Albert J R\} and David Komander and Huib Ovaa",

year = "2013",

month = feb,

day = "27",

doi = "10.1021/ja309802n",

language = "English",

volume = "135",

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journal = "Journal of the American Chemical Society",

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TY - JOUR

T1 - On terminal alkynes that can react with active-site cysteine nucleophiles in proteases

AU - Ekkebus, Reggy

AU - van Kasteren, Sander I

AU - Kulathu, Yogesh

AU - Scholten, Arjen

AU - Berlin, Ilana

AU - Geurink, Paul P

AU - de Jong, Annemieke

AU - Goerdayal, Soenita

AU - Neefjes, Jacques

AU - Heck, Albert J R

AU - Komander, David

AU - Ovaa, Huib

PY - 2013/2/27

Y1 - 2013/2/27

N2 - 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-1ß, a caspase-1 substrate.

AB - 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-1ß, a caspase-1 substrate.

U2 - 10.1021/ja309802n

DO - 10.1021/ja309802n

M3 - Article

C2 - 23387960

SN - 0002-7863

VL - 135

SP - 2867

EP - 2870

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

IS - 8

ER -

On terminal alkynes that can react with active-site cysteine nucleophiles in proteases

Abstract

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