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Probing enzymatic activity – a radical approach

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Abstract

Deubiquitinating enzymes (DUBs) are known to have numerous important interactions with the ubiquitin cascade and their dysregulation is associated with several diseases, including cancer and neurodegeneration. They are an important class of enzyme, and activity-based probes have been developed as an effective strategy to study them. Existing activity-based probes that target the active site of these enzymes work via nucleophilic mechanisms. We present the development of latent ubiquitin-based probes that target DUBs via a site selective, photoinitiated radical mechanism. This approach differs from existing photocrosslinking probes as it requires a free active site cysteine. In contrast to existing cysteine reactive probes, control over the timing of the enzyme–probe reaction is possible as the alkene warhead is completely inert under ambient conditions, even upon probe binding. The probe's reactivity has been demonstrated against recombinant DUBs and to capture endogenous DUB activity in cell lysate. This allows more finely resolved investigations of DUBs.

Graphical abstract: Probing enzymatic activity – a radical approach

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Article information


Submitted
17 Oct 2019
Accepted
03 Feb 2020
First published
06 Feb 2020

This article is Open Access
All publication charges for this article have been paid for by the Royal Society of Chemistry

Chem. Sci., 2020, Advance Article
Article type
Edge Article

Probing enzymatic activity – a radical approach

N. C. Taylor, G. Hessman, H. B. Kramer and J. F. McGouran, Chem. Sci., 2020, Advance Article , DOI: 10.1039/C9SC05258E

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