Fluorescently Labelled ATP Analogues for Direct Monitoring of Ubiquitin Activation.
ATP
PET
UBA1
fluorescent probes
ubiquitin
Journal
Chemistry (Weinheim an der Bergstrasse, Germany)
ISSN: 1521-3765
Titre abrégé: Chemistry
Pays: Germany
ID NLM: 9513783
Informations de publication
Date de publication:
15 May 2020
15 May 2020
Historique:
received:
02
03
2020
revised:
06
03
2020
pubmed:
11
3
2020
medline:
19
8
2020
entrez:
11
3
2020
Statut:
ppublish
Résumé
Simple and robust assays to monitor enzymatic ATP cleavage with high efficiency in real-time are scarce. To address this shortcoming, we developed fluorescently labelled adenosine tri-, tetra- and pentaphosphate analogues of ATP. The novel ATP analogues bear - in contrast to earlier reports - only a single acridone-based dye at the terminal phosphate group. The dye's fluorescence is quenched by the adenine component of the ATP analogue and is restored upon cleavage of the phosphate chain and dissociation of the dye from the adenosine moiety. Thereby the activity of ATP-cleaving enzymes can be followed in real-time. We demonstrate this proficiency for ubiquitin activation by the ubiquitin-activating enzymes UBA1 and UBA6 which represents the first step in an enzymatic cascade leading to the covalent attachment of ubiquitin to substrate proteins, a process that is highly conserved from yeast to humans. We found that the efficiency to serve as cofactor for UBA1/UBA6 very much depends on the length of the phosphate chain of the ATP analogue: triphosphates are used poorly while pentaphosphates are most efficiently processed. Notably, the novel pentaphosphate-harbouring ATP analogue supersedes the efficiency of recently reported dual-dye labelled analogues and thus, is a promising candidate for broad applications.
Identifiants
pubmed: 32154932
doi: 10.1002/chem.202001091
pmc: PMC7317923
doi:
Substances chimiques
Fluorescent Dyes
0
UBA6 protein, human
0
Adenosine Triphosphate
8L70Q75FXE
Ubiquitin-Activating Enzymes
EC 6.2.1.45
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
6279-6284Subventions
Organisme : Deutsche Forschungsgemeinschaft
ID : SFB 969
Informations de copyright
© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
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