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Chembiochem. 2014 Nov 24;15(17):2598-2604. doi: 10.1002/cbic.201402368. Epub 2014 Sep 24.

A fluorimetric readout reporting the kinetics of nucleotide-induced human ribonucleotide reductase oligomerization.

Author information

1
Department of Chemistry and Chemical Biology Cornell University, Ithaca, NY 14853.
2
Department of Biochemistry Weill Cornell Medical College, New York, NY, 10065.
#
Contributed equally

Abstract

Human ribonucleotide reductase (hRNR) is a target of nucleotide chemotherapeutics in clinical use. The nucleotide-induced oligomeric regulation of hRNR subunit α is increasingly being recognized as an innate and drug-relevant mechanism for enzyme activity modulation. In the presence of negative feedback inhibitor dATP and leukemia drug clofarabine nucleotides, hRNR-α assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typically trigger α-dimerization. Currently, both knowledge of and tools to interrogate the oligomeric assembly pathway of RNR in any species in real time are lacking. We therefore developed a fluorimetric assay that reliably reports on oligomeric state changes of α with high sensitivity. The oligomerization-directed fluorescence quenching of hRNR-α, covalently labeled with two fluorophores, allows for direct readout of hRNR dimeric and hexameric states. We applied the newly developed platform to reveal the timescales of α self-assembly, driven by the feedback regulator dATP. This information is currently unavailable, despite the pharmaceutical relevance of hRNR oligomeric regulation.

KEYWORDS:

feedback inhibition; fluorescence reporter assay; human ribonucleotide reductase; oligomeric regulation; stopped-flow kinetics

PMID:
25256246
PMCID:
PMC4271543
DOI:
10.1002/cbic.201402368
[Indexed for MEDLINE]
Free PMC Article

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