A Fluorimetric Readout Reporting the Kinetics of Nucleotide-Induced Human Ribonucleotide Reductase Oligomerization

作者：Fu Yuan; Lin Hong Yu; Wisitpitthaya Somsinee; Blessing William A; Aye Yimon^*

来源：ChemBioChem, 2014, 15(17): 2598-2604.

DOI：10.1002/cbic.201402368

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

Human ribonucleotide reductase (hRNR) is a target of nucleotide chemotherapeutics in clinical use. The nucleotide-induced oligomeric regulation of hRNR subunit a 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-alpha assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typically trigger alpha-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 alpha with high sensitivity. The oligomerization-directed fluorescence quenching of hRNR-alpha, 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 alpha self-assembly, driven by the feedback regulator dATP. This information is currently unavailable, despite the pharmaceutical relevance of hRNR oligomeric regulation.

• 出版日期2014-11-24