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A Fluorimetric Readout Reporting the Kinetics of Nucleotide‐induced Human Ribonucleotide Reductase Oligomerization
Author(s) -
Fu Yuan,
Lin Hongyu,
Wisitpitthaya Somsinee,
Blessing William,
Aye Yimon
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.722.6
Subject(s) - ribonucleotide reductase , nucleotide , ribonucleotide , chemistry , enzyme kinetics , kinetics , biophysics , biochemistry , enzyme , protein subunit , stereochemistry , biology , active site , physics , quantum mechanics , gene
Human ribonucleotide reductase (hRNR) is one of the primary targets of nucleotide cancer drugs in clinical use. The nucleotide‐induced oligomeric regulation of hRNR subunit a is increasingly recognized as an innate as well as a drug‐relevant mechanism to enzyme activity modulations. In the presence of negative feedback inhibitor dATP and leukemia drug clofarabine nucleotides [ClFD(T)P], hRNR‐a assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typically trigger a dimerization. To date, both knowledge about and tools to interrogate the oligomeric assembly pathway of RNR in any species in real time are lacking. To fill this void, we developed a fluorimetric assay that can reliably report on the oligomeric state changes of a with high sensitivity. The oligomerization‐directed fluorescence quenching of hRNR‐a covalently labeled with two different fluorophores enables the direct readout of hRNR dimeric and hexameric states. Proof of concept was demonstrated with all three known hexamerization inducers: dATP and ClFD(T)P.We then applied the newly developed platform to reveal the timescales of a self‐assembly pathway driven by the feedback regulator dATP. Dimerization of hRNR‐awas found to be the rate‐limiting step along the oligomerization pathway. (Dimerization rate constant: 6×10 4 M ‐1 s ‐1 ) Kinetics of the subsequent steps were estimated to be an order of magnitude faster. The study sheds new light on kinetic understanding of hRNR‐a oligomeric regulation in response to feedback inhibitors.