Clofarabine 5′-di and -triphosphates inhibit human ribonucleotide reductase by altering the quaternary structure of its large subunit
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Author(s)
Aye, Yimon
Stubbe, JoAnne
Date Issued
May 2011
Journal
Proceedings of the National Academy of Sciences of the United States of America
Publisher
National Academy of Sciences (U.S.)
Citation
Aye, Y., and J. Stubbe. “Clofarabine 5’-di and -triphosphates inhibit human ribonucleotide reductase by altering the quaternary structure of its large subunit.” Proceedings of the National Academy of Sciences 108.24 (2011): 9815-9820. ©2011 by the National Academy of Sciences.
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Final published version
Abstract
Human ribonucleotide reductases (hRNRs) catalyze the conversion of nucleotides to deoxynucleotides and are composed of alpha- and beta-subunits that form active alpha nbeta m (n, m = 2 or 6) complexes. alpha binds NDP substrates (CDP, UDP, ADP, and GDP, C site) as well as ATP and dNTPs (dATP, dGTP, TTP) allosteric effectors that control enzyme activity (A site) and substrate specificity (S site). Clofarabine (ClF), an adenosine analog, is used in the treatment of refractory leukemias. Its mode of cytotoxicity is thought to be associated in part with the triphosphate functioning as an allosteric inhibitor of hRNR. Studies on the mechanism of inhibition of hRNR by ClF di- and triphosphates (ClFDP and ClFTP) are presented. ClFTP is a reversible inhibitor (Ki = 40 nM) that rapidly inactivates hRNR. However, with time, 50% of the activity is recovered. D57N-alpha, a mutant with an altered A site, prevents inhibition by ClFTP, suggesting its A site binding. ClFDP is a slow-binding, reversible inhibitor (Ki=17 nM; t1/2 = 23 min). CDP protects alpha from its inhibition. The altered off-rate of ClFDP from E•ClFDP∗ by ClFTP (A site) or dGTP (S site) and its inhibition of D57N-alpha together implicate its C site binding. Size exclusion chromatography of hRNR or alpha alone with ClFDP or ClFTP, ± ATP or dGTP, reveals in each case that α forms a kinetically stable hexameric state. This is the first example of hexamerization of alpha induced by an NDP analog that reversibly binds at the active site.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Massachusetts Institute of Technology. Department of Chemistry
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DOI of Published Version
https://doi.org/10.1073/pnas.1013274108
