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Updated: Jul 12, 2026
10:26
Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations
Published on: November 7, 2019
Enhanced subunit interactions with gemcitabine-5'-diphosphate inhibit ribonucleotide reductases
Jun Wang1, Gregory J S Lohman, JoAnne Stubbe
1Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Summary
Substoichiometric inhibition of ribonucleotide reductases (RNRs) by F2CDP leads to covalent modification and altered complex formation. This mechanism explains RNR inactivation by low drug concentrations, impacting cancer treatment.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Ribonucleotide reductases (RNRs) are essential enzymes for DNA synthesis, catalyzing nucleotide to deoxynucleotide conversion.
- Class I RNRs feature alpha and beta subunits with varying quaternary structures (e.g., α2β2, α6β6).
- 2',2'-difluoro-2'-deoxycytidine (F2C) is a cancer therapeutic; its phosphorylated form, F2CDP, inhibits RNRs.
Purpose of the Study:
- Investigate the mechanism of RNR inhibition by F2CDP.
- Determine the structural consequences of F2CDP-mediated inactivation in E. coli and human RNRs.
- Elucidate how substoichiometric F2CDP amounts lead to complete enzyme inactivation.
Main Methods:
- Enzyme kinetics assays with radiolabeled F2CDP ([1'-(3)H]-F2CDP and [5-(3)H]-F2CDP).
- Size exclusion chromatography to analyze RNR complex formation.
- Isolation and characterization of inactivated RNR complexes.
Main Results:
- F2CDP acts as a substoichiometric, mechanism-based inhibitor (0.5 eq F2CDP/α) of both E. coli and human RNRs.
- Inactivation involves covalent sugar labeling of RNR (0.5 eq/α), cytosine release, and a 40% loss of β2 activity.
- E. coli RNR forms an α2β2 complex, while human RNR forms an α6β6 complex upon inactivation.
- F2CDP and ATP significantly enhance subunit interactions, stabilizing the inactivated complexes.
Conclusions:
- F2CDP causes substoichiometric inactivation of RNR through covalent modification and altered quaternary structure.
- The formation of stable, higher-order complexes (α6β6 in humans) explains complete inactivation with low inhibitor concentrations.
- Understanding this mechanism provides insights into RNR regulation and potential therapeutic strategies.
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