In Vitro Characterization of Enzymatic Properties and Inhibition of the p53R2 Subunit of Human Ribonucleotide Reductase

Jimin Shao, Bingsen Zhou, Lijun Zhu, Weihua Qiu, Yate Ching Yuan, Bixin Xi, Yun Yen

Research output: Contribution to journalArticle

76 Citations (Scopus)

Abstract

p53R2 is a newly identified subunit of ribonucleotide reductase (RR) and plays a crucial role in supplying precursors for DNA repair in a p53-dependent manner. In our current work, all three human RR subunit proteins (p53R2, hRRM2, and hRRM1) were prokaryotically expressed and highly purified. Using an in vitro [3H]CDP reduction assay, the activity of RR reconstituted with either p53R2 or hRRM2 was found to be time, concentration, and hRRM1 dependent. The kinetic activity of p53R2-containing RR was about 20-50% lower than that of hRRM2-containing RR. Using a synthetic heptapeptide to inhibit RR activity, it was shown that p53R2 bound to hRRM1 through the same COOH-terminal heptapeptide as hRRM2. However, hRRM2 had a 4.76-fold higher binding affinity for hRRM1 than p53R2, which may explain the reduced RR activity of p53R2 relative to hRRM2. Of interest, p53R2 was 158-fold more susceptible to the iron chelator deferoxamine mesylate than hRRM2, although the iron content of the two proteins determined by atomic absorption spectrometer was almost the same. To the contrary, p53R2 was 2.50-fold less sensitive than hRRM2 to the radical scavenger hydroxyurea, whereas EPR showed similar spectra of the tyrosyl radical in two proteins. Triapine, a new RR inhibitor, was equally potent for p53R2 and hRRM2. These inhibition studies showed that the iron center and tyrosyl radical are involved in RR activity for both p53R2 and hRRM2. The susceptibility differences to RR inhibitors between p53R2 and hRRM2 may lead to a new direction in drug design for human cancer treatment.

Original languageEnglish
Pages (from-to)1-6
Number of pages6
JournalCancer Research
Volume64
Issue number1
DOIs
Publication statusPublished - Jan 1 2004
Externally publishedYes

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

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