Inactivation of Escherichia coli Ribonucleotide Reductase by 2‘-Deoxy-2‘-mercaptouridine 5‘-Diphosphate. Electron Paramagnetic Resonance Evidence for a Transient Protein Perthiyl Radical

Ribonucleotide reductase catalyzes a key step in DNA biosynthesis and repair, supplying the cell with the four common deoxyribonucleotides. It is thus the target of antiproliferative agents. The enzyme consists of two subunits named protein R1 and protein R2. R1 provides the sites for the nucleotide...

Full description

Saved in:
Bibliographic Details
Published inBiochemistry (Easton) Vol. 35; no. 26; pp. 8595 - 8602
Main Authors Covès, Jacques, Le Hir de Fallois, Loïc, Le Pape, Laurent, Décout, Jean-Luc, Fontecave, Marc
Format Journal Article
LanguageEnglish
Published United States American Chemical Society 02.07.1996
Subjects
Online AccessGet full text

Cover

Loading…
More Information
Summary:Ribonucleotide reductase catalyzes a key step in DNA biosynthesis and repair, supplying the cell with the four common deoxyribonucleotides. It is thus the target of antiproliferative agents. The enzyme consists of two subunits named protein R1 and protein R2. R1 provides the sites for the nucleotide substrates and redox-active cysteines required for catalysis. R2 harbors a tyrosyl radical essential for activity. We show here that 2‘-deoxy-2‘-mercaptouridine 5‘-diphosphate, a substrate analog, is a very efficient inactivator of ribonucleotide reductase (K i = 35 μM, k inact = 0.18 s-1). Inactivation is due to specific scavenging of the protein R2 tyrosyl radical. This unique feature sets this compound apart from other mechanism-based inhibitors such as 2‘-azido- or 2‘-chloro-2‘-deoxyribonucleotide which induce partial or total protein R1 inactivation. During reaction, a transient organic radical was detected by EPR spectroscopy. Its g anisotropy (gz = 2.0620, gy = 2.0265, and gx = 2.0019) and its hyperfine structure are consistent with a perthiyl RSS• radical. The loss of the hyperfine structure by deuterium labeling of the β protons of R1 cysteines unambiguously shows that the perthiyl radical is located on protein R1. We thus conclude that inactivation of ribonucleotide reductase by 2‘-deoxy-2‘-mercaptouridine 5‘-diphosphate is due to an irreversible transfer of the radical located on protein R2 to a cysteine residue of protein R1.
Bibliography:ark:/67375/TPS-WPDTN2J1-N
istex:A9BEA5EFAC75F86A7DE95ADCC2588CB8F7EBD908
Abstract published in Advance ACS Abstracts, June 1, 1996.
Financial support from Rhône-Poulenc Rorer is gratefully acknowledged.
ObjectType-Article-2
SourceType-Scholarly Journals-1
ObjectType-Feature-1
content type line 23
ObjectType-Article-1
ObjectType-Feature-2
ISSN:0006-2960
1520-4995
DOI:10.1021/bi960355o