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 site s for the nucleotide substrates and redox-active cysteines required fo r catalysis. R2 harbors a tyrosyl radical essential for activity. We s how here that 2'-deoxy-2'-mercaptouridine 5'-diphosphate, a substrate analog, is a very efficient inactivator of ribonucleotide reductase (K -i = 35 mu M, 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 tot al protein R1 inactivation. During reaction, a transient organic radic al was detected by EPR spectroscopy. Its g anisotropy (g(z) = 2.0620, g(y) = 2.0265, and g(x) = 2.0019) and its hyperfine structure are cons istent with a perthiyl RSS(.) radical. The loss of the hyperfine struc ture by deuterium labeling of the beta protons of R1 cysteines unambig uously shows that the perthiyl radical is located on protein R1. We th us conclude that inactivation of ribonucleotide reductase by 2'-deoxy- 2'-mercaptouridine 5'-diphosphate is due to an irreversible transfer o f the radical located on protein R2 to a cysteine residue of protein R 1.