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 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 (Ki = 35 microM, Kinact = 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 beta 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.
Covès
J
J
Laboratoire d'Etudes Dynamiques et Structurales de la Sélectivité, Université Joseph Fourier, Grenoble, France.
Le Hir de Fallois
L
L
Le Pape
L
L
Décout
J L
JL
Fontecave
M
M
D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D005609:Free Radicals; D010100:Oxygen / Q000737:chemistry; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013873:Thionucleotides / Q000494:pharmacology*; D014530:Uridine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology
C100424:2'-deoxy-2'-mercaptouridine 5'-diphosphate; D004791:Enzyme Inhibitors; D005609:Free Radicals; D013438:Sulfhydryl Compounds; D013873:Thionucleotides; D014530:Uridine Diphosphate; D012264:Ribonucleotide Reductases; D010100:Oxygen