Reactivity of the tyrosyl radical of Escherichia coli ribonucleotide reductase -- control by the protein.
Ribonucleotide reductase is a key enzyme for DNA synthesis. Its small component, named protein R2, contains a tyrosyl radical essential for activity. Consequently, radical scavengers are potential antiproliferative agents. In this study, we show that the reactivity of the tyrosyl radical towards phenols, hydrazines, hydroxyurea, dithionite and ascorbate can be finely tuned by relatively small modifications of its hydrophobic close environment. For example, in this hydrophobic pocket, Leu77-->Phe mutation resulted in a protein with a much higher susceptibility to radical scavenging by hydrophobic agents. This might suggest that the protein is flexible enough to allow small molecules to penetrate in the radical site. When mutations keeping the hydrophobic character are brought further from the radical (for example Ile74-->Phe) the reactivity of the radical is instead very little affected. When a positive charge was introduced (for example Ile74-->Arg or Lys) the protein was more sensitive to negatively charged electron donors such as dithionite. These results allow us to understand how tyrosyl radical sites have been optimized to provide a good stability for the free radical.
Gerez
C
C
Laboratoire d'Etudes Dynamiques et Structurales de la Selectivité, Université Joseph Fourier, CNRS UMR 5616, Chimie-Recherche, Grenoble, France.
Elleingand
E
E
Kauppi
B
B
Eklund
H
H
Fontecave
M
M
D019943:Amino Acid Substitution; D001205:Ascorbic Acid / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004227:Dithionite / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D016166:Free Radical Scavengers / Q000378:metabolism; D005609:Free Radicals / Q000378:metabolism; D006834:Hydrazines / Q000378:metabolism; D006918:Hydroxyurea / Q000378:metabolism; D007700:Kinetics; D007930:Leucine; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010636:Phenols / Q000378:metabolism; D010649:Phenylalanine; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D016166:Free Radical Scavengers; D005609:Free Radicals; D006834:Hydrazines; D010636:Phenols; D011994:Recombinant Proteins; C066447:tyrosyl radical Y(D); D004227:Dithionite; C029424:hydrazine; D014443:Tyrosine; D010649:Phenylalanine; D012264:Ribonucleotide Reductases; D007930:Leucine; D001205:Ascorbic Acid; D006918:Hydroxyurea