title: Evidence by mutagenesis that Tyr(370) of the mouse ribonucleotide reductase R2 protein is the connecting link in the intersubunit radical transfer pathway.
abstract: Ribonucleotide reductase catalyzes all de novo synthesis of deoxyribonucleotides. The mammalian enzyme consists of two non-identical subunits, the R1 and R2 proteins, each inactive alone. The R1 subunit contains the active site, whereas the R2 protein harbors a binuclear iron center and a tyrosyl free radical essential for catalysis. It has been proposed that the radical properties of the R2 subunit are transferred approximately 35 A to the active site of the R1 protein, through a coupled electron/proton transfer along a conserved hydrogen-bonded chain, i.e. a radical transfer pathway (RTP). To gain a better insight into the properties and requirements of the proposed RTP, we have used site-directed mutagenesis to replace the conserved tyrosine 370 in the mouse R2 protein with tryptophan or phenylalanine. This residue is located close to the flexible C terminus, known to be essential for binding to the R1 protein. Our results strongly indicate that Tyr(370) links the RTP between the R1 and R2 proteins. Interruption of the hydrogen-bonded chain in Y370F inactivates the enzyme complex. Alteration of the same chain in Y370W slows down the RTP, resulting in a 58 times lower specific activity compared with the native R2 protein and a loss of the free radical during catalysis.
authors.affiliation: Department of Medical Biosciences, Medical Biochemistry, Umeâ University, SE-901 87 Umeâ, Sweden.
authors.forename: U
authors.initials: U
authors.lastname: Rova
authors.forename: A
authors.initials: A
authors.lastname: Adrait
authors.forename: S
authors.initials: S
authors.lastname: Pötsch
authors.forename: A
authors.initials: A
authors.lastname: Gräslund
authors.forename: L
authors.initials: L
authors.lastname: Thelander
mesh_terms: D000818:Animals; D002384:Catalysis; D005609:Free Radicals; D007501:Iron; D051379:Mice; D016297:Mutagenesis, Site-Directed; D012264:Ribonucleotide Reductases / Q000737:chemistry*; D013329:Structure-Activity Relationship; D014443:Tyrosine
chemical_list: D005609:Free Radicals; D014443:Tyrosine; D007501:Iron; D012264:Ribonucleotide Reductases