Evidence by mutagenesis that Tyr(370) of the mouse ribonucleotide reductase R2 protein is the connecting link in the intersubunit radical transfer pathway.
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.
Rova
U
U
Department of Medical Biosciences, Medical Biochemistry, Umeâ University, SE-901 87 Umeâ, Sweden.
Adrait
A
A
Pötsch
S
S
Gräslund
A
A
Thelander
L
L
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
D005609:Free Radicals; D014443:Tyrosine; D007501:Iron; D012264:Ribonucleotide Reductases