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- Behravan, Gity, et al.
(författare)
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Formation of a free radical of the sulfenylimine type in the mouse ribonucleotide reductase reaction with 2'-azido-2'-deoxycytidine 5'-diphosphate
- 1995
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Ingår i: Biochimica et Biophysica Acta, Gene Structure and Expression. - : Elsevier BV. - 0167-4781 .- 1879-2634. ; 1264:3, s. 323-329
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Tidskriftsartikel (refereegranskat)abstract
- Mouse and Escherichia coli ribonucleotide reductases (RR) both belong to the same class of RR, where the enzyme consists of two non-identical subunits, proteins R1 and R2. A transient free radical was observed by EPR spectroscopy in the mouse RR reaction with the suicidal inhibitor 2′-azido-2′-deoxycytidine 5′-diphosphate. The detailed hyperfine structure of the EPR spectrum of the transient radical is somewhat different for the mouse and previously studied E. coli enzymes. When the positive allosteric effector ATP was replaced by the negative effector dATP, no transient radical was observed, showing that ‘normal' binding of the inhibitor to the substrate binding site is required. Using the mouse protein R2 mutants W 103Y and D266A, where the mutations have been shown to specifically block long range electron transfer between the active site of the R1 protein to the iron/radical site in protein R2, no evidence of transient radical was found. Taken together, the data suggest that the radical is located at the active site in protein R1, and is probably of the sulfenylimine type
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| 2. |
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| 3. |
- Rova, Ulrika, et al.
(författare)
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Evidence by mutagenesis that Tyr370 of the mouse ribonucleotide reductase R2 protein is the connecting link in the intersubunit radical transfer pathway
- 1999
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Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 274:34, s. 23746-23751
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Tidskriftsartikel (refereegranskat)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 ~35 Å 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 Tyr370 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.
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| 4. |
- Rova, Ulrika, et al.
(författare)
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Evidence by site-directed mutagenesis supports long-range electron transfer in mouse ribonucleotide reductase
- 1995
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 34:13, s. 4267-4275
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Tidskriftsartikel (refereegranskat)abstract
- Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, each inactive alone. The R1 protein binds the ribonucleotide substrates while the R2 protein contains a binuclear iron center and a tyrosyl free radical, essential for activity. The crystal structures of the corresponding Escherichia coli proteins suggest that the distance from the active site in R1 to the tyrosyl radical buffed in R2 is about 35 Å. Therefore, an electron pathway was suggested between the active site and the tyrosyl radical. Such a pathway could include a conserved tryptophan on the suggested RI interaction surface of R2 and a conserved aspartic acid hydrogen bonded both to the tryptophan and to a histidine iron ligand. To find experimental support for such an electron pathway, we have replaced the conserved tryptophan in mouse R2 with phenylalanine or tyrosine and the aspartic acid with alanine. All the mutated R2 proteins were shown to bind metal with the same affinity as native R2 and to form the binuclear iron center. In addition, the W103Y and D266A proteins formed a normal tyrosyl free radical while only low amounts of radical were observed in the W103F protein. Neither the kinetic rate constants nor the equilibrium dissociation constant of the R1/R2 complex was affected by the mutations as shown by BIAcore biosensor technique. However, all mutant R2 proteins were completely inactive in the enzymatic assay, supporting the hypothesis that the tryptophan and aspartic acid residues are important links in an amino acid residue specific long-range electron transfer.
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| 5. |
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| 6. |
- Schmidt, Peter Paul, et al.
(författare)
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Kinetic evidence that a radical transfer pathway in protein R2 of mouse ribonucleotide reductase is involved in generation of the tyrosyl free radical
- 1998
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Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 273:34, s. 21463-21472
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Tidskriftsartikel (refereegranskat)abstract
- Class I ribonucleotide reductases consist of two subunits, R1 and R2. The active site is located in R1; active R2 contains a diferric center and a tyrosyl free radical (Tyr()), both essential for enzymatic activity. The proposed mechanism for the enzymatic reaction includes the transport of a reducing equivalent, i.e. electron or hydrogen radical, across a 35-Å distance between Tyr() in R2 and the active site in R1, which are connected by a hydrogen-bonded chain of conserved, catalytically essential amino acid residues. Asp266 and Trp103 in mouse R2 are part of this radical transfer pathway. The diferric/Tyr() site in R2 is reconstituted spontaneously by mixing iron-free apoR2 with Fe(II) and O2. The reconstitution reaction requires the delivery of an external reducing equivalent to form the diferric/Tyr() site. Reconstitution kinetics were investigated in mouse apo-wild type R2 and the three mutants D266A, W103Y, and W103F by rapid freeze-quench electron paramagnetic resonance with ≤4 Fe(II)/R2 at various reaction temperatures. The kinetics of Tyr() formation in D266A and W103Y is on average 20 times slower than in wild type R2. More strikingly, Tyr() formation is completely suppressed in W103F. No change in the reconstitution kinetics was found starting from Fe(II)-preloaded proteins, which shows that the mutations do not affect the rate of iron binding. Our results are consistent with a reaction mechanism using Asp266 and Trp103 for delivery of the external reducing equivalent. Further, the results with W103F suggest that an intact hydrogen-bonded chain is crucial for the reaction, indicating that the external reducing equivalent is a H(). Finally, the formation of Tyr() is not the slowest step of the reaction as it is in Escherichia coli R2, consistent with a stronger interaction between Tyr() and the iron center in mouse R2. A new electron paramagnetic resonance visible intermediate named mouse X, strikingly similar to species X found in E. coli R2, was detected only in small amounts under certain conditions. We propose that it may be an intermediate in a side reaction leading to a diferric center without forming the neighboring Tyr().
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