Abstract
The enzyme, ribonucleotide reductase, catalyses the formation of deoxyribonucleotides from ribonucleotides, a reaction essential for DNA synthesis in all living cells. The Escherichia coli ribonucleotide reductase, which is the prototype of all known eukaryotic and virus-coded enzymes, consists of two nonidentical subunits, proteins B1 and B2. The B2 subunit contains an antiferromagnetically coupled pair of ferric ions and a stable tyrosyl free radical. EPR studies show that the tyrosyl radical, formed by loss of ferric ions and a stable tyrosyl free radical. EPR studies show that the tyrosyl radical, formed by loss of an electron, has its unpaired spin density delocalized in the aromatic ring of tyrosine. Effects of iron-radical interaction indicate a relatively close proximity between the iron center and the radical. The EPR signal of the radical can be studied directly in frozen packed cells of E. coli or mammalian origin, if the cells are made to overproduce ribonucleotide reductase. The hypothetic role of the tyrosyl free radical in the enzymatic reaction is not yet elucidated, except in the reaction with the inhibiting substrate analogue 2'-azido-CDP. In this case, the normal tyrosyl radical is destroyed with concomitant appearance of a 2'-azido-CDP-localized radical intermediate. Attempts at spin trapping of radical reaction intermediates have turned out negative. In E. coli the activity of ribonucleotide reductase may be regulated by enzymatic activities that interconvert a nonradical containing form and the fully active protein B2. In synchronized mammalian cells, however, the cell cycle variation of ribonucleotide reductase, studied by EPR, was shown to be due to de novo protein synthesis. Inhibitors of ribonucleotide reductase are of medical interest because of their ability to control DNA synthesis. One example is hydroxyurea, used in cancer therapy, which selectively destroys the tyrosyl free radical.
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Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Akerblom L., Ehrenberg A., Gräslund A., Lankinen H., Reichard P., Thelander L. Overproduction of the free radical of ribonucleotide reductase in hydroxyurea-resistant mouse fibroblast 3T6 cells. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2159–2163. doi: 10.1073/pnas.78.4.2159. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Atkin C. L., Thelander L., Reichard P., Lang G. Iron and free radical in ribonucleotide reductase. Exchange of iron and Mössbauer spectroscopy of the protein B2 subunit of the Escherichia coli enzyme. J Biol Chem. 1973 Nov 10;248(21):7464–7472. [PubMed] [Google Scholar]
- Averett D. R., Lubbers C., Elion G. B., Spector T. Ribonucleotide reductase induced by herpes simplex type 1 virus. Characterization of a distinct enzyme. J Biol Chem. 1983 Aug 25;258(16):9831–9838. [PubMed] [Google Scholar]
- Barlow T., Eliasson R., Platz A., Reichard P., Sjöberg B. M. Enzymic modification of a tyrosine residue to a stable free radical in ribonucleotide reductase. Proc Natl Acad Sci U S A. 1983 Mar;80(6):1492–1495. doi: 10.1073/pnas.80.6.1492. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Berglund O. Ribonucleoside diphosphate reductase induced by bacteriophage T4. III. Isolation and characterization of proteins B1 and B2. J Biol Chem. 1975 Sep 25;250(18):7450–7455. [PubMed] [Google Scholar]
- Blakley R. L., Orme-Johnson W. H., Bozdech J. M. Mechanism of Lactobacillus leichmannii ribonucleotide reductase studied with Coalpha-[alpha-(Aden-9-yl)]-Cobeta-adenosylcobamide (Pseudocoenzyme B12) as coenzyme. Biochemistry. 1979 May 29;18(11):2335–2339. doi: 10.1021/bi00578a031. [DOI] [PubMed] [Google Scholar]
- Carlson J., Fuchs J. A., Messing J. Primary structure of the Escherichia coli ribonucleoside diphosphate reductase operon. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4294–4297. doi: 10.1073/pnas.81.14.4294. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ehrenberg A., Reichard P. Electron spin resonance of the iron-containing protein B2 from ribonucleotide reductase. J Biol Chem. 1972 Jun 10;247(11):3485–3488. [PubMed] [Google Scholar]
- Engström Y., Eriksson S., Thelander L., Akerman M. Ribonucleotide reductase from calf thymus. Purification and properties. Biochemistry. 1979 Jul 10;18(14):2941–2948. doi: 10.1021/bi00581a004. [DOI] [PubMed] [Google Scholar]
- Eriksson S., Gräslund A., Skog S., Thelander L., Tribukait B. Cell cycle-dependent regulation of mammalian ribonucleotide reductase. The S phase-correlated increase in subunit M2 is regulated by de novo protein synthesis. J Biol Chem. 1984 Oct 10;259(19):11695–11700. [PubMed] [Google Scholar]
- Eriksson S., Sjöberg B. M., Hahne S. Ribonucleoside diphosphate reductase from Escherichia coli. An immunological assay and a novel purification from an overproducing strain lysogenic for phage lambdadnrd. J Biol Chem. 1977 Sep 10;252(17):6132–6138. [PubMed] [Google Scholar]
- Fasanella E. L., Gordy W. Electron spin resonance of an irradiated single crystal of L-tyrosine-HC. Proc Natl Acad Sci U S A. 1969 Feb;62(2):299–304. doi: 10.1073/pnas.62.2.299. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Galloway D. A., Swain M. A. Organization of the left-hand end of the herpes simplex virus type 2 BglII N fragment. J Virol. 1984 Mar;49(3):724–730. doi: 10.1128/jvi.49.3.724-730.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gibson T., Stockwell P., Ginsburg M., Barrell B. Homology between two EBV early genes and HSV ribonucleotide reductase and 38K genes. Nucleic Acids Res. 1984 Jun 25;12(12):5087–5099. doi: 10.1093/nar/12.12.5087. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gräslund A., Ehrenberg A., Thelander L. Characterization of the free radical of mammalian ribonucleotide reductase. J Biol Chem. 1982 May 25;257(10):5711–5715. [PubMed] [Google Scholar]
- Holmgren A. Thioredoxin. Annu Rev Biochem. 1985;54:237–271. doi: 10.1146/annurev.bi.54.070185.001321. [DOI] [PubMed] [Google Scholar]
- Joelson T., Uhlin U., Eklund H., Sjöberg B. M., Hahne S., Karlsson M. Crystallization and preliminary crystallographic data of ribonucleotide reductase protein B2 from Escherichia coli. J Biol Chem. 1984 Jul 25;259(14):9076–9077. [PubMed] [Google Scholar]
- Knappe J., Neugebauer F. A., Blaschkowski H. P., Gänzler M. Post-translational activation introduces a free radical into pyruvate formate-lyase. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1332–1335. doi: 10.1073/pnas.81.5.1332. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lankinen H., Gräslund A., Thelander L. Induction of a new ribonucleotide reductase after infection of mouse L cells with pseudorabies virus. J Virol. 1982 Mar;41(3):893–900. doi: 10.1128/jvi.41.3.893-900.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Larsen I. K., Sjöberg B. M., Thelander L. Characterization of the active site of ribonucleotide reductase of Escherichia coli, bacteriophage T4 and mammalian cells by inhibition studies with hydroxyurea analogues. Eur J Biochem. 1982 Jun 15;125(1):75–81. doi: 10.1111/j.1432-1033.1982.tb06653.x. [DOI] [PubMed] [Google Scholar]
- Levitzki A., Koshland D. E., Jr The role of negative cooperativity and half-of-the-sites reactivity in enzyme regulation. Curr Top Cell Regul. 1976;10:1–40. doi: 10.1016/b978-0-12-152810-2.50008-5. [DOI] [PubMed] [Google Scholar]
- McLauchlan J., Clements J. B. DNA sequence homology between two co-linear loci on the HSV genome which have different transforming abilities. EMBO J. 1983;2(11):1953–1961. doi: 10.1002/j.1460-2075.1983.tb01684.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Petersson L., Gräslund A., Ehrenberg A., Sjöberg B. M., Reichard P. The iron center in ribonucleotide reductase from Escherichia coli. J Biol Chem. 1980 Jul 25;255(14):6706–6712. [PubMed] [Google Scholar]
- Reichard P., Ehrenberg A. Ribonucleotide reductase--a radical enzyme. Science. 1983 Aug 5;221(4610):514–519. doi: 10.1126/science.6306767. [DOI] [PubMed] [Google Scholar]
- Sahlin M., Gräslund A., Ehrenberg A., Sjöberg B. M. Structure of the tyrosyl radical in bacteriophage T4-induced ribonucleotide reductase. J Biol Chem. 1982 Jan 10;257(1):366–369. [PubMed] [Google Scholar]
- Sjöberg B. M., Eklund H., Fuchs J. A., Carlson J., Standart N. M., Ruderman J. V., Bray S. J., Hunt T. Identification of the stable free radical tyrosine residue in ribonucleotide reductase. A sequence comparison. FEBS Lett. 1985 Apr 8;183(1):99–102. doi: 10.1016/0014-5793(85)80962-5. [DOI] [PubMed] [Google Scholar]
- Sjöberg B. M., Gräslund A., Eckstein F. A substrate radical intermediate in the reaction between ribonucleotide reductase from Escherichia coli and 2'-azido-2'-deoxynucleoside diphosphates. J Biol Chem. 1983 Jul 10;258(13):8060–8067. [PubMed] [Google Scholar]
- Sjöberg B. M., Gräslund A., Loehr J. S., Loehr T. M. Ribonucleotide reductase: a structural study of the dimeric iron site. Biochem Biophys Res Commun. 1980 Jun 16;94(3):793–799. doi: 10.1016/0006-291x(80)91304-2. [DOI] [PubMed] [Google Scholar]
- Sjöberg B. M., Loehr T. M., Sanders-Loehr J. Raman spectral evidence for a mu-oxo bridge in the binuclear iron center of ribonucleotide reductase. Biochemistry. 1982 Jan 5;21(1):96–102. doi: 10.1021/bi00530a017. [DOI] [PubMed] [Google Scholar]
- Sjöberg B. M., Reichard P., Gräslund A., Ehrenberg A. The tyrosine free radical in ribonucleotide reductase from Escherichia coli. J Biol Chem. 1978 Oct 10;253(19):6863–6865. [PubMed] [Google Scholar]
- Sjöberg B. M., Reichard P. Nature of the free radical in ribonucleotide reductase from Escherichia coli. J Biol Chem. 1977 Jan 25;252(2):536–541. [PubMed] [Google Scholar]
- Standart N. M., Bray S. J., George E. L., Hunt T., Ruderman J. V. The small subunit of ribonucleotide reductase is encoded by one of the most abundant translationally regulated maternal RNAs in clam and sea urchin eggs. J Cell Biol. 1985 Jun;100(6):1968–1976. doi: 10.1083/jcb.100.6.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stubbe J. A., Kozarich J. W. Fluoride, pyrophosphate, and base release from 2'-deoxy-2'-fluoronucleoside 5'-diphosphates by ribonucleoside-diphosphate reductase. J Biol Chem. 1980 Jun 25;255(12):5511–5513. [PubMed] [Google Scholar]
- Stubbe J. A. Mechanism of B12-dependent ribonucleotide reductase. Mol Cell Biochem. 1983;50(1):25–45. doi: 10.1007/BF00225278. [DOI] [PubMed] [Google Scholar]
- Stubbe J., Ackles D. On the mechanism of ribonucleoside diphosphate reductase from Escherichia coli. Evidence for 3'-C--H bond cleavage. J Biol Chem. 1980 Sep 10;255(17):8027–8030. [PubMed] [Google Scholar]
- Stubbe J., Ator M., Krenitsky T. Mechanism of ribonucleoside diphosphate reductase from Escherichia coli. Evidence for 3'-C--H bond cleavage. J Biol Chem. 1983 Feb 10;258(3):1625–1631. [PubMed] [Google Scholar]
- Thelander L., Eriksson S., Akerman M. Ribonucleotide reductase from calf thymus. Separation of the enzyme into two nonidentical subunits, proteins M1 and M2. J Biol Chem. 1980 Aug 10;255(15):7426–7432. [PubMed] [Google Scholar]
- Thelander L., Gräslund A. Mechanism of inhibition of mammalian ribonucleotide reductase by the iron chelate of 1-formylisoquinoline thiosemicarbazone. Destruction of the tyrosine free radical of the enzyme in an oxygen-requiring reaction. J Biol Chem. 1983 Apr 10;258(7):4063–4066. [PubMed] [Google Scholar]
- Thelander L., Gräslund A., Thelander M. Continual presence of oxygen and iron required for mammalian ribonucleotide reduction: possible regulation mechanism. Biochem Biophys Res Commun. 1983 Feb 10;110(3):859–865. doi: 10.1016/0006-291x(83)91040-9. [DOI] [PubMed] [Google Scholar]
- Thelander L., Larsson B. Active site of ribonucleoside diphosphate reductase from Escherichia coli. Inactivation of the enzyme by 2'-substituted ribonucleoside diphosphates. J Biol Chem. 1976 Mar 10;251(5):1398–1405. [PubMed] [Google Scholar]
- Thelander L. Reaction mechanism of ribonucleoside diphosphate reductase from Escherichia coli. Oxidation-reduction-active disulfides in the B1 subunit. J Biol Chem. 1974 Aug 10;249(15):4858–4862. [PubMed] [Google Scholar]
- Thelander L., Reichard P. Reduction of ribonucleotides. Annu Rev Biochem. 1979;48:133–158. doi: 10.1146/annurev.bi.48.070179.001025. [DOI] [PubMed] [Google Scholar]
- Thelander L., Sjöberg B. R., Eriksson S. Ribonucleoside diphosphate reductase (Escherichia coli). Methods Enzymol. 1978;51:227–237. doi: 10.1016/s0076-6879(78)51032-x. [DOI] [PubMed] [Google Scholar]
- Thelander M., Gräslund A., Thelander L. Subunit M2 of mammalian ribonucleotide reductase. Characterization of a homogeneous protein isolated from M2-overproducing mouse cells. J Biol Chem. 1985 Mar 10;260(5):2737–2741. [PubMed] [Google Scholar]