Ribonucleotide Reductase from <i>Escherichia coli</i>: Demonstration of a Highly Active Form of the Enzyme

Eriksson, Staffan

doi:10.1111/j.1432-1033.1975.tb02232.x

Cited by 30 publications

(14 citation statements)

References 13 publications

(12 reference statements)

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“…The ratios between the two activities were constant over the entire pH range measured with the highest activity values around pH 8.0 (data not shown). A similar pH optimum was observed previously for the wild type protein (50). If the pH dependence of ribonucleotide reductase activity is contributed by the position 441 residue, our results indicates that a glutamic and an aspartic side chain contribute similarly.…”

Section: Enzyme Activity Of Mutant R1 Proteins As Compared With Wildsupporting

confidence: 90%

A New Mechanism-based Radical Intermediate in a Mutant R1 Protein Affecting the Catalytically Essential Glu441 inEscherichia coli Ribonucleotide Reductase

Persson

Eriksson

Katterle

et al. 1997

Journal of Biological Chemistry

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The invariant active site residue Glu 441 in protein R1 of ribonucleotide reductase from Escherichia coli has been engineered to alanine, aspartic acid, and glutamic acid. Each mutant protein was structurally and enzymatically characterized. Glu 441 contributes to substrate binding, and a carboxylate side chain at position 441 is essential for catalysis. The most intriguing results are the suicidal mechanism-based reaction intermediates observed when R1 E441Q is incubated with protein R2 and natural substrates (CDP and GDP). In a consecutive reaction sequence, we observe at least three clearly discernible steps: (i) a rapid decay (k 1 > 1. Ribonucleotide reductase is an essential enzyme of all living cells and catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides. Several classes of ribonucleotide reductases with different subunit composition and cofactor requirements are known, but they all share a radical-based reaction mechanism (1).The aerobic class Ia ribonucleotide reductase from Escherichia coli is the best characterized enzyme. It consists of two components denoted protein R1 and protein R2, each of which is a homodimer. Protein R1 contains redox-active cysteines essential for catalysis. Cysteines 225, 439, and 462 are located at the active site, where all four physiological substrates (CDP, UDP, GDP, or ADP) can bind. R1 also contains two different allosteric sites that bind nucleoside triphosphate effector molecules. One site regulates the overall enzyme activity, and the other site determines the substrate specificity (2, 3). Protein R2 contains a stable tyrosyl free radical at position 122 and an adjacent dinuclear iron center (4 -6). The tyrosyl radical is essential for catalysis.The separate three-dimensional structures of protein R1 and of protein R2 are known (6 -9). A model-built holoenzyme complex of the R1 and R2 structures indicates that the distance between the active site in R1 and Tyr 122 in R2 is about 30 -40 Å (8). Chains of conserved hydrogen-bonded residues leading from the active site of R1 in the direction of Tyr 122 in R2, and vice versa, have been identified and are believed to be part of a radical transfer pathway between the two sites (1, 6 -9). Mutational analysis of the residues postulated to be involved in radical transfer between R1 and R2 during catalysis supports this hypothesis (4, 10 -14).

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Section: Enzyme Activity Of Mutant R1 Proteins As Compared With Wildsupporting

confidence: 90%

A New Mechanism-based Radical Intermediate in a Mutant R1 Protein Affecting the Catalytically Essential Glu441 inEscherichia coli Ribonucleotide Reductase

Persson

Eriksson

Katterle

et al. 1997

Journal of Biological Chemistry

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“…Such results show that the enzyme in the cell performs its function more efficiently and suggest that the isolated proteins represent a degenerate species. Evidence for this derives also from the finding that permeabilized cells of E. coli (138) and cell lysates on cellophane discs (139) show up to 50-fold higher activities than extracts prepared by alumina grinding. Similarly, freshly prepared "gentle lysates" give a high activity that rapidly decays on aging (139).…”

Section: Co Rrelation Of In VI Tro and In Vi Vo Activitiesmentioning

confidence: 93%

“…Evidence for this derives also from the finding that permeabilized cells of E. coli (138) and cell lysates on cellophane discs (139) show up to 50-fold higher activities than extracts prepared by alumina grinding. Similarly, freshly prepared "gentle lysates" give a high activity that rapidly decays on aging (139). Such bacterial extracts, as well as the cellophane system, show much less sig moidicity in the assay, and activity is not further stimulated by addition of an excess of complementary subunit.…”

Section: Co Rrelation Of In VI Tro and In Vi Vo Activitiesmentioning

confidence: 93%

Reduction of Ribonucleotides

Thelander¹,

Reichard²

1979

Annu. Rev. Biochem.

1,124

580

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“…Although the Trx and Grx concentrations (55,56) seem high enough in the cell to predict that they can drive RNR activity, the activity of the enzyme in vivo must have higher turnover particularly with the Grx system. The highly active enzyme in vivo remains to be isolated and characterized, as is also true for E. coli (6,17,57).…”

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confidence: 99%

Molecular Mechanisms of Thioredoxin and Glutaredoxin as Hydrogen Donors for Mammalian S Phase Ribonucleotide Reductase

Avval¹,

Holmgren

2009

Journal of Biological Chemistry

131

122

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Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in deoxyribonucleotide synthesis essential for DNA replication and repair. RNR in S phase mammalian cells comprises a weak cytosolic complex of the catalytic R1 protein containing redox active cysteine residues and the R2 protein harboring the tyrosine free radical. Each enzyme turnover generates a disulfide in the active site of R1, which is reduced by C-terminally located shuttle dithiols leaving a disulfide to be reduced. Electrons for reduction come ultimately from NADPH via thioredoxin reductase and thioredoxin (Trx) or glutathione reductase, glutathione, and glutaredoxin (Grx), but the mechanism has not been clarified for mammalian RNR. Using recombinant mouse RNR, we found that Trx1 and Grx1 had similar catalytic efficiency (k cat /K m ). With 4 mM GSH, Grx1 showed a higher affinity (apparent K m value, 0.18 M) compared with Trx1 which displayed a higher apparent k cat , suggesting its major role in S phase DNA replication. Surprisingly, Grx activity was strongly dependent on GSH concentrations (apparent K m value, 3 mM) and a Grx2 C40S mutant was active despite only one cysteine residue in the active site. This demonstrates a GSH-mixed disulfide mechanism for glutaredoxin catalysis in contrast to the dithiol mechanism for thioredoxin. This may be an advantage with the low levels of RNR for DNA repair or in tumor cells with high RNR and no or low Trx expression. Our results demonstrate mechanistic differences between the mammalian and canonical Escherichia coli RNR enzymes, which may offer an explanation for the nonconserved shuttle dithiol sequences in the C terminus of the R1. Ribonucleotide reductase (RNR),3 the essential enzyme generating the four deoxyribonucleoside triphosphates (dNTPs) required for DNA synthesis (1), plays a critical role in the high fidelity DNA replication and repair. The class Ia enzyme, present in all eukaryotes (from yeast to mammals), some bacteria, and some viruses, is composed of two subunits: R1 and R2 (2). The R2 protein (␤) is a homodimer (2 ϫ 45 kDa), and each polypeptide harbors a tyrosyl free radical that is generated and stabilized by an iron center (1, 2).The R1 protein (␣) contains the substrate-binding site, the active site including three cysteine residues, two redox active shuttle cysteines, and allosteric sites. The allosteric sites control substrate specificity and enzyme activity via binding of nucleotide triphosphates (3). In the absence of nucleotide effectors, the mammalian R1 is a 90-kDa monomer. Binding of effectors to the specificity site induces the formation of R1 dimers that interact with the R2 dimer, forming an active ␣ 2 ␤ 2 complex. However, the R1 hexamer induced by ATP/dATP (4) is suggested to form the major complex of RNR with the R2 dimer (5).In enzyme turnover, a disulfide is formed in the active site, from the catalytically active SH group of the cysteines; consequently the 2Ј-OH of the ribonucleoside, is replaced with a hydrogen atom (6). As shown in Escherichia coli RNR, whic...

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Ribonucleotide Reductase from Escherichia coli: Demonstration of a Highly Active Form of the Enzyme

Cited by 30 publications

References 13 publications

A New Mechanism-based Radical Intermediate in a Mutant R1 Protein Affecting the Catalytically Essential Glu441 inEscherichia coli Ribonucleotide Reductase

A New Mechanism-based Radical Intermediate in a Mutant R1 Protein Affecting the Catalytically Essential Glu441 inEscherichia coli Ribonucleotide Reductase

Reduction of Ribonucleotides

Molecular Mechanisms of Thioredoxin and Glutaredoxin as Hydrogen Donors for Mammalian S Phase Ribonucleotide Reductase

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