Deoxyribonucleoside diphosphate

FIGURE 2.4 Biosynthesis of 2-deoxyribonucleotides.

enzyme ribonucleotide reductase (RNR), also known as nucleoside diphosphate reductase (NDPR).

3.1. Structure and catalytic cycle of RNR

RNR plays a central role in cell growth and proliferation by ensuring a balanced supply of nucleotide precursors for DNA synthesis. The most extensively studied RNR is that from Escherichia coli, which is considered as a suitable prototype for the mammalian enzyme. In eukaryotes, RNR has two subunits containing each a tyrosine that generates and stabilizes a tyrosyl radical through a redox process that transforms the initial Fe (II) complex into a binuclear oxo-bridged Fe (III) complex. A high-resolution X-ray diffraction study has shown that the first iron atom is pentacoordinate, although it maintains an octahedral structure, while the second one is hexacoordinate2 (Fig. 2.5).

Although the Tyr-122 radical triggers the reductive process, it is too far away from the catalytic site. Therefore, it must generate a second radical in the vicinity of the substrate, probably a thiyl radical from Cys-439. The cysteine radical then abstracts the C3'-H atom of the nucleoside diphosphate substrate and generates the anion-radical 2.1, with prior or simultaneous deprotonation of the C3'-OH group by the Glu-441 residue of the enzyme. Two cysteine residues, probably Cys-225 and Cys-462, form the redox-active sulfhydryl pair responsible for the reduction of this radical. Thus, protonation of the C2'-OH and subsequent elimination of a molecule of water yields a cation that is stabilized by migration of the unpaired electron from C-30 to C-20 to give 2.2. The Cys-462 mercapto group transfers a proton and one electron to this radical to give 2.3, with concomitant formation of a disulfide anion radical, which then transfers one electron to the carbonyl group in 2.3, leading to 2.4. Radical 2.4 is transformed into 2.5 by a mechanism reverse to the one that produced 2.1, and the active center of the enzyme is finally regenerated by reduction of the newly formed disulfide unit by thioredoxin, a ubiquitous protein that has a pair of proximal cysteine residues, which reacts with the oxidized form of RNR via disulfide exchange (Fig. 2.6).3

It is interesting to note that the enzymatic reaction of RNR is initiated by the formation of free radical 2.2, even though the reactions leading to reductive elimination of the C2'-OH group are ionic. The reason for this type of mechanism may be the enhanced stability of 2.2 through the stabilizing effect of the radical at C-3 on the intermediate carbocation formed at C-2, as shown by the resonance structures in Fig. 2.7.


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