Ribonucleotide reductases free radical mechanisms

Ribonucleotide reductase is the enzyme that catalyzes synthesis of deoxyribonucleoside diphophosphates (dNDPs) from ribonucleoside diphosphates (rNDPs). Ribonucleotide reductase reduces the hydroxyl at carbon 2 of the ribose sugar in the rNDP to a hydrogen, forming a deoxyribose sugar and a corresponding dNDP. A free-radical mechanism is involved in the reaction. Three classes of ribonucleotide reductases are known. 

Ribonucleotide reductase, the enzyme catalyzing the synthesis of dNDPs from rNDPs, reduces the hydroxyl at carbon 2 to a hydrogen via a free radical mechanism. The following three classes of ribonucleotide reductases are known ... 

Ribonucleotide reductase is notable in that its reaction mechanism provides the best-characterized example of the involvement of free radicals in biochemical transformations, once thought to be rare in biological systems. The enzyme in E. coli and most eukaryotes is a dimer, with subunits designated R1 and R2 (Fig. 22-40). The R1 subunit contains two lands of regulatory sites, as described below. The two active sites of the enzyme are formed at the interface between the R1 and R2 subunits. At each active site, R1 contributes two sulfhydryl groups required for activity and R2 contributes a stable tyrosyl radical. The R2 subunit also has a binuclear iron (Fe3+) cofactor that helps generate and stabilize the tyrosyl radicals (Fig. 22-40). The tyrosyl radical is too far from the active site to interact directly with the site, but it generates another radical at the active site that functions in catalysis. 

Studies on three different iron—sulfur enzyme systems which all require S-adenosylmethionine (SAM) — lysine 2,3-aminomutase, pyruvate-formate lyase, and anaerobic ribonucleotide reductase — have led to the identification of SAM as a major source of free radicals in living cells (for a recent review, see Atta et ak, 2010). As in the dehydratases, these systems have a [4Fe—4S] centre chelated by only three cysteines with one accessible coordination site. The cluster is active only in the reduced state [4Fe—4S] and appears to combine the two roles described previously, serving both as a ligand for substrate binding and as a redox catalyst (Figure 13.19). Their mechanism again requires that the exposed iron atom of the cluster shifts towards octahedral geometry as it binds... 

Figure 25.11 Ribonucleotide reductase mechanism. (1) An electron is transferred from a cysteine residue on R1 to a tyrosine radical on R2. generating a highly reactive cysteine thiyl radical. (2) This radical abstracts a hydrogen atom from C-3 of the ribose unil. (3) The radica at C-3 releases OH from the C-2 carbon atom. Combined with a proton from a second cysteine residue, the OH is eliminated as water. (4) A hydride ion is transferred from a third cysteine residue with the concomitant formation of a disulfide bond. (5) The C-3 radical recaptures the originally abstracted hydrogen atom. (6) An electron is transferred from R2 to reduce the thiyl radical, which also accepts a proton. The deoxyribonucleotide is free to leave Rl. The disulfide formed in the active site must be reduced to begin another cycle.

R1. It contains the active site. The two subunits make up the small subunit of the protein called R2, which contains the free radical. A clue to the mechanism of action of the enzyme (tyrosine free radical) is shown in Figure 22.14. Hydroxyurea, an inhibitor of ribonucleotide reductase, destroys the free radical. 

DNA synthesis depends on a balanced supply of the four deoxyribonucleotides [1]. In all living organisms, with no exception so far, this is achieved by reduction of the corresponding ribonucleotides (substrates can be either ribonucleoside diphosphates NDP or ribonucleoside triphosphates NTP) by NADPH (Scheme 10-1), through a complex free radical chemistry. The substrate specificity is modulated by a sophisticated allosteric mechanism which makes it possible for a single protein to regulate the reduction of all four conunon ribonucleotides. This aspect will not be discussed here. Three well-characterized classes of ribonucleotide reductases (RNRs) have been described so far, which all are radical metalloenzymes [2-5]. 

The ribonucleotide reductases (RNRs) catalyze the deoxygenation of nucleotides in the ratedetermining step of the biosynthesis of DNA. These enzymes have been categorized into three classes, each of which incorporates a metal site that initiates nucleotide reduction via processes that involve free radicals, in particular cysteinyl radicals. " Extensive studies of these processes have provided detailed insights into their mechanisms, as described in comprehensive reviews. Here we focus on the most thoroughly characterized class I enzymes, 4oi which in their resting state contain a stable tyrosyl radical in close proximity, but not directly coordinated, to a diiron cluster. 

Graslund A. 2002. Ribonucleotide reductase Kinetic methods for demonstrating radical transfer pathway in protein R2 of mouse enzyme in generation of tyrosyl free radical. In Enzyme kinetics and mechanism, Pt F detection and characterization of enzyme reaction intermediates, pp. 399 14. New York Academic Press. 

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