Ribonucleotide reductase active site

The enzyme contains two catalytic sites, two regulatory sites and two specificity sites. The catalytic site binds the substrates, thioredoxin (reduced by NADPH + H+) and the nucleoside diphosphates. The allosteric regulatory site binds ATP as an activator in competition with dATP as an inhibitor. The specificity site binds dGTP, dTTP and dATP but not dCTP and modulates ribonucleotide reductase activity selectively for the four NDP substrates to balance the four dNTP pools. 

Enzyme from Mn-deficient cells showed no ribonucleotide reductase activity but could be activated by addition of Mn (215). Furthermore, Mn was incorporated into the B2 subunit when the bacteria were grown on MnCL-enriched medium (8). These experiments strongly implicate a manganese containing active site. 

The formation of deoxyribonucleotides requires ribonucleotide reductase activity, which catalyzes the reduction of ribose on nucleotide diphosphate substrates to 2 -deoxyribose. Substrates for the enzyme include adenosine diphosphate (ADP), guanosine diphosphate (GDP), cytidine diphosphate (CDP), and uridine diphosphate (UDP). Regulation of the enzyme is complex. There are two major allosteric sites. One controls the overall activity of the enzyme, whereas the other determines the substrate specificity of the enzyme. All deoxyribonucleotides are synthesized using this one enzyme. 

Lu S, Lihhy E, Saleh L, Xing G, Bollinger Jr JM, Moeime-Loccoz P. 2004. Characterization of NO adducts of the diiron center in protein R2 of Escherichia coli ribonucleotide reductase and site-directed variants implications for the O2 activation mechanism. J Biol Inorg Chem 9 818-827. 

Metalloenzymes with non-heme di-iron centers in which the two irons are bridged by an oxide (or a hydroxide) and carboxylate ligands (glutamate or aspartate) constitute an important class of enzymes. Two of these enzymes, methane monooxygenase (MMO) and ribonucleotide reductase (RNR) have very similar di-iron active sites, located in the subunits MMOH and R2 respectively. Despite their structural similarity, these metal centers catalyze very different chemical reactions. We have studied the enzymatic mechanisms of these enzymes to understand what determines their catalytic activity [24, 25, 39-41]. 

Ribonucleotide reductase is responsible for the conversion of the four biological ribonucleotides (RNA) into their corresponding deoxy forms (DNA). Although RNR is not an oxygenase during its primary catalyzed reaction (the conversion of ribonucleotides), it activates oxygen to generate a stable tyrosyl radical that is essential to the overall mechanism [46 49]. The common link between the chemistry of MMO and RNR is the activation of O2 by the di-iron active site. 

Interest in this class of coordination compounds was sparked and fueled by the discovery that radical cofactors such as tyrosyl radicals play an important role in a rapidly growing number of metalloproteins. Thus, in 1972 Ehrenberg and Reichard (1) discovered that the R2 subunit of ribonucleotide reductase, a non-heme metal-loprotein, contains an uncoordinated, very stable tyrosyl radical in its active site. In contrast, Whittaker and Whittaker (2) showed that the active site of the copper containing enzyme galactose oxidase (GO) contains a radical cofactor where a Cu(II) ion is coordinated to a tyrosyl radical. 

The function of the metal site in the oxygen-dependent radical enzymes galactose oxidase, amine oxidases, ribonucleotide reductase, and cytochrome c oxidase is inter alia to bind 02 in their reduced forms and undergo the appropriate redox chemistry to generate a metal-bound, activated oxygen species of variable nature. 

Figure 5. Active site structure of the met form of the E. coli R2 protein of ribonucleotide reductase as determined in a 2.2-A resolution X-ray crystallographic study (14, 102).

Figure 13.4 A proposed mechanism for all three classes of ribonucleotide reductases. Classes I and II RNRs require an active site Glu residue and a pair of redox-active Cys. Class HI RNRs lack the Glu and one of the Cys, and use formate as the reductant. (From Stubbe etal., 2001. Copyright 2001, with permission from Elsevier.)...

Studies on three different iron-sulfur enzyme systems, which all require S-adenosyl methionine—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. 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... 

The binuclear iron unit consisting of a (p,-oxo(or hydroxo))bis(p.-carboxylato)diiron core is a potential common structural feature of the active sites of hemerythrin, ribonucleotide reductase, and the purple acid phosphatases. Synthetic complexes having such a binuclear core have recently been prepared their characterization has greatly facilitated the comparison of the active sites of the various proteins. The extent of structural analogy among the different forms of the proteins is discussed in light of their spectroscopic and magnetic properties. It is clear that this binuclear core represents yet another stractural motif with the versatility to participate in different protein functions. 

The regulation of ribonucleotide reductase is complex. The substrate-specificity and activity of the enzyme are controlled by two allosteric binding sites (a and b) in the R1 subunits. ATP and dATP increase or reduce the activity of the reductase by binding at site a. Other nucleotides interact with site b, and thereby alter the enzyme s specificity. 

Magnesium is bound in the active site of D-xylose isomerase (Carrell et al., 1984, 1989 Farber et al., 1987 Key etal., 1988 Henrick a/., 1989). Here, the site at which two metals [from among Mg(II), Mn(ll), and Co(II)] bind is similar to that found for Fe(ll) in ribonucleotide reductase (Nordlund et al., 1990). The active site of xylose isomerase is shown in Fig. 28. Magnesium ions are preferred in ATP-utilizing enzyme reactions (Mildvan, 1987). 

Cyclic voltammetry has been also used for estimation of the rate constants for oxidation of water-soluble ferrocenes in the presence of HRP (131). There is a perfect match between the data obtained spectrophotometrically and electrochemically (Table IV), which proves that the cyclic voltammetry reveals information on the oxidation of ferrocenes by Compound II. It is interesting to note that an enzyme similar to HRP, viz. cytochrome c peroxidase, which catalyzes the reduction of H202 to water using two equivalents of ferrocytochrome c (133-136), is ca. 100 times more reactive than HRP (131,137). The second-order rate constant equals 1.4 x 106 M-1 s 1 for HOOCFc at pH 6.5 (131). There is no such rate difference in oxidation of [Fe(CN)e]4- by cytochrome c peroxidase and HRP (8). These comparisons should not however create an impression that the enzymatic oxidation of ferrocenes is always fast. The active-R2 subunit of Escherichia coli ribonucleotide reductase, which has dinuclear nonheme iron center in the active site, oxidizes ferrocene carboxylic acid and other water-soluble ferrocenes with a rate constant of... 

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. 

A likely mechanism for the ribonucleotide reductase reaction is illustrated in Figure 22-41. The 3 -ribonu-cleotide radical formed in step (T) helps stabilize the cation formed at the 2 carbon after the loss of H20 (steps and (3)). Two one-electron transfers accompanied by oxidation of the dithiol reduce the radical cation (step ). Step (5) is the reverse of step ( ) regenerating the active site radical (ultimately, the tyrosyl radical) and forming the deoxy product. The oxidized dithiol is reduced to complete the cycle (step ). Ini , coli, likely sources of the required reducing equivalents for this reaction are thioredoxin and glutaredoxin, as noted above. 

MECHANISM FIGURE 22-41 Proposed mechanism for ribonucleotide reductase. In the enzyme of . coli and most eukaryotes, the active thiol groups are on the R1 subunit the active-site radical (—X ) is on the R2 subunit and in . coli is probably a thiyl radical of Cys439 (see Fig. 22-40). Steps (T) through are described in the text. 

FIGURE 22-42 Regulation of ribonucleotide reductase by deoxynu-deoside triphosphates. The overall activity of the enzyme is affected by binding at the primary regulatory site (left). The substrate specificity of the enzyme is affected by the nature of the effector molecule bound... 

Ribonucleotide reductase is responsible for maintaining a balanced supply of the deoxyribonucleotides required for DNA synthesis. To achieve this, the regulation of the enzyme is complex. In addition to the single active site, there are two sites on the enzyme involved in regulating its activity (Figure 22.13). 

It was a surprise to discover that a mutant of E. coli lacking thioredoxin can still reduce ribonucleotides. In the mutant cells thioredoxin is replaced by glutaredoxin, whose active site disulfide linkage is reduced by glutathione rather than directly by NADPH. Oxidized glutathione is, in turn, reduced by NADPH and glutathione reductase. Thus, the end result is the same with respect to ribonucleotide reduction. 

A chain of hydrogen-bonded side chains apparently provides a pathway for transfer of an impaired electron from the active site to the Tyr 122 radical and from there to the radical generating center.363 The tyrosyl radical can be destroyed by removal of the iron by exposure to 02 or by treatment of ribonucleotide reductases with hydroxyurea, which reduces the radical and also destroys catalytic activity ... 

Figure 16-21 (A) Scheme showing the diiron center of the R2 subunit of E. coli ribonucleotide reductase. Included are the side chains of tyrosine 122, which loses an electron to form a radical, and of histidine 118, aspartate 237, and tryptophan 48. These side chains provide a pathway for radical transfer to the R1 subunit where the chain continues to tyrosines 738 and 737 and cysteine 429.354a c From Andersson et al.35ic (B) Schematic drawing of the active site region of the E. coli class IH ribonucleotide reductase with a plausible position for a model-built substrate molecule. Redrawn from Lenz and Giese373 with permission.

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